ffe

ctu

era,

e V

tate U

sity o

for re

ne 24

valuable insights into maternalfetal and trans-mucosal

transmission, design and testing of anti-retroviral agents,

Biek et al., 2003; Brown et al., 1994; Carpenter et al., 1996;

Troyer et al., 2004, 2005). Additionally, sequence compar-

ison of pol sequences from lion and puma lentiviruses (LLV,

-Pco) demonstrated

Virology 342 (2005) 6Introduction

Feline immunodeficiency virus (FIV), like HIV, infects

lymphocytes, results in T cell activation and CD4/CD8

inversion, and ultimately leads to the death of the host due

to immunological exhaustion (Ackley et al., 1990). In this

respect, FIV is unique in its similarity to the pathogenic

primate lentiviruses, as ungulate lentiviruses induce disease

conditions that are more typical of a chronic inflammatory

reaction (reviewed in Miller et al., 2000; Overbaugh et al.,

1997). FIV infection of the domestic cat has been

established as an animal model for AIDS, providing

pediatric AIDS, vaccine design, and lentiviral pathogenesis

(reviewed in Bendinelli et al., 1995; Elder et al., 1998;

Willett et al., 1997b).

Serologic surveys of 36 non-domestic feline species have

revealed a minimum of 20 species with antibodies that

cross-react with FIV antigens (Barr et al., 1995; Brown et

al., 1993; Carpenter and OBrien, 1995; Olmsted et al.,

1992; Troyer et al., 2005). Genetic characterization of the

lentiviruses associated with seroconversion in several of

these species has determined that they are distinct from each

other, related to domestic cat FIV, and have marked intra-

and inter-strain genetic heterogeneity (Barr et al., 1997;lions are hosts for apparently apathogenic lentiviruses (PLV, LLV) distinct from FIV. We compared receptor use among these viruses by: (1)

evaluating target cell susceptibility; (2) measuring viral replication following exposure to specific and non-specific receptor antagonists; and

(3) comparing Env sequence and structural motifs. Most isolates of LLV and PLV productively infected domestic feline T cells, but differed

from domestic cat FIV by infecting cells independent of CXCR4, demonstrating equivalent or enhanced replication following heparin

exposure, and demonstrating substantial divergence in amino acid sequence and secondary structure in Env receptor binding domains. PLV

infection was, however, inhibited by CD134/OX40 antibody. Thus, although PLV and LLV infection interfere with FIV superinfection, we

conclude that LLVand PLV utilize novel, more promiscuous mechanisms for cell entry than FIV, underlying divergent tropism and biological

properties of these viruses.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Feline; Lentivirus; Receptor; CXCR4; EnvelopeFeline immunodeficiency virus (FIV) causes fatal disease in domAbstract

estic cats via T cell depletion-mediated immunodeficiency. Pumas andFeline lentiviruses demonstrate di

envelope stru

Natalia Smirnovaa, Jennifer L. Troy

Mary Possb, Su

aDepartment of Microbiology, Immunology, and Pathology, Colorado SbDivision of Biological Sciences, Univer

Received 20 April 2005; returned to author

Available onli0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.virol.2005.07.024

* Corresponding author. Fax: +1 970 491 0523.

E-mail address: suev@lamar.colostate.edu (S. VandeWoude).rences in receptor repertoire and

ral elements

Jennifer Schisslera, Julie Terweea,

andeWoudea,*

niversity, 1619 Campus Delivery, Fort Collins, CO 80523-1619, USA

f Montana, Missoula, MT 59812, USA

vision 9 June 2005; accepted 20 July 2005

August 2005

0 76

www.elsevier.com/locate/yviroPLV; also referred to as FIV-Ple and FIVincreased diversity, saturation of synonymous sites, and an

increased proportion of substitutions at non-synonymous

irologsites when compared to sequences derived from FIV-

infected cats (Biek et al., 2003; Brown et al., 1994;

Carpenter et al., 1996, 1998; Troyer et al., 2004). These

observations suggest that LLV and PLV have been main-

tained in their host lineages longer than FIV has been in

domestic cat populations.

The clinical effects of the FIVs indigenous to wild and

captive non-domestic cat populations have not been well

studied. However, given high seroprevalence rates in the

absence of indications of decreased population fitness, it

appears likely that these infections are not responsible for

high levels of pathology in free-ranging populations (Biek et

al., 2003; Brown et al., 1994; Carpenter et al., 1996).

Additionally, several strains of PLV and LLV can produc-

tively infect domestic cats without resulting in disease,

suggesting that viral as well as host factors mediate

pathogenicity and disease outcome (VandeWoude et al.,

1997a, 2000, 2002, 2003). PLV-1695, an isolate originally

obtained from a British Columbia cougar, is infectious for

domestic cats both parenterally and oronasally, and mucosal

infection was contained in three of four cats in the absence

of specific markers of enhanced host immune response.

Moreover, this isolate demonstrated a greater propensity to

target tissues of the gastrointestinal tract than FIV (Terwee

et al., 2005). These observations support the supposition

that LLV and PLV are less pathogenic and, at least in a

domestic cat model, have significantly different biological

properties than FIV.

The virusreceptor interaction is the primary event in the

process of lentivirus infection, is a critical determinant of

cell tropism, and significantly influences the pathogenesis of

disease. For example, human immunodeficiency virus

targets helper T cells preferentially as these cells express

the viral receptor CD4 (Klatzmann et al., 1984; Maddon et

al., 1986), and variation at the level of viral receptor

specificity underlies the classification of HIV isolates into

syncytium-inducing (SI, X4-adapted strains; Feng et al.,

1996) and non-syncytium-inducing (NSI, R5 strains;

Alkhatib et al., 1996; Choe et al., 1996; Deng et al., 1996;

Doranz et al., 1996; Dragic et al., 1996). It is generally

thought that R5 tropic strains are the initial infecting

subtype, and as a result, gastrointestinal tissues expressing

high levels of CCR5 are targeted during initial infection

(Apetrei et al., 2004). As disease progresses, X4-adapted

strains are increasingly isolated, and at end stage of disease

become the primary virus in circulation (Apetrei et al.,

2004). Pathogenicity has been linked to X4 and R5 usage in

both SIV and SIV-HIV recombinant (S/HIV) systems

(Nishimura et al., 2004), and a paucity of CD4+CCR5+

target cells has been correlated with lack of pathogenicity in

natural SIV infections of African Green Monkeys (Allan et

al., 2004). Receptor tropism has been mapped to the Env

region of HIV and SIV, particularly in the V3 loop (Carrillo

and Ratner, 1996; Chesebro et al., 1996; Johnston and

N. Smirnova et al. / VPower, 2002; Kato et al., 1999; Pohlmann et al., 2004;

Speck et al., 1997). Similarly, pathogenicity and cell tropismof FIV have also been linked to env V3 sequence (Hohdatsu

et al., 1996; Hosie et al., 2002; Johnston and Power, 2002;

Lerner and Elder, 2000; Pedersen et al., 2001; Verschoor et

al., 1995). As described above, PLV-1695 localizes in the

gastrointestinal tract tissues and draining lymph nodes in

cats infected parenterally or mucosally, unlike domestic cat

FIV, which is more highly tropic for thymus and bone

marrow (Terwee et al., 2005).

The chemokine receptor CXCR4 has been shown to act

as a co-receptor for FIV, suggesting conservation of

receptor usage between the feline and primate lentiviruses

(Willett et al., 1997a, 1997c, 1998). However, despite the

fact that FIV pathogenicity closely mimics that of HIV, FIV

infection has not been demonstrated to involve interaction

with CD4 and there are no firm data to support a role for

CCR5 in infection of feline cells (Johnston and Power,

1999; Johnston et al., 2001; Lerner and Elder, 2000).

However, CD134/OX40, a member of the tumor necrosis

factor/nerve growth factor-receptor superfamily, has been

identified as a high-affinity receptor for field strains of FIV

(de Parseval et al., 2004a; Shimojima et al., 2004); and

binding mechanics between FIV Env and CD134 mimic

that of HIV Env and CD4 (de Parseval et al., 2005). CD134

is expressed primarily on activated CD4+ T cells, plays a

role in CD4+ expansion, and has been implicated in

modulating cell adhesion in HTLV-1 infection (Uchiyama,

1997; Weinberg et al., 2004). In contrast to FIVs isolated

from cats during the asymptomatic stage of disease, tissue

culture-adapted FIVs appear to utilize CXCR4 as a sole

receptor, as do FIVs isolated from cats with end stage

immunodeficiency disease (Haining et al., 2004). These

findings illustrate a virusreceptor interaction that varies

with pathogenicity as observed in the primate lentivirus

system. For these reasons, we have compared viral

receptor interactions and env sequences between patho-

genic domestic cat FIV and the presumably less pathogenic

non-domestic feline lentiviruses.

Our initial experimental data suggested that FIV and

PLV have overlapping receptor repertoires, as viral

interference has been observed in vitro and in vivo, and

lion and puma lentiviruses were able to infect domestic cat

PBMC and cell lines (VandeWoude et al., 1997a, 1997b).

The aim of this study was to further characterize receptor

use by these apparently less pathogenic, more highly host-

adapted viruses. We have investigated the use of CD134,

CXCR4, CD4, and CD25 by FIV, PLV, and LLV. We have

also evaluated non-specific binding interactions via heparin

and cyanovirin-N (CVN-N) interference. The data pre-

sented here demonstrate that the non-domestic feline

lentiviruses use different receptors and binding modalities

than FIV. Comparisons of FIV- and PLV-Env amino acid

sequences reveal substantial differences in biochemical

properties in the V3 loop, providing a rationale for

variation in receptor use and suggesting a plausible

y 342 (2005) 6076 61mechanism for the observed biological differences noted

among the feline lentiviruses.

Results

Non-domestic cat lentiviruses replicate in domestic cat cells

We used a representative panel of primary feline

lymphoid cells, lymphoid cell lines, and adherent cells to

assess in vitro growth characteristics of several strains of

LLV and PLV. As previously described, several isolates of

PLV and LLV are able to grow on domestic cat cells in vitro

(Table 1; VandeWoude et al., 2003, 1997a). The feline

lymphoid cell lines 3201 and Mya-1 cells were more likely

to support growth of non-domestic cat lentiviruses than

adherent cell lines. LLV was more difficult to propagate in

vitro than PLV, though several isolates were recovered

following co-cultivation with Mya-1 cells. PLV-1695 and

FIV-C-PG were also able to replicate when grown on puma-

derived PBMC stocks, and conversely, FIV-C-PG was

replication competent on puma-origin PBMC. Thus, domes-

effect of AMD3100 on infection with LLV and PLV.

Twenty-four hour exposure to high concentrations of

AMD3100 efficiently and reproducibly inhibited FIV

infection in a dose-dependent manner as previously reported

(Figs. 1A and 3A, Tables 2 and 3). Figs. 1B and C

demonstrate that comparatively, LLV and PLV are not

inhibited by AMD3100 in a dose-dependent manner. In

order to compare results across all three viruses despite

differences in assay technique (ELISA vs. reverse-tran-

scriptase) and wide variation in raw data ranges, viral

growth in the presence of inhibitor is presented as a

percentage of growth in the presence of no AMD control

in Fig. 1D and Figs. 2, 4, 5, and 6. Values from the raw data

for these figures, and subsequent statistical comparisons of

percent inhibition, are presented in Table 3.

Comparisons of time course data between FIV and PLV

exposed to lower concentrations of AMD3100 demonstrated

that although the duration of FIV growth inhibition persisted

ivoa

= 25 background, nd = not done. Results ba In vivo virulence, see (VandeWoude et al., 1997b; VandeWoude et al., Tb 3201feline lymphosarcoma.c CrFKCrandell feline kidney.d Mya-1IL-2-dependent feline T cells.for several weeks post-inoculation (Fig. 2A), PLV infection

was only inhibited at doses above 0.25 Ag/ml, and then onlytransiently (Fig. 2B). In order to evaluate the possibility that

this transient inhibition was attributable to puma virus

adaptation to domestic cat cells, AMD3100 inhibition was

tested on puma-derived PBMC alongside Mya-1 cells. As

previously noted in Mya-1 cells, FIV-C-PG growth was

completely inhibited, and PLV-1695 growth was transiently

inhibited at the highest concentration. AMD3100 also

blocked FIV infection of puma PBMC; however, in contrast,

PLV-1695 growth was not inhibited by AMD3100 in puma

cells at any concentration (Fig. 3).

Taken collectively, these results reveal that: (1)

AMD3100 potently blocks FIVCXCR4 interactions;

dilute concentrations of this drug present for 1 h are

capable of significant reduction in viral replication greater

than 2 weeks post-exposure; (2) high concentrations of

AMD3100 are capable of inducing transient reductions in

In vitro tropism

Lion PBMC Cat PBMC 3201b CrFKc Mya-1d

nd +++ +++ ++++ +++

nd +++ +++nd +++ ++ ++nd +++ ++++ ++ ++++ nd ++

+++ ++ ++++ + ++ ++++ +++ + + ++++ nd ++

2 background; + = 25 background, ++ = 510 background, +++ =on at least duplicate estimations monitored for 28 days after infection.e et al., 2005).

irologN. Smirnova et al. / VPLV (and possibly LLV) cellular entry on domestic cat-

derived cells; and (3) FIV infection of puma PBMC-

derived cells is CXCR4 dependent, while PLV infection is

Fig. 1. High dose AMD3100 inhibits FIV more significantly than LLV- or

PLV-growth. (AC) Triplicate wells containing 3201 Tcells were exposed to

a range of high concentrations of AMD3100, then inoculated with 10 TCID50FIV-B-2542, LLV-458, or PLV-1695. Cells were washed at 24 h and re-

exposed to the same AMD concentrations as on day 0, providing an extended

period of inhibitor exposure to maximize inhibition of PLV and LLV.

Supernatants were collected bi-weekly for 3 weeks and assayed as described

in Materials and methods. FIV growth was inhibited at all concentrations as

illustrated in panel A for day 10 post-infection (PI). Replication of LLVand

PLV was transiently inhibited (LLVon day 14 and PLVon day 10 PI only),

but the trend was not statistically significant (B, C). Because y-axis scale

range did not allow single graph comparisons, data were converted to %-no-

inhibitor control (as described in Materials and methods) in panel D and in

Figs. 2, 4, 5, and 6. Bars represent average and standard deviation of triplicate

assays. Indicates P < 0.001 (see Table 3).not, as evidenced by response to the presence to CXCR4

antagonist AMD3100.

In order to evaluate the possibility that LLV and PLV

attached to CXCR4 in a manner not disrupted by

AMD3100, we attempted to block viral entry with anti-

CXCR4 antibody. In contrast to AMD3100 interference, this

agent did not inhibit infection by any of the feline

lentiviruses. Others have observed that FIV infection was

not inhibited by anti-CXCR4 antibody, a phenomenon

attributed to the disparate sites of antibody vs. FIV binding

(de Parseval et al., 2004b). SDF1-a, the natural ligand forCXCR4, inhibited FIV growth as expected, albeit to a lesser

extent than AMD3100. Others have reported weaker

inhibition of FIV growth by SDF1-a than by AMD3100,which may be observed because of cellular CXCR4

upregulation following exposure to its ligand (Hosie et al.,

1998). PLV growth was not abrogated in the presence of

SDF1-a in multiple trials. LLV was only transientlyinhibited by SDF1-a in 2/5 trials, again demonstrating thatinfection of domestic cat cells by non-domestic FIVs is less

sensitive to disruption of CXCR4 binding than the domestic

cat virus (Table 2).

Blocking CD134 significantly inhibits FIV, transiently

inhibits PLV, and does not inhibit LLV in vitro

The recent identification of CD134 as a primary FIV

receptor (de Parseval et al., 2004a; Shimojima et al., 2004)

led us to investigate whether this molecule also served as a

receptor for LLVor PLV cell infection. Several experiments

were conducted to determine the cross reactivity of human

anti-CD134 with domestic cat CD134. Mean fluorescence

intensity (MFI) correlated with antibody concentration over

a range of 1 to 20 Ag/ml on Mya-1 cells (Fig. 4A).Approximately 13% of Mya-1 cells were labeled using 5

Ag/ml antibody, whereas less than 5% of cells were labeledwhen the concentration was decreased to 2 Ag/ml (data notshown).

Because of a generalized suppression of cell growth

following addition of antibody (which precluded analysis in

concentrations greater than 5 Ag/ml), isotype matchedirrelevant antibody (rather than no antibody) was used in

similar dilutions as non-specific immunoglobulin control.

Growth of FIV-C-PG was consistently and significantly

inhibited on Mya-1 cells by anti-CD134 at a concentration

of 5 Ag/ml compared to irrelevant isotype control antibody(P < 0.001), but was not inhibited at 2 Ag/ml (Fig. 4B,Table 3). PLV-1695 was also significantly inhibited by

CD134 antibody at 5 Ag/ml (P < 0.001, Fig. 4B, Table 3)but not 2 Ag/ml, though percent inhibition was less than forFIV. LLV-458 was not significantly inhibited by 5 Ag/ml ofanti-CD134 antibody (P = 0.058; Fig. 4B). These data are

similar to observations of AMD3100 inhibition, i.e. strong

support for FIV use of CD134 for cellular entry that cannot

y 342 (2005) 6076 63be replicated in identical inhibition experiments with PLV

or LLV.

irologTable 2

Summary of inhibition studies

Inhibitor Virus Cell type Trials Result

AMD-3100 FIV-B-2542 3201, Mya 3/3 Dose-dependent

inhibition

LLV-458 3201, Mya 2/2 Transient inhibition

PLV-1695 3201, Mya 5/5 Transient inhibition

FIV-C-PG Puma 1/1 Complete inhibition

PLV-1695 Puma 5/5 Transient inhibition

SDF-1a FIV-B-2542 3201,FePBMC

3/3 Dose-dependent

inhibition

FIV-C-PG Mya 1/1 Dose-dependent

inhibition

LLV-458 3201, 3/5 No inhibition

N. Smirnova et al. / V64Saturation of CD4 and CD25 surface molecules does not

inhibit FIV, LLV, or PLV infection

Given that blocking of either CXCR4 or CD134 did not

fully inhibit the non-domestic lentiviruses, other surface

molecules were assayed for a possible role in viral binding.

Mya-1 cells are the only cell type found thus far that are

permissive for all three feline lentiviruses (FIV, PLV, and

LLV). These cells are CD4+CD25+ (data not shown).

Therefore, the possible role of these surface molecules in

viral entry was determined. Incubation of Mya-1 cells with

high, medium, or low concentrations of anti-CD4 and anti-

CD25 either alone or concurrently failed to inhibit FIV, PLV,

and LLV infection (Tables 2 and 3).

FePBMC

LLV-458 Mya,

FePBMC

2/5 Transient inhibition

PLV-1695 3201, Mya,

FePBMC

5/5 No inhibition

Anti-CD134 FIV-C-PG Mya 1/1 Dose-dependent

inhibition

PLV-1695 Mya 1/1 Dose-dependent

inhibition

LLV-458 Mya 1/1 Marginal

inhibition

Anti-CD4 FIV-C-PG Mya 1/1 No inhibition

LLV-458 Mya 1/1 No inhibition

PLV-1695 Mya, 3201 3/3 No inhibition

Anti-CD25 FIV-C-PG Mya 1/1 No inhibition

LLV-458 Mya 1/1 No inhibition

PLV-1695 Mya 1/1 No inhibition

Anti-X4 PLV-1695 Mya 2/2 No inhibition

TAK 779 PLV-1695 3201 2/2 No inhibition

Cyanovirin FIV-C-PG Mya 2/2 Dose-dependent

inhibition

PLV-1695 Mya 2/2 No inhibition

LLV-458 Mya 1/1 Dose-dependent

inhibition

Heparin FIV-C-PG Mya 1/1 Dose-dependent

inhibition

LLV-458 Mya, 3201 4/4 Enhancement

PLV Mya, 3201 3/3 Enhancement

Each trial consisted of samples run in triplicate or in sets of five

(cyanovirin, anti-CD134, anti-CD25) at each inhibitor concentration.

Transient inhibition is defined as dose-dependent inhibition noted only at

the earliest timepoint that no-inhibitor control supernatants were RT

positive. Enhancement is defined as inhibitor RT values significantly

greater than no inhibitor control at multiple timepoints post-inoculation.Heparin inhibits FIV, but enhances LLV and PLV infection

In addition to specific receptor binding, non-specific

interactions between lentiviral envelope and target cell

membranes are involved in HIV, SIV, and FIV infection.

For example, heparin binding has been shown to play a role in

both HIV- and FIV-cellular entry, purportedly through

interactions between basic gp120 (gp95) with anionic cellular

heparin sulfates (de Parseval and Elder, 2001; Ibrahim et al.,

1999; Roderiquez et al., 1995; Tanabe-Tochikura et al.,

1992). To better define non-specific interactions that might

assist infection with LLV and PLV, we infected cells in the

presence of heparin. FIV-C-PG infection was inhibited in a

dose-dependent manner (P < 0.05) by heparin as previously

reported for FIV-34TF10 and FIV-Pet (de Parseval and Elder,

2001). In striking contrast, LLV and PLV infection was not

inhibited, and in fact, viral growth in the face of heparin

exceeded no-heparin controls in 4 of 4 (LLV) or 3 of 3 (PLV)

trials (Fig. 5, Tables 2 and 3), suggesting substantial

differences in non-specific binding interactions between

domestic and non-domestic FIVs.

High mannose glycosylation contributes to FIVand LLV, but

not PLV, Envreceptor interactions

CVN-N, an antibiotic which blocks high-mannose

glycosylation sites on viral Env, interferes with both HIV

and FIV receptor binding and subsequent infection in a non-

specific manner (Barrientos et al., 2003; Bolmstedt et al.,

2001; Dey et al., 2000; Mori and Boyd, 2001; OKeefe et

al., 2000, 2003), and has recently been shown to interfere

with vaginal and rectal transmission of SIV (Tsai et al.,

2003, 2004). This activity is based upon binding of the

molecule to envelope glycoproteins (specifically high-

mannose oligosaccharides) which interferes with Env

receptor binding and fusion. Fig. 6 demonstrates the ability

of CVN-N to inhibit infection of LLV on Mya-1 cells,

though results were not strictly dose dependent. FIV and

PLV infection was only inhibited at one dose and timepoint

(Fig. 6, Table 3). However, growth of all three viruses was

blunted in the presence of any concentration of CVN-N.

These results demonstrate that CVN-N stringency and

efficiency of in vitro FIV-, LLV-, or PLV-growth inhibition

are highly variable compared to the specific receptor

antagonists AMD3100 and anti-CD134.

FIV Env sequence and biochemical properties differ

substantially from non-domestic cat lentiviral Env

Given that Env has been shown to be the principal

lentiviral receptor-binding domain, we compared Env

among 2 PLV strains (PLV-1695 and PLV-14), and 2 FIV

strains (Petaluma and C-PG). Addition of lentiviral-Env

sequence obtained from a Pallas cat FIV-like virus

y 342 (2005) 6076(Otocolobus manul, FIV-Oma) assisted in bridging align-

ments of highly divergent regions of FIV- and PLV-Env.

Table 3

Statistical analysis of inhibition studies represented in figures

Virus AMD (Ag/ml) Viral growth Dose vs. control 2-tailed Students t Sequential dose 2-tailed Students t

FIV 0 0.781 T 0.084

0.3 0.098 T 0.006 P < 0.001 P < 0.05

1.25 0.076 T 0.008 P < 0.001 P < 0.015 0.034 T 0.004 P < 0.001

PLV 0 13,061 T 4626

0.3 7533 T 3377 nsd nsd

1.25 9215 T 3261 nsd nsd5 8901 T 4135 nsd

LLV 0 1138 T 1044

0.3 435 T 296 nsd nsd

1.25 362 T 195 nsd nsd5 310 T 36 nsd

Virus Antibody Ag/ml Viral growth Dose vs. control 2-tailed Students t Sequential dose 2-tailed Students t

FIV Control 2 1.48 T 0.41

Anti-CD134 1.20 T 0.27 nsdControl 5 1.17 T 0.11

Anti-CD134 0.34 T 0.17 P < 0.001 P < 0.001

PLV Control 2 335 T 82Anti-CD134 352 T 51 nsd

Control 5 394 T 66

Anti-CD134 218 T 40 P < 0.01 P < 0.05

LLV Control 2 677 T 325Anti-CD134 698 T 263 nsd

Control 5 567 T 120

Anti-CD134 403 T 113 nsd nsd

Virus Treatment Viral growth Dose vs. control 2-tailed Students t Sequential dose 2-tailed Students t

FIV Control 1.845 T 0.055

Anti-CD4+anti-CD25 1.900 T 0.133 nsd nsd

PLV Control 816 T 152Anti-CD4+anti-CD25 1013 T 418 nsd nsd

LLV Control 768 T 118

Anti-CD4+anti-CD25 585 T 184 nsd nsd

Virus Heparin (Ag/ml) Viral growth Dose vs. control 2-tailed Students t Sequential dose 2-tailed Students t

FIV 0 0.374 T 0.260

2.5 0.165 T 0.159 P < 0.05* nsd

5 0.029 T 0.018 P < 0.05* nsd

20 0.022 T 0.055 nsdPLV 0 511 T 550

2.5 Not done

5 1020 T 1150 nsd nsd

20 646 T 809 nsd nsdLLV 0 331 T 52

2.5 773 T 491 P < 0.10* nsd

5 508 T 227 nsd20 508 T 105 P < 0.05* nsd

Virus CV-N (Ag/ml) Viral growth Dose vs. control 2-tailed Students t Sequential dose 2-tailed Students t

FIV 0 2.19 T 0.25

1 1.72 T 0.70 nsd nsd10 0.38 T 0.39 P < 0.001 P < 0.01

PLV 0 1683 T 304

1 776 T 599 P < 0.05 nsd

10 1316 T 772 nsd nsdLLV 0 1473 T 444

1 1226 T 684 nsd nsd

10 497 T 601 P < 0.05 nsd

Statistical comparisons are made using 2-tailed two-sample homoscedastic Students t test except where indicated. Values for FIV represent OD450nm ELISA

absorbance values. Values for PLV and LLV represent cpm (counts per minute) in reverse-transcriptase assays.

* Indicates 1-tailed two-sample homoscedastic Students t test.

N. Smirnova et al. / Virology 342 (2005) 6076 65

positive charge noted in this region among domestic cat

FIVs (+8 to +12). Three to 6 additional N-glycosylation

sites were predicted in non-domestic lentiviruses in com-

parison with FIV, and 56 fewer cysteine residues were

present in PLV vs. FIV. A similar trend was noted in V3, the

region implicated as receptor binding domain for FIV

(Hohdatsu et al., 1996; Sodora et al., 1994; Verschoor

et al., 1995; Willett and Hosie, 1999). In contrast, the more

conserved regions flanking V3V5 were more positively

charged in non-domestic lentiviruses than FIV, and con-

tained similar numbers of cysteine residues and putative

N-glycosylation sites (Fig. 7, Table 4). Such differences

indicate that substantial divergence occurs in secondary and

tertiary structures at the receptor-binding region of PLV- vs.

FIV-Env, providing an explanation for the differential

susceptibilities to receptor inhibitors, and supporting the

hypothesis that PLV uses a different receptor to gain cellular

entry than FIV.

Discussion

irology 342 (2005) 6076Fig. 2. Low dose AMD3100 inhibits FIV-, but only transiently inhibits

PLV-growth. Triplicate wells containing 3201 T cells were exposed to a

N. Smirnova et al. / V66Sequences were aligned to compare structural regions,

particularly those known to be important for receptor

binding. Domestic cat FIV Env differed from non-domestic

cat lentiviruses at approximately 77% of the amino acid

sites. However, the sequence divergence was not uniform in

that regions of relative homology (3050%) were inter-

rupted by long tracts of unalignable sequence (12%homology). Despite substantial sequence disparity, three

hydrophobic regions were conserved in similar positions

across all feline lentiviruses, allowing more probable align-

ments within the non-homologous regions (Fig. 7). The

V3V5 region of PLV shared least homology with FIV Env

(corresponding to aa 365 to 627 in the env alignment and

defined in Pancino et al., 1993). In FIV, this region has been

shown to contain the CXCR4 binding site (Willett et al.,

1997a), neutralizing antibody binding sites (Lombardi et al.,

1994, 1995; Tozzini et al., 1993) and several epitopes

important for cell tropism and cell line adaptation (Hohdatsu

et al., 1996; Vahlenkamp et al., 1997; Verschoor et al.,

1995). Within the V3V5 region, several biochemical

differences are notable that suggest corresponding differ-

ences in secondary structure and function (Table 4). Total

charge was negative in all three non-domestic lentivirus

isolates (1 to 4); this differs substantially from the highly

The data presented here strongly support the conclusion

that lion and puma lentiviruses have different mechanismsrange of mid-to-low range concentrations of AMD3100, then inoculated

with 10 TCID50 FIV-B-2542 (A) and PLV-1695 (B). Cells were washed at 1

h and fed with AMD-free media. Supernatants were collected and processed

as described in Materials and methods. As previously described, FIV was

inhibited in a dose-dependent manner at all concentrations over a 2 week

growth period (A). PLV replication was initially inhibited in an

approximately does-dependent manner (days 810 PI, B) but the trend

did not persist after day 15 PI (growth exceeds no AMD control, B). Bars

represent the average % inhibition of triplicate assays. *Indicates P < 0.05

(see Table 3).Fig. 3. AMD 3100 inhibits FIV-, but not PLV-, growth in domestic cat cells

and puma-derived cells. Mya-1 (domestic cat) cells or puma PBMC-derived

cells (2 105) were exposed to AMD 3100 at concentrations 5 Ag/ml, 1.25Ag/ml, and 0.3 Ag/ml, then inoculated with 10 TCID50 FIV-C-PG (A) andPLV-1695 (B). Experiments were performed in quintiplicate. Cells were

incubated overnight (18 h), washed, and re-exposed to the same

concentrations of AMD 3100 as on day 0. Supernatants were collected

bi-weekly and subjected to reverse transcriptase assay. RT values shown are

from day 10 (Mya-1 cell) or day 14 (puma cells) PI. (A) FIV growth was

significantly inhibited at all concentrations in both cell types. (B) PLV

growth was slightly inhibited at the highest AMD dose in Mya-1 cells, andnot for any AMD concentrations on puma cells. Indicates P < 0.01;

*indicates P < 0.05 (see Table 3).

Fig. 5. Heparin inhibits FIV- but enhances LLV- and PLV-growth. Nave

Mya-1 cells were incubated with decreasing dilutions of heparin for 1 h.

FIV-C-PG, LLV-458, and PLV-1695 supernatant stocks were then plated on

Mya-1 cells and processed as described in Materials and methods. Data are

shown for day 7 PI for LLV and FIV, day 14 PI for PLV. Bars represent

average of % inhibition in three assays. Whereas FIV replication was

irology 342 (2005) 6076 67N. Smirnova et al. / Vfor cell entry than those of domestic cat FIV as manifested

by the following findings: (a) AMD3100 and SDF-1a onlytransiently inhibited PLV and LLV replication relative to

FIV in domestic cat cells, and, in contrast to FIV-C-PG,

PLV-1695 replication was not impaired by AMD3100 in

puma-derived PBMC; (b) replication of LLV and PLV on

Mya-1 cells was not inhibited by heparin; and (c) V3V5

Env differs substantially between FIV and two strains of

PLV in amino acid sequence, charge, potential post-trans-

lational modification sites, and cysteine content. The role of

CD134 binding in LLV and PLV infection is less clear and

discussed further below.

Puma and lion lentiviruses infected domestic cat PBMC

and T cell lines, particularly Mya-1 cells, demonstrating the

capacity of LLV and PLV to adapt to use domestic cat cell

surface moieties to gain cellular entry. The Mya-1 cell line

was derived from feline blood mononuclear cells, and is

readily infected with primary isolates of FIV (Miyazawa et

Fig. 4. Anti-CD134 inhibits FIV- and PLV- but not LLV-growth. (A) Mya-1

cells were incubated with dilutions of anti-human CD134 (OX40, Ancell

Corporation: clone Ber Act 35) or Isotype control (Mouse IgG1, Ancell

Corporation: clone MOPC 31C) for 30 min at 4 -C, washed, and stained

using Anti-Mouse IgG FITC, and subjected to flow cytometry as described

in Materials and methods. Mean fluorescence intensity decreased with anti-

CD134 concentration. Twelve fifteen percent of Mya-1 cells were labeled

at 5 Ag/ml compared to

intracellular enzymes responsible for interruption of trans-

species viral replication (Gaddis et al., 2004; Mariani et al.,

2003; Schrofelbauer et al., 2004).

Primary strains of FIV are dependent upon both CD134

and CXCR4 for cell entry (de Parseval et al., 2004a,

2004b; Egberink et al., 1999; Hosie et al., 1998;

Richardson et al., 1999; Shimojima et al., 2004), restricting

their growth to CXCR4+ CD134+ target cells. Tissue

culture adaptation can occur, rendering strains CD134

independent, explaining the variation in cell susceptibilities

secondary structure, provides plausible explanation for the

results of inhibition experiments presented here. Our obser-

vation of enhanced growth may occur because neutralization

of anionic heparan sulfates by heparin facilitates binding of a

neutral or anionically charged V3 loop found on PLV, and

possibly LLV, isolates.

Glycosylation of HIV Env is considered a major compo-

nent of cell tropism and receptor targeting, and CVN-N has

been shown to inhibit in vitro FIV infection, inferring a

similar mechanistic property (Dey et al., 2000). Despite the

show

use o

from

sites a

boxed

grey

>25%

d C1

). Epit

fined

N. Smirnova et al. / Virology 342 (2005) 607668noted in Table 1. We found that anti-CD134 and the

CXCR4 inhibitor, AMD3100 markedly disrupted in vitro

infection by FIV-C-PG. Though CXCR4 blocking tran-

siently inhibited LLV and PLV replication, in neither case

was the degree of blocking as consistent and marked as for

FIV. The fact that AMD3100 was ineffective at blocking

PLV-1695 infection in puma-derived cells suggests that

adaptation of PLV in domestic cat cells may confer some

low-level degree of CXCR4 dependency that is rapidly

reversed under pressure of an antagonist. We noted dose-

dependent inhibition of PLV growth in the presence of

anti-CD134 antibody. In light of recent reports that the

binding site for anti-CD134 monoclonal antibody clone

Ber Act 35 does not overlap with FIV-Env binding sites on

CD134 for other FIV strains (de Parseval et al., 2005), and

that two strains of PLV (PLV-14, CoLV) were able to

infect X4CD134 AH927 cells (B. Willett, personalcommunication), further investigations using other strat-

egies will be required to clarify the precise association

between LLV and PLV usage of CXCR4 and CD134.

Heparin binding did not interfere with LLV and PLV

infection, in contrast to its ability to block FIV infection in

vitro. The HIV/FIV blocking activity of heparin is thought to

be dependent upon electrostatic interactions between the

basic FIV V3 loop and acidic cell-bound heparin sulfates

(Roderiquez et al., 1995). The V3 binding domain amino acid

sequences of two highly divergent PLVs (PLV-14 and PLV-

1695) have net charges substantially more acidic than FIV. de

Parseval and Elder found that FIV strains with net charges of

+6 or +8 in V3 were more significantly inhibited by heparin

than strains with charges of +4 (de Parseval and Elder, 2001).

Assuming that the V3 loop of PLV functions as a potential

cellular binding domain, the neutral to anionic charge of this

region, combined with the highly divergent amino acid and

Fig. 7. Predicted amino acid alignment of domestic and non-domestic FIVs

correlative sites from FIV-Pet Env. These numbers differ between strains beca

672 (610 and 611 in the FIV-pet reference sequence) indicated the transition

Cysteine residues, FC_, are indicated by pink, and predicted N-glycosylationcontain white letters. Fully conserved amino acid residues are dark grey and

across taxa (>0.5 in the Gonnet PAM250 matrix; Yang, 1997) are in medium

the Gonnet PAM250 matrix; Yang, 1997) are in light grey. Regions that have

are indicated by white boxes above the amino acid sequence rows and labele

sequences (light green, entire variable region; dark green, V3 binding region

rows; stronger binding sites are darker orange. Other structural features de(PID), alpha-helix, Trp-rich motif, transmembrane region) have also been indicated

region (residues 365627), where non-domestic feline lentiviruses have lower chfact that PLV-1695 Env sequence predicts a significant

number of potential glycosylation sites, CVN-N inhibition

experiments reported were unable to conclusively demon-

strate dose-dependent inhibition of either this virus or LLV.

These ambivalent results are likely attributable to variation in

glycosylation patterns of the virus stocks used, since this

variable is dependent upon the cell in which viral propagation

occurs (Levy, 1998). It is also possible that the selectivity of

CV-N for terminal Man(12)Man moieties on high-mannose

Man8 or Man9 glycans precludes inhibition of glycosylation

signatures of the virus stocks used in this study (Bewley and

Otero-Quintero, 2001).

Because the LLV and PLV strains we used for blocking

studies readily infect domestic cat cells, it is difficult to

ascribe our observations to variation between surface

molecules on domestic vs. non-domestic T cells. It is

possible that adaptations in viral genomic sequence and

structure occur when PLV or LLV infect domestic cat cells,

suggested by our experiments in puma-derived PBMC.

Observations by Biek et al. (2003) have demonstrated that

sequence diversity within PLV is very low, and preliminary

studies analyzing a series of PLV-1695 isolates across pol

has not detected sequence selection in tissue culture relative

to wild type virus (Poss et al., data not shown), so such

adaptation events appear to be rare. SIV receptor use has

been assessed based upon infection of cells displaying

human receptor molecules, even though these viruses

originated in African monkeys, and are often been main-

tained in, or adapted to, macaque cells (Chen et al., 1997;

Liu et al., 2000; Vodros et al., 2001, 2003). While lion and

puma candidate receptor proteins require testing to validate

observations made using domestic cat cells, analogy to

primate lentivirus receptor usage would support the validity

of the data presented here.

s variable regions of structural homology. Numbers across the top are for

f amino acid mismatches/insertions. A vertical line between residues 671 and

the surface protein (gp120;SU) to the transmembrane protein (gp41:TM).

re shown in yellow. Hydrophobic regions are indicated by black boxes that

with bold letters. Sites that contain strongly conserved amino acid changes

, and those with weakly conserved amino acid changes across taxa (

N. Smirnova et al. / Virology 342 (2005) 6076 69

domestic cat FIV. Further studies comparing amino acid

structure of LLV-Env to PLV and FIV-Oma will allow an

8

0

4

4

3

ddy-G

ley et

r each

amino

presen

irologWe have previously observed viral interference between

PLV and FIV (VandeWoude et al., 2002), and have here

noted similar in vitro tropisms, leading us to initially

hypothesize that these viruses used similar receptors. The

work presented here does not support this hypothesis. Other

mechanisms underlying viral interference could include: (a)

suppression of superinfection by innate immune mecha-

nisms, similar to observations that HIV-2 prevents super-

infection by R5 strains of HIV-1 by inducing h-chemokineproduction, or by actions of intracellular nucleases (Gaddis

et al., 2004; Mariani et al., 2003; Schrofelbauer et al., 2004);

(b) selection of PLV laboratory-cultured isolates with altered

mechanisms for cell entry relative to primary strains

(discussed above); (c) different receptor binding sites on

CD134 or CXCR4 for PLV vs. FIV, attributable to

variations in puma vs. domestic cat molecules; or (d)

disruption of primary PLV viral receptor expression

following initial infection with FIV. Viral glycosylation

sites and point mutations in Env substantially influence

receptor usage among HIV strains (McCaffrey et al., 2004;

Nabatov et al., 2004). It is also possible that the biological

viral isolates used here contained viral quasi-species with

Table 4

Comparison of Env amino acid sequence properties

V3 binding V3 V3V5

376416 354419 354567

T N C T N C T N

FIVA-PET 6 0 2 7 1 3 12

FIVC-PG 5 0 2 5 1 3 8 1

OMA 3 0 1 5 4 1 2 1PLV-14 3 0 1 4 4 1 4 1PLV-1695 4 0 1 4 5 1 1 1FIVA-Pet = FIV Petaluma strain (Olmsted et al., 1989); FIV-C-PG = FIVC-Pa

et al., 1997) FIV sequence; PLV-14 = Florida Panther FIV sequence (Lang

Amino acid residues corresponding to FIV-Pet sequence are indicated unde

homology among all depicted strains, FPC3 = region demonstrating high

between amino terminus of V5 loop and beginning of conserved region, re

N. Smirnova et al. / V70varying receptor affinities due to slight variation in sequence

and N-glycosylation, which do not reflect all subtypes of

PLV and LLV.

Perhaps the most intriguing finding presented here is

the marked differences in FIV, PLV, and FIV-Oma Env

amino acid sequences and alignments. Alterations in the

V3 loop of FIV and HIV have been strongly correlated

with changes in host cell susceptibility, receptor affinity,

neutralizing antibody binding sites, and pathogenicity

(Bjorndal et al., 1997; Carrillo and Ratner, 1996; Chesebro

et al., 1996; Nielsen and Yang, 1998; Nishimura et al.,

1996; Pohlmann et al., 2004; Siebelink et al., 1995; Speck

et al., 1997; Verschoor et al., 1995). Therefore, the high

degree of nucleotide and amino acid divergence noted here

provides an attractive explanation for differential receptor

use by non-domestic cat lentiviruses vs. FIV. It is

interesting that FIV-Oma demonstrates an intermediate

alignment between FIV and PLV, despite the fact that theopportunity to further study this trend using an African-

origin FIV. The lack of inhibition of LLV and PLV by

agents that reproducibly inhibit FIV or HIV, together with

substantial differences noted in predicted Env structure will

provide models to relate changes in receptor use and Env

structure to lentiviral pathogenicity.

Materials and methods

Cells and viruses

Heparinized blood samples were obtained from cats in a

specific pathogen free breeding colony housed in anPallas cat is an Asian cat species vs. the North American

dwelling puma. That the V3V5 region of PLV more

closely aligns with that of the Pallas cat suggests these

older and more host-adapted viruses might have evolved

convergently to use similar receptor mechanisms which

vary from the more recently emerged and virulent

FPC2 V5-FPC3 FPC3

283331 568658 659687

C T N C T N C T N C

14 2 1 3 0 0 0 1 0 0

13 1 1 3 0 0 0 1 0 0

8 1 2 3 8 0 0 6 0 0

8 1 1 3 9 0 0 4 0 08 1 1 3 5 0 0 3 0 0ammer strain (de Rozieres et al., 2004); OMA= Pallas cat (O. manul; Barr

al., 1994); PLV-1695 = British Columbia cougar FIV sequence (see text).

column heading; FPC2 = region demonstrating high amino acid sequence

acid sequence homology among all depicted strains; V5-FPC3 = region

ting an area of intermediate homology among depicted strains.

y 342 (2005) 6076AAALAC International accredited animal facility. Periph-

eral blood mononuclear cells (PBMC) were isolated from

heparinized blood by ficoll gradient centrifugation (Quack-

enbush et al., 1990). Lion and puma nave PBMC were

provided by Dr. Stephen OBrien as viably frozen field

samples; Drs. Don Hunter and Caroline Krumm provided

anti-coagulated blood collected from a free-ranging puma.

The feline lymphoid cell lines 3201 (Hardy et al., 1980) and

Mya-1 (Miyazawa et al., 1990, 1992) were grown under

standard conditions (Quackenbush et al., 1990) using

commercially obtained media (Invitrogen Life Sciences,

Carlsbad, CA). Puma-derived PBMC infection experiments

were performed on cells isolated from EDTA anti-coagu-

lated blood using Histopaque (Sigma) and were cultured for

2 months in RPMI-1640 supplemented with Glutamax I,

non-essential amino acids, sodium pyruvate, glucose,

sodium bicarbonate, l-glucose, antibiotics, concanavalin A

at 5 Ag/ml, and recombinant human IL-2 at 100 units/ml.

irologConcanavalin A exposure was discontinued after 8 dou-

blings (2 months). Cells used in experiments described had

been in culture for approximately 4 months and replicated

continuously when grown in IL-2.

PLV-1695 (gift of Dr. Ken Langelier) was amplified in

feline PBMC or Mya-1 cells as previously described

(Terwee et al., 2005; VandeWoude et al., 1997b). LLV-

458 and PLV-14 supernatants were obtained from Dr.

Stephen J. OBrien, and stocks were amplified in domestic

cat PBMC or Mya-1 cells in vitro (VandeWoude et al.,

1997a, 1997b). Supernatants were harvested at peak RT

activity (Willey et al., 1988). FIV-B-2542 and FIV-C-Paddy

Gammer (FIVC-PG) stocks were generated in feline PBMC

or Mya-1 cells using standard conditions (Quackenbush et

al., 1990). These two field strains of FIV have been shown

in previous studies to infect cats mucosally and trans-

placentally and cause immunological and clinical decline

(Allison and Hoover, 2003; Burkhard et al., 1997; Diehl et

al., 1996; Obert and Hoover, 2000; Pedersen et al., 2001;

Rogers and Hoover, 1998, 2002). FIV-GL8 and FIV-PET

were prepared as previously described (Willett et al.,

1997a). Stocks were titrated on the targeted cell type for

each assay using standard techniques (Harper, 1993) and

used in inhibition experiments at concentrations of 2040

TCID50.

In vitro tropism studies

Supernatants grown in nave cat, lion, or puma PBMC

were co-cultivated on feline PBMC, CrFK, 3201, or Mya-

1 cells. Cells were plated in 24-well plates in the

appropriate media at a concentration of 2 106/ml. Viralstocks were added to constitute 10% total media volume.

Twenty-five to 50% of the supernatants were collected and

cells re-fed with fresh media bi-weekly for 4 weeks.

Supernatants were subjected to microtiter RT assay as

previously described (VandeWoude et al., 1997a; Willey et

al., 1988).

Inhibitors

AMD3100 was a gift from Dr. Lou Hegedus (Colorado

State University, Fort Collins, CO, Department of Chem-

istry). Recombinant human SDF-1a was obtained fromPeprotech (Rocky Hill, NJ). Mouse monoclonal antibodies

were obtained as follows: anti-human CXCR4, catalogue #

MAB-172, R&D Systems (Minneapolis, MN); anti-feline

CD4 clone 3-4F4, Southern Biotech (Birmingham AL);

anti-human CD134 (OX40) clone Ber Act 35; mouse IgG1

(clone MOPC 31C, Ancell Corporation, Bayport, MN);

and IgG2a (Unlb Clone HOPC-1 Southern Biotech,

Birmingham AL) isotype control. Porcine heparin (sodium

salt), Grade I-A was purchased from Sigma Aldrich (St

Louis, MO). Cyanovirin-N (CVN, purified recombinant

N. Smirnova et al. / Vexpressed in E. coli) was a gift from Dr. John Elder (The

Scripps Research Institute, La Jolla, California, Departmentof Molecular Biology). Anti-feline CD25 monoclonal

antibody was a gift of Dr. Wayne Tompkins (North

Carolina State University, Raleigh, NC, Immunology

Program).

Inhibition assays

Inhibition of virus-CXCR4 binding was attempted using

the CXCR4 inhibitor AMD3100, monoclonal antibody

anti-CXCR4 172, and SDF-1a. Virus was plated inconstant concentration against serial dilutions of inhibitor,

or in one AMD3100 experiment, virus was titrated against

a constant concentration of inhibitor. CD4, CD25 and

CD134 inhibition studies were performed with anti-feline

CD4 or CD25 monoclonal antibody or anti-human CD134.

Serial-dilutions of heparin and CVN were used for non-

specific blocking studies as outlined below. Anti-CD4,

anti-CD25, and anti-CD134 binding limits for serial

dilutions were determined by flow cytometric analysis as

described below. Positive controls consisted of FIV when

appropriate (i.e. CXCR4 and CD134 inhibition) or flow

cytometric analysis of cells labeled with the concentrations

of antibodies used (i.e. CD4, CD25, CD134). Negative

controls consisted of virus without inhibitor, cells without

virus, or isotype control antibodies (for CD4, CD25,

CD134, CXCR4).

The standard format for inhibition assays was as

follows: (1) target cells (Mya-1, 3201, or PBMC) were

plated at 2 105 cells/well in replicates of 35 inlymphocyte or 3201 media; (2) inhibitors were added to

2 final concentration in ranges shown previously toinhibit FIV infection (AMD3100, SDF1a, heparin, CVN;Donzella et al., 1998; Egberink et al., 1999) or in

concentrations determined using flow cytometry to eval-

uate limiting dilution (anti-CD134, anti-CD4, anti-CD25);

(3) 2040 TCID50 viral supernatant stocks were added to

wells in an equal volume to each well, or nave

supernatant was added as negative control; cells were

incubated at 37 -C with 5% CO2 for 24 h, then washedand re-fed with media alone. In CVN, CD25, CD4, and

CD134 inhibition studies, protocols were modified so that

cells were exposed to inhibitor for 1 h at 37 -C followedby addition of virus and a 2-h, 4 -C incubation. Cellswere then again incubated at 37 -C for 4 h. We employedthis protocol to synchronize viral-cell binding in each

culture and eliminate any potential viral binding kinetic

differences in the experiments. Cells were then centri-

fuged at 400 g, washed and re-plated with media; CVNwas re-added to cultures in the original exposure

concentration.

Supernatants were collected bi-weekly starting at day 3

and assayed for virus as described below for most samples;

in some cases FIV supernatants were collected daily.

Samples were considered positive when OD450 nm read-

y 342 (2005) 6076 71ings or RT values exceeded 2.5 media control in at leasttwo samples, and was confirmed at later timepoints during

GATTT 3V and 3PLV14R, 5VACAGATATCTTTGAAGG-GACACAT 3V. PCR products were gel isolated and cloned

irologsampling. Ranges for ELISA and RT assays varied widely

between viruses and assays, which precluded multi-viral

comparisons on one graph. Therefore, percent inhibition

was calculated as [((NRx Neg) (Rx Neg)) / (NRx Neg)] * 100, where NRx = cells with virus and no inhibitor;

Neg = cells with no virus; Rx = cells with virus and

inhibitor. Percent of no-inhibitor control was calculated as

100% (percent inhibition). Percent inhibition wasdetermined starting at timepoints where positive controls

were clearly above background as described until 34

weeks post-inoculation. Multiple replications were per-

formed for several inhibitors as listed in Table 2 to verify

reproducibility of results using multiple viral strains or cell

types. Statistics presented in Table 3 were calculated using

raw data subjected to two-sample homoscedastic Students t

test analysis (Microsoft Excel, Microsoft Corporation,

Redmond, WA).

Viral detection

The presence of FIV in culture supernatants was detected

using p24 antigen capture ELISA (Dreitz et al., 1995).

Previous studies have demonstrated that PLV and LLV are

not detected by this ELISA assay (VandeWoude et al.,

1997a). LLV and PLV growth was detected using a reverse

transcriptase microtiter assay as described with the excep-

tion that 3 Al of RT reaction was spotted on Wallac DEAEnylon microbeta counter filter paper and read with a Wallac

microbeta counter (Wallac/Perkin Elmer, Wellesley, MA;

Willey et al., 1988).

Flow cytometry

2.0 105 Mya-1 cells were plated and centrifuged at800 g in a 96-well-v-bottom plate (Costar, Corning Inc,Corning NY). Mouse monoclonal anti-human CD134 (1

mg/ml), anti-feline CD4 (0.5 mg/ml), or anti-feline CD25

(cell culture supernatant) described above was diluted in

flow buffer at concentrations from 1:50 to 1:1000 (anti-

CD134) or 1:100 to 1:10000 (anti-CD4, anti-CD25) in a

final volume of 0.2 ml with 2 105 cells. Reactions wereincubated at 4 -C for 30 min, then washed twice. Cellswere incubated with 1:100 dilution of fluorescein iso-

thiocyanate (FITC)-conjugated sheep anti-mouse IgG

(Sigma, St Louis, MO), for 30 min. After washing,

samples were passed through an EPICS XL-MCL flow

cytometer (Beckman Coulter, Miami, FL). Cells stained

with isotype control antibodies were used to set analysis

gates. Results following acquisition of 10,000 events were

analyzed using FlowJo software (Tree Star, Inc., San

Carlos, CA).

PLV-1695 env sequencing

N. Smirnova et al. / V72Genomic DNA was extracted with QIAamp DNA blood

mini kit (Qiagen, Valencia CA) from Mya-1 cells persis-into pDrive (Qiagen) and used to transform TOP10 cells

(Invitrogen Corp, Carlsbad CA). Plasmid DNA was recov-

ered from transformed colonies and sequenced.

Comparison of domestic and non-domestic feline lentiviral

Env regions

env nucleotide sequences for FIV-Petaluma (accession

no. M25381; Olmsted et al., 1989), FIV-C36 (accession no.

AY600517; de Rozieres et al., 2004), Pallas cat lentivirus,

FIV-Oma (accession no. U56928; Barr et al., 1997), and

PLV-14 (accession no. U03982; Langley et al., 1994) were

obtained from GenBank. Predicted amino acid sequences

were aligned with those of PLV-1695 (see above) in Clustal

X (Thompson et al., 1997) using the PAM 350 protein

weight matrix (Yang, 1997) and realigned manually. The

predicted secondary structure of these amino acid sequences

including N-glycosylation sites (Gupta et al., in preparation;

Gupta and Brunak, 2002; Gupta et al., 2002), protein

domains (Corpet et al., 1998), disulfide bonds (Rost et

al., 2004), hydrophobic regions, and net charge was

determined using the PredictProtein server; http://

www.embl-heidelberg.de/predictprotein/, NetNGlyc 1.0

server; http://www.cbs.dtu.dk/services/NetNGlyc/, and Iso-

electric Plot; http://www-biol.univ-mrs.fr/d_abim/d_docs/

doc-tab-pk.html.

Acknowledgments

We thank Drs. Brian Willett and Margaret Hosie for

discussions regarding PLV receptor use, and for manuscript

review. Drs. J. Elder, L. Hegedus, W. Tompkins, K.

Langelier, D. Hunter, and C. Krumm and S.J. OBrien

generously contributed reagents, cells, and virus stocks, and

we thank Dr. E.A. Hoover and the Feline Retrovirus

Research Laboratory at CSU for provision of laboratory

infrastructure support, and for manuscript review. We thank

Jennifer Yactor, Kerry Sondgeroth, and Joy Zartman for

technical assistance.tently infected with PLV-1695. Nested PCR were employed

to amplify a 4.5 kb fragment that encompasses the 3V codingregions of the PLV genome. Reactions employed ExTaq

DNA polymerase (Takara Mirus Bio, Madison WI) accord-

ing to manufacturers protocol. PCR conditions were 3 min

at 94 -C, and then 35 cycles of 94 -C for 30 s, 30 s annealingat 54 -C for first round PCR and 52 -C for second rounds, a70 -C extension for 4 min 30 s, and a final 10 min extensionat 70 -C. First round PCR primers were 3PLV11F, 5VGCCGGAACCTACAGACCCCTTAT 3V and 3PLV12R, 5VTTTGTTCTGCCCATTCTCCTATTG 3V and second roundprimers were 3770F, 5VAGAGAAGAAGATGCTAGATAT-

y 342 (2005) 6076This work was supported by Public Health Service grants

R29 AI41871 and R01 AI49765 from NIAID, DAIDS, the

73 (4), 25762586.

irologBrown, E.W., Miththapala, S., O Brien, S.J., 1993. Prevalence of exposure

to feline immunodeficiency virus in exotic Felid species. J. Zoo Wildl.

Med. 24 (3), 357364.

Brown, E.W., Yuhki, N., Packer, C., OBrien, S.J., 1994. A lion lentivirusColorado State University College of Veterinary Medicine

and Biomedical Science Research Council, and the Merck-

Meriel Summer Fellowship Program.

References

Ackley, C.D., Yamamoto, J.K., Levy, N., Pedersen, N.C., Cooper, M.D.,

1990. Immunologic abnormalities in pathogen-free cats experimen-

tally infected with feline immunodeficiency virus. J. Virol. 64 (11),

56525655.

Alkhatib, G., Combadiere, C., Broder, C.C., Feng, Y., Kennedy, P.E.,

Murphy, P.M., Berger, E.A., 1996. CC CKR5: a RANTES, MIP-1alpha,

MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1.

Science 272 (5270), 19551958.

Allan, J.S., Wilson, B., Potter, J., Hodara, V., Giavedoni, L., White, R.,

2004. 22nd Annual Symposium on Nonhuman Primate Models for

AIDS, San Antonio, TX.

Allison, R.W., Hoover, E.A., 2003. Feline immunodeficiency virus is

concentrated in milk early in lactation. AIDS Res. Hum. Retroviruses

19 (3), 245253.

Apetrei, C., Robertson, D.L., Marx, P.A., 2004. The history of SIVS and

AIDS: epidemiology, phylogeny and biology of isolates from naturally

SIV infected non-human primates (NHP) in Africa. Front. Biosci. 9,

225254.

Barr, M.C., Zou, L., Holzschu, D.L., Phillips, L., Scott, F.W., Casey, J.W.,

Avery, R.J., 1995. Isolation of a highly cytopathic lentivirus from a

nondomestic cat. J. Virol. 69 (11), 73717374.

Barr, M.C., Zou, L., Long, F., Hoose, W.A., Avery, R.J., 1997. Proviral

organization and sequence analysis of feline immunodeficiency virus

isolated from a Pallas cat. Virology 228 (1), 8491.

Barrientos, L.G., OKeefe, B.R., Bray, M., Sanchez, A., Gronenborn, A.M.,

Boyd, M.R., 2003. Cyanovirin-N binds to the viral surface glycopro-

tein, GP1,2 and inhibits infectivity of Ebola virus. Antiviral Res. 58 (1),

4756.

Bendinelli, M., Pistello, M., Lombardi, S., Poli, A., Garzelli, C., Matteucci,

D., Ceccherini-Nelli, L., Malvaldi, G., Tozzini, F., 1995. Feline

immunodeficiency virus: an interesting model for AIDS studies and

an important cat pathogen. Clin. Microbiol. Rev. 8 (1), 87112.

Bewley, C.A., Otero-Quintero, S., 2001. The potent anti-HIV protein

cyanovirin-N contains two novel carbohydrate binding sites that

selectively bind to Man (8) D1D3 and Man (9) with nanomolar affinity:

implications for binding to the HIV envelope protein gp120. J. Am.

Chem. Soc. 123 (17), 38923902.

Biek, R., Rodrigo, A.G., Holley, D., Drummond, A., Anderson Jr., C.R.,

Ross, H.A., Poss, M., 2003. Epidemiology, genetic diversity, and

evolution of endemic feline immunodeficiency virus in a population of

wild cougars. J. Virol. 77 (17), 95789589.

Bjorndal, A., Deng, H., Jansson, M., Fiore, J.R., Colognesi, C., Karlsson, A.,

Albert, J., Scarlatti, G., Littman, D.R., Fenyo, E.M., 1997. Coreceptor

usage of primary human immunodeficiency virus type 1 isolates varies

according to biological phenotype. J. Virol. 71 (10), 74787487.

Bolmstedt, A.J., OKeefe, B.R., Shenoy, S.R., McMahon, J.B., Boyd, M.R.,

2001. Cyanovirin-N defines a new class of antiviral agent targeting N-

linked, high-mannose glycans in an oligosaccharide-specific manner.

Mol. Pharmacol. 59 (5), 949954.

Brelot, A., Heveker, N., Adema, K., Hosie, M.J., Willett, B., Alizon, M.,

1999. Effect of mutations in the second extracellular loop of CXCR4 on

its utilization by human and feline immunodeficiency viruses. J. Virol.

N. Smirnova et al. / Vrelated to feline immunodeficiency virus: epidemiologic and phyloge-

netic aspects. J. Virol. 68 (9), 59535968.Burkhard, M.J., Obert, L.A., ONeil, L.L., Diehl, L.J., Hoover, E.A., 1997.

Mucosal transmission of cell-associated and cell-free feline immunode-

ficiency virus. AIDS Res. Hum. Retroviruses 13 (4), 347355.

Caney, S.M., Day, M.J., Gruffydd-Jones, T.J., Helps, C.R., Hirst, T.R.,

Stokes, C.R., 2002. Expression of chemokine receptors in the

feline reproductive tract and large intestine. J. Comp. Pathol. 126 (4),

289302.

Carpenter, M.A., OBrien, S.J., 1995. Coadaptation and immunodefi-

ciency virus: lessons from the Felidae. Curr. Opin. Genet. Dev. 5 (6),

739745.

Carpenter, M.A., Brown, E.W., Culver, M., Johnson, W.E., Pecon-Slattery,

J., Brousset, D., OBrien, S.J., 1996. Genetic and phylogenetic

divergence of feline immunodeficiency virus in the puma (Puma

concolor). J. Virol. 70 (10), 66826693.

Carpenter, M.A., Brown, E.W., MacDonald, D.W., OBrien, S.J., 1998.

Phylogeographic patterns of feline immunodeficiency virus genetic

diversity in the domestic cat. Virology 251 (2), 234243.

Carrillo, A., Ratner, L., 1996. Human immunodeficiency virus type 1

tropism for T-lymphoid cell lines: role of the V3 loop and C4 envelope

determinants. J. Virol. 70 (2), 13011309.

Chakrabarti, L.A., 2004. The paradox of simian immunodeficiency virus

infectino in sooty mangebys: active viral replication without disease

progression. Front. Biosci. 9, 521539.

Chen, Z., Zhou, P., Ho, D.D., Landau, N.R., Marx, P.A., 1997. Genetically

divergent strains of simian immunodeficiency virus use CCR5 as a

coreceptor for entry. J. Virol. 71 (4), 27052714.

Chesebro, B., Wehrly, K., Nishio, J., Perryman, S., 1996. Mapping of

independent V3 envelope determinants of human immunodeficiency

virus type 1 macrophage tropism and syncytium formation in

lymphocytes. J. Virol. 70 (12), 90559059.

Choe, H., Farzan, M., Sun, Y., Sullivan, N., Rollins, B., Ponath, P.D., Wu,

L., Mackay, C.R., LaRosa, G., Newman, W., Gerard, N., Gerard, C.,

Sodroski, J., 1996. The b-chemokine receptors CCR3 and CCR5

facilitate infection by primary HIV-1 isolates. Cell 85 (7), 11351148.

Corpet, F., Gouzy, J., Kahn, D., 1998. The ProDom database of protein

domain families. Nucleic Acids Res. 26 (1), 323326.

de Parseval, A., Elder, J.H., 2001. Binding of recombinant feline

immunodeficiency virus surface glycoprotein to feline cells: role of

CXCR4, cell-surface heparans, and an unidentified non-CXCR4

receptor. J. Virol. 75 (10), 45284539.

de Parseval, A., Chatterji, U., Sun, P., Elder, J.H., 2004a. Feline

immunodeficiency virus targets activated CD4+ T cells by using

CD134 as a binding receptor. Proc. Natl. Acad. Sci. U.S.A. 101 (35),

1304413049.

de Parseval, A., Ngo, S., Sun, P., Elder, J.H., 2004b. Factors that increase

the effective concentration of CXCR4 dictate feline immunodefi-

ciency virus tropism and kinetics of replication. J. Virol. 78 (17),

91329143.

de Parseval, A., Chatterji, U., Morris, G., Sun, P., Olson, A.J., Elder, J.H.,

2005. Structural mapping of CD134 residues critical for interaction with

feline immunodeficiency virus. Nat. Struct. Mol. Biol. 12 (1), 6066.

de Rozieres, S., Mathiason, C.K., Rolston, M.R., Chatterji, U., Hoover,

E.A., Elder, J.H., 2004. Characterization of a highly pathogenic

molecular clone of feline immunodeficiency virus clade C. J. Virol. 78

(17), 89718982.

Deng, H.K., Lui, R., Ellmeier, W., Choe, S., Unutmaz, D., Burkhart, M., Di

Marzio, P., Marmon, S., Sutton, R.E., Hill, C.M., Davis, C.B., Peiper,

S.C., Schall, T.J., Littman, D.R., Landau, N.R., 1996. Identification of a

major co-receptor for primary isolates of HIV-1. Nature 381, 661666.

Dey, B., Lerner, D.L., Lusso, P., Boyd, M.R., Elder, J.H., Berger, E.A.,

2000. Multiple antiviral activities of cyanovirin-N: blocking of human

immunodeficiency virus type 1 gp120 interaction with CD4 and

coreceptor and inhibition of diverse enveloped viruses. J. Virol. 74

(10), 45624569.

Diehl, L.J., Mathiason-DuBard, C.K., ONeil, L.L., Hoover, E.A., 1996.

y 342 (2005) 6076 73Plasma viral RNA load predicts disease progression in an accelerated

feline immunodeficiency virus model. J. Virol. 70, 25032507.

irologDonzella, G.A., Schols, D., Lin, S.W., Este, J.A., Nagashima, K.A.,

Maddon, P.J., Allaway, G.P., Sakmar, T.P., Henson, G., De Clercq, E.,

Moore, J.P., 1998. AMD3100, a small molecule inhibitor of HIV-1 entry

via the CXCR4 co-receptor. Nat. Med. 4 (1), 7277.

Doranz, B.J., Rucker, J., Yi, Y., Smyth, R.J., Samson, M., Peiper, S.C.,

Parmentier, M., Collman, R.B., Doms, R.W., 1996. A dual-tropic

primary HIV-1 isolate that uses fusin and the beta-chemokine

receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 85

(7), 11491158.

Dragic, T., Litwin, V., Allaway, G.P., Martin, S.R., Huang, Y., Nagashima,

K.A., Cayanan, C., Maddon, P.J., Koup, R.A., Moore, J.P., Paxton,

W.A., 1996. HIV-1 entry into CD4+ cells is mediated by the chemokine

receptor CC-CKR-5. Nature 381 (6584), 667673.

Dreitz, M.J., Dow, S.W., Fiscus, S.A., Hoover, E.A., 1995. Developmental

of monoclonal antibodies and capture immunoassays for feline

immunodeficiency virus. Am. J. Vet. Res. 56, 764768.

Egberink, H.F., De Clercq, E., Van Vliet, A.L., Balzarini, J., Bridger, G.J.,

Henson, G., Horzinek, M.C., Schols, D., 1999. Bicyclams, selective

antagonists of the human chemokine receptor CXCR4, potently inhibit

feline immunodeficiency virus replication. J. Virol. 73 (8), 63466352.

Elder, J.H., Dean, G.A., Hoover, E.A., Hoxie, J.A., Malim, M.H., Mathes,

L., Neil, J.C., North, T.W., Sparger, E., Tompkins, M.B., Tompkins,

W.A., Yamamoto, J., Yuhki, N., Pedersen, N.C., Miller, R.H., 1998.

Lessons from the cat: feline immunodeficiency virus as a tool to

develop intervention strategies against human immunodeficiency virus

type 1. AIDS Res. Hum. Retroviruses 14 (9), 797801.

Feng, Y., Broder, C.C., Kennedy, P.E., Berger, E.A., 1996. HIV-1 entry

cofactor: functional cDNA cloning of a seven-transmembrane, G

protein-coupled receptor. Science 272 (5263), 872877.

Gaddis, N.C., Sheehy, A.M., Ahmad, K.M., Swanson, C.M., Bishop, K.N.,

Beer, B.E., Marx, P.A., Gao, F., Bibollet-Ruche, F., Hahn, B.H., Malim,

M.H., 2004. Further investigation of simian immunodeficiency virus

Vif function in human cells. J. Virol. 78 (21), 1204112046.

Gupta, R., Brunak, S., 2002. Prediction of glycosylation across the

human proteome and the correlation to protein function. Pac. Symp.

Biocomput., 310322.

Gupta, R., Jensen, L.J., Brunak, S., 2002. Orphan protein function and its

relation to glycosylation. Ernst Schering Res. Found Workshop (38),

276294.

Gupta, R., Jung, E., Brunak, S., in preparation. Prediction of

N-glycosylation Sites in Human Proteins.

Haining, H., McMonagle, E.L., Knapp, E., Klein, D., Willett, B.J., Hosie,

M.J., 2004. 7th International Feline Retrovirus Research Symposium,

Pisa, Italy.

Hardy Jr., W.D., McClelland, A.J., Zuckerman, E.E., Snyder Jr., H.W.,

MacEwen, E.G., Francis, D., Essex, M., 1980. Development of virus

non-producer lymphosarcomas in pet cats exposed to FeLv. Nature 288

(5786), 9092.

Harper, D.R. (Ed.), 1993. Virology LabFax 1st edR In: Hames, B.D.,

Rickwood, D. (Eds.), The LabFax Series. Academic Press, London, UK.

Hohdatsu, T., Hirabayashi, H., Motokawa, K., Koyama, H., 1996.

Comparative study of the cell tropism of feline immunodeficiency virus

isolates of subtypes A, B and D classified on the basis of the env gene

V3V5 sequence. J. Gen. Virol. 77 (Pt. 1), 93100.

Hosie, M.J., Broere, N., Hesselgesser, J., Turner, J.D., Hoxie, J.A.,

Neil, J.C., Willett, B.J., 1998. Modulation of feline immunodefi-

ciency virus infection by stromal cell-derived factor. J. Virol. 72 (3),

20972104.

Hosie, M.J., Willett, B.J., Klein, D., Dunsford, T.H., Cannon, C.,

Shimojima, M., Neil, J.C., Jarrett, O., 2002. Evolution of

replication efficiency following infection with a molecularly cloned

feline immunodeficiency virus of low virulence. J. Virol. 76 (12),

60626072.

Ibrahim, J., Griffin, P., Coombe, D.R., Rider, C.C., James, W., 1999. Cell-

surface heparan sulfate facilitates human immunodeficiency virus Type

N. Smirnova et al. / V741 entry into some cell lines but not primary lymphocytes. Virus Res. 60

(2), 159169.Johnston, J., Power, C., 1999. Productive infection of human peripheral

blood mononuclear cells by feline immunodeficiency virus: implica-

tions for vector development. J. Virol. 73 (3), 24912498.

Johnston, J.B., Power, C., 2002. Feline immunodeficiency virus xenoin-

fection: the role of chemokine receptors and envelope diversity. J. Virol.

76 (8), 36263636.

Johnston, J.B., Olson, M.E., Rud, E.W., Power, C., 2001. Xenoinfection of

nonhuman primates by feline immunodeficiency virus. Curr. Biol. 11

(14), 11091113.

Joshi, A., Vahlenkamp, T.W., Garg, H., Tompkins, W.A., Tompkins, M.B.,

2004. Preferential replication of FIV in activated CD4(+)CD25(+)T

cells independent of cellular proliferation. Virology 321 (2), 307322.

Kato, K., Sato, H., Takebe, Y., 1999. Role of naturally occurring basic

amino acid substitutions in the human immunodeficiency virus type 1

subtype E envelope V3 loop on viral coreceptor usage and cell tropism.

J. Virol. 73 (7), 55205526.

Klatzmann, D., Champagne, E., Chamaret, S., Gruest, J., Guetard, D.,

Hercend, T., Gluckman, J.C., Montagnier, L., 1984. T-lymphocyte T4

molecule behaves as the receptor for human retrovirus LAV. Nature 312

(5996), 767768.

Langley, R.J., Hirsch, V.M., OBrien, S.J., Adger-Johnson, D., Goeken,

R.M., Olmsted, R.A., 1994. Nucleotide sequence analysis of puma

lentivirus (PLV-14): genomic organization and relationship to other

lentiviruses. Virology 202 (2), 853864.

Lerner, D.L., Elder, J.H., 2000. Expanded host cell tropism and cytopathic

properties of feline immunodeficiency virus strain PPR subsequent to

passage through interleukin-2-independent T cells. J. Virol. 74 (4),

18541863.

Levy, J.A., 1998. HIV and the Pathogenesis of AIDS, 2nd edR ASM Press,Washington, DC.

Linenberger, M.L., Deng, T., 1999. The effects of feline retroviruses

on cytokine expression. Vet. Immunol. Immunopathol. 72 (34),

343368.

Liu, H.Y., Soda, Y., Shimizu, N., Haraguchi, Y., Jinno, A., Takeuchi, Y.,

Hoshino, H., 2000. CD4-dependent and CD4-independent utilization of

coreceptors by human immunodeficiency viruses type 2 and simian

immunodeficiency viruses. Virology 278 (1), 276288.

Lombardi, S., Garzelli, C., Pistello, M., Massi, C., Matteucci, D.,

Baldinotti, F., Cammarota, G., da Prato, L., Bandecchi, P., Tozzini, F.,

1994. A neutralizing antibody-inducing peptide of the V3 domain of

feline immunodeficiency virus envelope glycoprotein does not induce

protective immunity. J. Virol. 68 (12), 83748379.

Lombardi, S., Massi, C., Tozzini, F., Zaccaro, L., Bazzichi, A., Bandecchi,

P., La Rosa, C., Bendinelli, M., Garzelli, C., 1995. Epitope mapping of

the V3 domain of feline immunodeficiency virus envelope glycoprotein

by monoclonal antibodies. J. Gen. Virol. 76 (Pt. 8), 18931899.

Maddon, P.J., Dalgleish, A.G., McDougal, J.S., Clapham, P.R., Weiss, R.A.,

Axel, R., 1986. The T4 gene encodes the AIDS virus receptor and is

expressed in the immune system and the brain. Cell 47 (3), 333348.

Mariani, R., Chen, D., Schrofelbauer, B., Navarro, F., Konig, R., Bollman,

B., Munk, C., Nymark-McMahon, H., Landau, N.R., 2003. Species-

specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell 114

(1), 2131.

Masuoka, K., Toyosaki, T., Tohya, Y., Norimine, J., Kai, C., Mikami, T.,

1992. Monoclonal antibodies to feline lymphocyte membranes recog-

nize the leukocyte-common antigen (CD45R). J. Vet. Med. Sci. 54 (5),

865870.

McCaffrey, R.A., Saunders, C., Hensel, M., Stamatatos, L., 2004. N-linked

glycosylation of the V3 loop and the immunologically silent face of

gp120 protects human immunodeficiency virus type 1 SF162 from

neutralization by anti-gp120 and anti-gp41 antibodies. J. Virol. 78 (7),

32793295.

Miller, R.J., Cairns, J.S., Bridges, S., Sarver, N., 2000. Human immuno-

deficiency virus and AIDS: insights from animal lentiviruses. J. Virol.

74 (16), 71877195.

y 342 (2005) 6076Miyazawa, T., Furuya, T., Itagaki, S., Tohya, Y., Takahashi, E., Mikami, T.,

1989. Establishment of a feline T-lymphoblastoid cell line highly

irologsensitive for replication of feline immunodeficiency virus. Arch. Virol.

108 (12), 131135.

Miyazawa, T., Kawaguchi, Y., Furuya, T., Itagaki, S., Takahashi, E.,

Mikami, T., 1990. Continuous production of feline immunodeficiency

virus in a feline T-lymphoblastoid cell line (MYA-1 cells). Nippon

Juigaku Zasshi 52 (4), 887890.

Miyazawa, T., Toyosaki, T., Tomonaga, K., Norimine, J., Ohno, K.,

Hasegawa, A., Kai, C., Mikami, T., 1992. Further characterization of a

feline T-lymphoblastoid cell line (MYA-1 cells) highly sensitive for

feline immunodeficiency virus. J. Vet. Med. Sci. 54 (1), 173175.

Mori, T., Boyd, M.R., 2001. Cyanovirin-N, a potent human immunodefi-

ciency virus-inactivating protein, blocks both CD4-dependent and CD4-

independent binding of soluble gp120 (sgp120) to target cells, inhibits

sCD4-induced binding of sgp120 to cell-associated CXCR4, and

dissociates bound sgp120 from target cells. Antimicrob. Agents

Chemother. 45 (3), 664672.

Muller, M.C., Barre-Sinoussi, F., 2003. SIVagm: genetic and biological

features associated with replication. Front. Biosci. 8, D1170D1185.

Nabatov, A.A., Pollakis, G., Linnemann, T., Kliphius, A., Chalaby, M.I.,

Paxton, W.A., 2004. Intrapatient alterations in the human immunode-

ficiency virus type 1 gp120 V1V2 and V3 regions differentially

modulate coreceptor usage, virus inhibition by CC/CXC chemokines,

soluble CD4, and the b12 and 2G12 monoclonal antibodies. J. Virol. 78

(1), 524530.

Nielsen, R., Yang, Z., 1998. Likelihood models for detecting positively

selected amino acid sites and applications to the HIV-1 envelope gene.

Genetics 148 (3), 929936.

Nishimura, Y., Nakamura, S., Goto, N., Hasegawa, T., Pang, H., Goto, Y.,

Kato, H., Youn, H.Y., Endo, Y., Mizuno, T., Momoi, Y., Ohno, K.,

Watari, T., Tsujimoto, H., Hasegawa, A., 1996. Molecular character-

ization of feline immunodeficiency virus genome obtained directly from

organs of a naturally infected cat with marked neurological symptoms

and encephalitis. Arch. Virol. 141 (10), 19331948.

Nishimura, Y., Miyazawa, T., Ikeda, Y., Izumiya, Y., Nakamura, K., Cai,

J.S., Sato, E., Kohmoto, M., Mikami, T., 1998. Molecular cloning and

expression of feline CD3epsilon. Vet. Immunol. Immunopathol. 65 (1),

4350.

Nishimura, Y., Igarashi, T., Donau, O.K., Buckler-White, A., Buckler, C.,

Lafont, B.A., Goeken, R.M., Goldstein, S., Hirsch, V.M., Martin, M.A.,

2004. Highly pathogenic SHIVs and SIVs target different CD4+ T cell

subsets in rhesus monkeys, explaining their divergent clinical courses.

Proc. Natl. Acad. Sci. U.S.A. 101 (33), 1232412329.

OKeefe, B.R., Shenoy, S.R., Xie, D., Zhang, W., Muschik, J.M., Currens,

M.J., Chaiken, I., Boyd, M.R., 2000. Analysis of the interaction

between the HIV-inactivating protein cyanovirin-N and soluble forms of

the envelope glycoproteins gp120 and gp41. Mol. Pharmacol. 58 (5),

982992.

OKeefe, B.R., Smee, D.F., Turpin, J.A., Saucedo, C.J., Gustafson,

K.R., Mori, T., Blakeslee, D., Buckheit, R., Boyd, M.R., 2003.

Potent anti-influenza activity of cyanovirin-N and interactions

with viral hemagglutinin. Antimicrob. Agents Chemother. 47 (8),

25182525.

Obert, L.A., Hoover, E.A., 2000. Feline immunodeficiency virus clade C

mucosal transmission and disease courses. AIDS Res. Hum. Retro-

viruses 16 (7), 677688.

Olmsted, R.A., Hirsch, V.M., Purcell, R.H., Johnson, P.R., 1989. Nucleo-

tide sequence analysis of feline immunodeficiency virus: genome

organization and relationship to other lentiviruses. Proc. Natl. Acad.

Sci. U.S.A. 86 (20), 80888092.

Olmsted, R.A., Langley, R., Roelke, M.E., Goeken, R.M., Adgerjohnson,

D., Goff, J.P., Albert, J.P., Packer, C., Laurenson, M.K., Caro, T.M.,

Scheepers, L., Wildt, D.E., Bush, M., Martenson, J.S., Obrien, S.J.,

1992. Worldwide prevalence of lentivirus infection in wild feline

species-epidemiologic and phylogenetic aspects. J. Virol. 66 (10),

60086018.

N. Smirnova et al. / VOverbaugh, J., Luciw, P.A., Hoover, E.A., 1997. Models for AIDS

pathogenesis: simian immunodeficiency virus, simian-human immuno-deficiency virus and feline immunodeficiency virus infections. Aids 11

(Suppl. A), S47S54.

Pancino, G., Fossati, I., Chappey, C., Castelot, S., Hurtrel, B., Moraillon,

A., Klatzmann, D., Sonigo, P., 1993. Structure and variations of feline

immunodeficiency virus envelope glycoproteins. Virology 192 (2),

659662.

Pedersen, N.C., Leutenegger, C.M., Woo, J., Higgins, J., 2001.

Virulence differences between two field isolates of feline immuno-

deficiency virus (FIV-APetaluma and FIV-CPGammar) in young

adult specific pathogen free cats. Vet. Immunol. Immunopathol. 79

(12), 5367.

Pohlmann, S., Davis, C., Meister, S., Leslie, G.J., Otto, C., Reeves, J.D.,

Puffer, B.A., Papkalla, A., Krumbiegel, M., Marzi, A., Lorenz, S.,

Munch, J., Doms, R.W., Kirchhoff, F., 2004. Amino acid 324 in the

simian immunodeficiency virus SIVmac V3 loop can confer CD4

independence and modulate the interaction with CCR5 and alternative

coreceptors. J. Virol. 78 (7), 32233232.

Quackenbush, S.L., Donahue, P.R., Dean, G.A., Myles, M.H., Ackley,

C.D., Cooper, M.D., Mullins, J.I., Hoover, E.A., 1990. Lymphocyte

subset alterations and viral determinants of immunodeficiency disease

induction by the feline leukemia virus FeLV-FAIDS. J. Virol. 64 (11),

54655474.

Richardson, J., Pancino, G., Merat, R., Leste-Lasserre, T., Moraillon, A.,

Schneider-Mergener, J., Alizon, M., Sonigo, P., Heveker, N., 1999.

Shared usage of the chemokine receptor CXCR4 by primary and

laboratory-adapted strains of feline immunodeficiency virus. J. Virol. 73

(5), 36613671.

Roderiquez, G., Oravecz, T., Yanagishita, M., Bou-Habib, D.C., Mostow-

ski, H., Norcross, M.A., 1995. Mediation of human immunodeficiency

virus type 1 binding by interaction of cell surface heparan sulfate

proteoglycans with the V3 region of envelope gp120-gp41. J. Virol. 69

(4), 22332239.

Rogers, A.B., Hoover, E.A., 1998. Maternal fetal feline immunodeficiency

virus transmission: timing and tissue tropisms. J. Infect. Dis. 178 (4),

960967.

Rogers, A.B., Hoover, E.A., 2002. Fetal feline immunodeficiency virus is

prevalent and occult. J. Infect. Dis. 186 (7), 895904.

Rost, B., Yachdav, G., Liu, J., 2004. The predict protein server. Nucleic

Acids Res. 32, W321W326 (Web Server issue).

Schrofelbauer, B., Chen, D., Landau, N.R., 2004. A single amino

acid of APOBEC3G controls its species-specific interaction with

virion infectivity factor (Vif). Proc. Natl. Acad. Sci. U.S.A. 101 (11),

39273932.

Shimojima, M., Nishimura, Y., Miyazawa, T., Kato, K., Tohya, Y., Akashi,

H., 2003. CD56 expression in feline lymphoid cells. J. Vet. Med. Sci. 65

(7), 769773.

Shimojima, M., Miyazawa, T., Ikeda, Y., McMonagle, E.L., Haining, H.,

Akashi, H., Takeuchi, Y., Hosie, M.J., Willett, B.J., 2004. Use of

CD134 as a primary receptor by the feline immunodeficiency virus.

Science 303 (5661), 11921195.

Siebelink, K.H., Karlas, J.A., Rimmelzwaan, G.F., Osterhaus, A.D., Bosch,

M.L., 1995. A determinant of feline immunodeficiency virus involved

in Crandell feline kidney cell tropism. Vet. Immunol. Immunopathol. 46

(12), 6169.

Sodora, D.L., Shpaer, E.G., Kitchell, B.E., Dow, S.W., Hoover, E.A.,

Mullins, J.I., 1994. Identification of three feline immunodeficiency

virus (FIV) env gene subtypes and comparison of the FIV and human

immunodeficiency virus type 1 evolutionary patterns. J. Virol. 68 (4),

22302238.

Speck, R.F., Wehrly, K., Platt, E.J., Atchison, R.E., Charo, I.F., Kabat, D.,

Chesebro, B., Goldsmith, M.A., 1997. Selective employment of

chemokine receptors as human immunodeficiency virus type 1

coreceptors determined by individual amino acids within the envelope

V3 loop. J. Virol. 71 (9), 71367139.

Tanabe-Tochikura, A., Tochikura, T.S., Blakeslee Jr., J.R., Olsen, R.G.,

y 342 (2005) 6076 75Mathes, L.E., 1992. Anti-human immunodeficiency virus (HIV)

agents are also potent and selective inhibitors of feline immunodefi-

ciency virus (FIV)-induced cytopathic effect: development of a new

method for screening of anti-FIV substances in vitro. Antivir. Res. 19

(2), 161172.

Terwee, J.A., Yactor, J.K., Sondgeroth, K.S., Vandewoude, S., 2005. Puma

lentivirus is controlled in domestic cats after mucosal exposure in the

absence of conventional indicators of immunity. J. Virol. 79 (5),

27972806.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G.,

1997. The CLUSTAL_X windows interface: flexible strategies for

multiple sequence alignment aided by quality analysis tools. Nucleic

Acids Res. 25 (24), 48764882.

Tozzini, F., Matteucci, D., Bandecchi, P., Baldinotti, F., Siebelink, K.,

Osterhaus, A., Bendinelli, M., 1993. Neutralizing antibodies in cats

infected with feline immunodeficiency virus. J. Clin. Microbiol. 31 (6),

VandeWoude, S., Hageman, C.L., OBrien, S.J., Hoover, E.A., 2000.

Proceedings, 5th Intl. Feline Retrovirus Research Symposium, Cannes,

France.

VandeWoude, S., Hageman, C.A., OBrien, S.J., Hoover, E.A., 2002.

Nonpathogenic lion and puma lentiviruses impart resistance to super-

infection by virulent feline immunodeficiency virus. J. Acquir. Immune

Defic. Syndr. 29 (1), 110.

VandeWoude, S., Hageman, C.L., Hoover, E.A., 2003. Domestic cats

infected with lion or puma lentivirus develop anti-feline immunodefi-

ciency virus immune responses. J. Acquir. Immune Defic. Syndr. 34 (1),

2031.

Verschoor, E.J., Boven, L.A., Blaak, H., van Vliet, A.L., Horzinek, M.C.,

de Ronde, A., 1995. A single mutation within the V3 envelope

neutralization domain of feline immunodeficiency virus determines its

tropism for CRFK cells. J. Virol. 69 (8), 47524757.

N. Smirnova et al. / Virology 342 (2005) 607676Troyer, J.L., Pecon-Slattery, J., Roelke, M.E., Black, L., Packer, C.,

OBrien, S.J., 2004. Patterns of feline immunodeficiency virus multiple

infection and genome divergence in a free-ranging population of

African lions. J. Virol. 78 (7), 37773791.

Troyer, J.L., Pecon-Slattery, J., Roelke, M.E., Johnson, W.E., VandeWoude,

S., Vazquez-Salat, N., Brown, M., Frank, L., Woodroffe, R., Winter-

bach, C., Winterbach, H., Hemson, G., Bush, M., Alexander, K.A.,

OBrien, S.J., 2005. Seroprevalence and genomic divergence of

circulating strains of feline immunodeficiency virus (FIV) among

felidae and hyaenidae species. J. Virol. (in revision).

Tsai, C.C., Emau, P., Jiang, Y., Tian, B., Morton, W.R., Gustafson, K.R.,

Boyd, M.R., 2003. Cyanovirin-N gel as a topical microbicide prevents

rectal transmission of SHIV89.6P in macaques. AIDS Res. Hum.

Retroviruses 19 (7), 535541.

Tsai, C.C., Emau, P., Jiang, Y., Agy, M.B., Shattock, R.J., Schmidt, A.,

Morton, W.R., Gustafson, K.R., Boyd, M.R., 2004. Cyanovirin-N

inhibits AIDS virus infections in vaginal transmission models. AIDS

Res. Hum. Retroviruses 20 (1), 1118.

Uchiyama, T., 1997. Human T cell leukemia virus type I (HTLV-I) and

human diseases. Annu. Rev. Immunol. 15, 1537.

Vahlenkamp, T.W., Verschoor, E.J., Schuurman, N.N., van Vliet, A.L.,

Horzinek, M.C., Egberink, H.F., de Ronde, A., 1997. A single amino

acid substitution in the transmembrane envelope glycoprotein of

feline immunodeficiency virus alters cellular tropism. J. Virol. 71 (9),

71327135.

Vahlenkamp, T.W., Tompkins, M.B., Tompkins, W.A., 2004. Feline

immunodeficiency virus infection phenotypically and functionally

activates immunosuppressive CD4+CD25+ T regulatory cells. J.

Immunol. 172 (8), 47524761.

VandeWoude, S., OBrien, S.J., Hoover, E.A., 1997a. Infectivity of lion and

puma lentiviruses for domestic cats. J. Gen. Virol. 78 (Pt. 4), 795800.

VandeWoude, S., OBrien, S.J., Langelier, K., Hardy, W.D., Slattery, J.P.,

Zuckerman, E.E., Hoover, E.A., 1997b. Growth of lion and puma

lentiviruses in domestic cat cells and comparisons with FIV. Virology

233 (1), 185192.Vodros, D., Thorstensson, R., Biberfeld, G., Schols, D., De Clercq, E.,

Fenyo, E.M., 2001. Coreceptor usage of sequential isolates from