spatial patterns of protein expression in focal infections of human cytomegalovirus

Spatial Patterns of Protein Expressionin Focal Infections of HumanCytomegalovirus

Vy Lam,1 Karl W. Boehme,2 Teresa Compton,2 John Yin1

1Department of Chemical and Biological Engineering,1415 Engineering Dr., University of Wisconsin, Madison, Wisconsin;telephone: (608) 265-3779; fax: (608) 262-5434; e-mail: [email protected] of Oncology, McArdle Cancer Research Center, 1400 University Avenue,University of Wisconsin, Madison, Wisconsin

Received 16 May 2005; accepted 24 October 2005

Published online 27 February 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20786

Abstract: Human cytomegalovirus (HCMV) is amedicallysignificant human pathogen that infects a wide range ofcell and tissue types. During infection, HCMV activates avariety of signal transduction pathways that induceprofound changes in cellular processes and dramaticallyaffect cellular gene expression patterns. To better definehow these virus-host interactions affect the local micro-environment and influence the spatial and temporalspread of HCMV, we initiated HCMV focal infections onnormal human dermal fibroblast monolayers and mon-itored viral gene expression patterns and infection spreadover 45 days. To establish baseline temporal measure-ments of HCMV infection and spread in cell monolayers,we characterized the influence of three experimentalvariables on viral gene expression: cell plating density,the presence of serum, and neutralization of cellularantiviral responses with an antibody against interferon-b.We found that high cell plating density or the inclusion ofserum correlated with enhanced HCMV infection spread.Dramatic differences in the expression pattern of the viralimmediate early 2 (IE2) gene were observed under theseconditions as compared to low plating density or theabsence of serum. In the latter case round, uniform fociwere observed with a clear wave of IE2 expressionvisible in advance of a late stage viral protein, envelopeglycoprotein B. By contrast, larger irregular fociwith armsof IE2 expressionwereobserved in thepresenceof serum.Addition of the antibody had little effect on the rate ofspread, which is consistent with the knowledge thatHCMV represses antiviral responses during infection.This experimental system provides a useful means tovisualize and quantify complex virus-host interactions.� 2006 Wiley Periodicals, Inc.

Keywords: human cytomegalovirus (HCMV); virus-hostinteractions; infection spread

INTRODUCTION

In vitro methods to elucidate mechanisms and dynamics of

virus-host interactions at the cellular and molecular level

have largely employed synchronous infections to examine

processes associated with a single round of infection.

Experimental protocols for such ‘‘one-step’’ or ‘‘single-

cycle’’ virus cultures typically mix an excess of virus

particles with cultured cells to insure that most or all of the

cells are rapidly infected. By sampling such infections over

time and quantifying levels of virus particles or viral

intermediate components, these studies have elucidated the

dynamics of viral entry, genome replication, protein expres-

sion, and progeny assembly. At the same time, detection of

changes in cellular components can reveal virus-mediated

alterations to the cell cycle, triggering of self-destructive

apoptotic pathways, or activation of cellular defensive

responses. Despite their central role in the study of

single infection cycles, synchronous infections are limited

in their capacity to shed light on processes that extend to time

scales of multiple infection cycles. Such processes include

the spread of virus from infected to non-infected cells,

propagation of cell–cell signaling induced by the infection,

and resistance of the cell population to the spread of the virus.

To better understand how cells and cell populations

interact with viruses over periods that span multiple rounds

of infection, we are developing in vitro systems where viruses

reproduce and gradually spread across a layer of susceptible

cells (Duca et al., 2001; Endler et al., 2003, 2005; Lam et al.,

2005; Lee et al., 1997; Lee and Yin, 1996a,b; Yin, 1991,

1993). For mammalian viral systems we have developed a

focal infection technique where host cell monolayers are

overlaid with agar and inoculated focally with virus (Duca

et al., 2001; Lam et al., 2005). The solidified agar limits

dispersion of the virus particles and thus maintains a spatial

segregation between infected and non-infected cells. Over

time, successive cycles of viral replication and spread to

locally susceptible non-infected cells drives propagation

�2006 Wiley Periodicals, Inc.

Correspondence to: Dr. John Yin

Contract grant sponsors: National Science Foundation; National Insti-

tutes of Health; U.S. Public Health Service.

Contract grant numbers: EIA-0331337; 5T32 GM08349; RO1 AI34998,

RO1 AI54915.

of the infection across the cell monolayer. The extent of

infection spread at any time post infection can be visualized

by immunofluorescent labeling of virus or host components,

followed by imaging. By collecting and analyzing images at

different times post infection one can deduce the rate of virus

spread. In our initial studies of an RNA virus, vesicular

stomatitis virus (VSV), infection spread in focal infections

was dependent on cell type, the titer of the initial inoculum,

and cell age at the time of inoculation (Duca et al., 2001). The

rate of VSV spread generally correlated with viral growth in

one-step virus cultures, however, induction of cellular

antiviral activities, such as interferon signaling, could be an

overriding factor in dictating both infection spread and viral

growth (Lam et al., 2005). We have further found that

different temporal stages of infection can be simultaneously

viewed at different locations within the leading edge of a

spreading infection front (Duca et al., 2001; Endler et al.,

2003); early events are visible at the very leading edge while

later events trail behind. We anticipate that focal infections of

DNAviruses, which generally exhibit more distinctive stages

of early, middle, and late gene expression than RNA viruses,

may open new perspectives on the dynamics of viral infection

that are often missed by single-round methods. Here we

employ the focal infection method to study the spread of

human cytomegalovirus (HCMV), a DNA virus.

Human cytomegalovirus (HCMV) is a ubiquitous member

of the herpesvirus family that infects between 50% and 90%

of the human population. Primary infection of normal

healthy individuals is typically asymptomatic. Although

the virus is readily controlled by the host immune system, it is

never fully cleared from the body and maintains a lifelong

relationship with the host as a latent infection. HCMV can

enter almost every human cell type examined, and as such is

able to infect almost every organ system in the body.

Accordingly, HCMV disease can manifest itself in a myriad

of ailments including hepatitis, gastrointestinal disorders,

and retinitis. Congenital HCMV infection is a major cause of

birth defects, often resulting in severe neurological and

cognitive disorders (Pass et al., 1980; Ramsay et al., 1991).

Immunosuppressed individuals such as transplant recipients

and those afflicted with AIDS are also affected by HCMV,

either through primary infection, reinfection, or reactivation

from latency (Ljungman, 1996; Pass, 2001).

HCMV is a large virus with a double-stranded DNA

genome that putatively encodes more than 200 proteins

(Chee et al., 1990). The virion contains a 250 kbp genome

housed within a protein capsid. The capsid is surrounded by a

herpesvirus-specific structure known as the tegument, which

is comprised of numerous viral proteins and phosphopro-

teins. Several tegument components act as transcription

factors that facilitate viral replication. A host-derived lipid

envelope, into which numerous virally-encoded glycopro-

teins are incorporated, encloses the virion. In infected cells,

HCMV genes are expressed in three stages: immediate early,

delayed early, and late (Mocarski and Courcelle, 2001). The

immediate early genes primarily encode regulatory proteins

that modulate viral and host gene expression. The delayed

early genes are involved with viral DNA replication, while

the late genes primarily encode structural proteins that are

incorporated into progeny virions.

A hallmark of HCMV infection is a dramatic reprogram-

ming of host cell gene expression (Browne et al., 2001;

Simmen et al., 2001; Zhu et al., 1997, 1998). Genes involved

in a myriad of cellular processes including host transcription

and translation, stress response, immune signaling, and

antiviral activity are modulated through a variety of

mechanisms (Browne et al., 2001; Zhu et al., 1998).

Engagement of viral entry receptors such as integrins and

the epidermal growth factor receptor as well as induction of

cellular antiviral responses directly affect host gene expres-

sion (Boehme et al., 2004; Boyle et al., 1999; Browne et al.,

2001; Compton et al., 2003; Feire et al., 2004; Wang et al.,

2003). Components of the virus tegument such as pp65 and

pp71 (Hensel et al., 1996; Lischka et al., 2003; Liu and

Stinski, 1992; Winkler and Stamminger, 1996) as well as

numerous viral gene products expressed during HCMV

infection are potent host gene expression regulators (Malone

et al., 1990; Pizzorno et al., 1988). HCMV infection can also

indirectly modulate host gene expression profiles through the

autocrine and paracrine activity of interferons, inflammatory

cytokines, and other secreted factors. The complexity of

potential interactions between the numerous viral and

cellular components and pathways creates significant

challenges in understanding how these interactions con-

tribute to a coordinated response within single cells and

across cell populations.

As an initial test of HCMV focal infections on normal

human dermal fibroblasts (NHDF) we assessed the effects

of cell-plating density, serum in the agar overlay, and

intercellular interferon-b secretion on virus spread. The

extent of infection spread at various times post infection was

visualized by imaging the IE2-enhanced green fluorescence

protein (EGFP) fusion protein encoded by the virus (Sanchez

et al., 2002). Following the collection of each sequence of

expanding infection images we also visualized the spatial

distribution of viral envelope glycoprotein B expression by

immunofluorescent labeling. We found that HCMV spread

was enhanced by increasing cell density, as well as the

addition of serum to the agar overlay. As anticipated, we were

able to observe a clear wave of early HCMV protein (IE2)

expression ahead of glycoprotein B expression. However,

this pattern was unexpectedly and dramatically altered in the

presence of serum, where IE2 expression spread as asym-

metric arms. Together these results demonstrate how tissue

culture conditions can affect the development of HCMV

spatial and temporal patterns of gene expression and spread.

MATERIALS AND METHODS

Cell and Virus Culture

Normal human dermal fibroblasts (NHDF) (Clonetics, San

Diego, CA) were grown as monolayers at 378C in a

humidified atmosphere containing 5% CO2 in Dulbecco’s

1030 Biotechnology and Bioengineering, Vol. 93, No. 6, April 20, 2006

DOI 10.1002/bit

minimal essential medium (DMEM, Life Technologies,

Gaithersburg, MD) containing 10% fetal bovine serum (FBS,

Omega Scientific, Tarzana, CA) and 1% penicillin–strepto-

mycin–amphotericin B (Fungizone, Life Technologies).

Human cytomegalovirus with enhanced green fluores-

cence protein fused to the immediate early 2 protein (HCMV

IE2-EGFP) was obtained from Dr. Deborah Spector (Uni-

versity of California, San Diego). Virus stocks were prepared

from infected NHDF monolayers and concentrated by

centrifugation (19,000 rpm for 1 h at 48C in a Sorvall RC

5C Plus centrifuge with a SS-34 rotor) through a sucrose

cushion (20% w/v).

Focal Infection of Cell Monolayers

Cells were plated in six-well plates at 2.5� 104, 5� 104, and

1� 105 cells/well (in 2 mL of culture medium). Two days

post-seeding the culture medium was aspirated and replaced

with 4 mL of agar overlay. The agar overlay was prepared by

combining agar powder (Agar Nobel, Difco, Detroit, MI) at

0.6% (w/v) with nanopure water (approximately 10% of the

final volume) and heating in a microwave for 40 s to dissolve

the agar. The agar solution was combined with infection

medium (90% of the final volume) warmed to 378C. Two

different infection media were used for the agar overlay,

DMEM and DMEM supplemented with 2% FBS. The agar

overlay was allowed to solidify at room temperature for

30 min. Avirus deposition reservoir was made by punching a

hole in the center of the overlay using the tip of a Pasteur

pipette. The plug of agar inside the tip of the pipette was

subsequently removed by applying gentle suction through

the pipette. Virus was added to the reservoir at an MOI of 5.

Where indicated, 250 U of a rabbit polyclonal antibody

against human interferon beta (31410-1, PBL Biomedical

Laboratories, Piscataway, NJ) was added to the virus ino-

culum. The plates were incubated at 378C until the

predetermined imaging times.

Fixation and Immunocytochemistry

Focally infected monolayers were fixed at the last time point

of the experiment with 3 mL of fixative per monolayer. The

fixative consisted of 4% (w/v) paraformaldehyde (VWR,

West Chester, PA) and 5% (w/v) sucrose (Sigma, St. Louis,

MO) in 10 mM phosphate buffered saline (PBS) at pH 7.4.

After 3 h fixation, the agar overlays were removed and

the monolayers were rinsed twice with 2 mL/well of PBS

and stored in PBS at 48C until immunofluorescence label-

ing. Viral glycoprotein B (gB) was visualized by indirect

immunofluorescence labeling, as previously described

(Pietropaolo and Compton, 1999), using a primary mono-

clonal murine antibody (hybridoma 27–78) against HCMV

gB (Britt, 1984) diluted 1:100 (0.5 mL per monolayer) and a

Cy3-conjugated donkey anti-mouse secondary antibody

(Jackson Immunoresearch, West Grove, PA) diluted 1:300

(0.5 mL/well). 40,6-diamidino-2-phenylindole (DAPI,

Sigma) was included with the secondary antibody solution,

at 0.6 mM, to stain DNA. In samples where cellular actin

cytoskeleton was visualized, Alexa Fluor 488 conjugated

phalloidin (Molecular Probes/Invitrogen, Carlsbad, CA) was

diluted 1:100 in PBS and added to each sample (0.5 mL/well)

for 20 min. Monolayers were stored in 2 mL of PBS at 48Cuntil imaging.

Image Collection, Processing, and Quantification

Images of EGFP expression in the focal infection monolayers

were acquired using a Nikon TE300 inverted epifluorescent

microscope equipped with a Nikon mercury light source, a

Prior XYZ translation stage driven by Metamorph 6.0

software (Universal Imaging), and a monochrome SensSys

4.0 cooled CCD camera. The location and extent of infection

was manually identified in each well and the number of

microscope fields necessary to fully image the infected

area at 4� magnification was estimated (12–40 fields). The

system was then programmed to move the stage consecu-

tively across the entire infected area, acquire an image at each

position, and finally, combine the individual images into a

single montage.

Prior to quantification, all images from an experiment were

pre-processed using the same scaling factors to enhance

contrast and resized to be easily accommodated on a

computer screen. Image pre-processing was performed with

Adobe Photoshop 7.0 and quantification of the infection

areas was performed with Matlab 6.1. Background illumina-

tion was estimated and subtracted from each image before a

threshold level was manually set to optimize the inclusion of

signal positive pixels across all images of an experiment. In

each image neighboring signal positive pixels were linked as

one object and the total area of this object, in pixels, was

reported for the image. The infection radius for each image

was calculated from the area of this object where the radius

equals (area/p)1/2. The length of each radius was then

converted from pixels to millimeters using the scale bar on

the image. At each time point three to four replicates were

quantified and the mean was plotted as a function of time. The

standard error of the mean (SEM) was included at each data

point as error bars. Statistical analysis of the data was done

using the Student’s t-test. In all cases, the null hypothesis was

that there was no difference between the infection radii of

control and treated samples.

RESULTS

High Cell Plating Density and the Presence ofSerum Increased the Rate of HCMV Spread

To assess the effect of different culture conditions on viral

spread in focal infections, a recombinant HCMV (AD169

strain) with the enhanced green fluorescent protein (EGFP)

fused to the immediate early 2 gene (IE2) was employed

(Sanchez et al., 2002). IE2 is among the first genes expressed

by HCMV upon infection and is frequently used as a marker

for viral infection. Focal infections were carried out on

Lam et al.: Protein Patterns in Spreading HCMV Infections 1031

Biotechnology and Bioengineering. DOI 10.1002/bit

NHDF monolayers plated at three cell densities: 2.5� 104,

5� 104, and 1� 105 cells/well. At all three cell densities

infections were performed in both the absence and presence

of serum. Figure 1A shows micrographs of the fluorescence

signals observed in representative infected monolayers at

43 days post infection. The extent of spread at various times

over the course of the experiment was quantified (Fig. 1B and

C), and rates of radial expansion were estimated by linear

regression after 12 days. Data points at 5 and 8 days were

neglected since the infection foci were broadly distributed

within the site of inoculation and did not yet form a

contiguous area of infection.

Higher cell-plating densities yielded higher rates of

HCMV spread (Fig. 2). In the absence of serum, spread

rates varied three fold, from 19 mm/day in monolayers plated

at 2.5� 104 cells/well, to 59 mm/day in monolayers plated at

1� 105 cells/well (Table I). Addition of serum to the agar

overlay also resulted in faster spread rates, particularly in

monolayers plated at 5� 104 cells/well. At this cell density,

the presence of serum yielded higher rates by approximately

30 mm/day. In monolayers plated at 2.5� 104 and 1� 105

cells, the increases were less dramatic at 5 and 20 mm/day,

respectively. Statistical analysis of the data show that the

enhanced spread rates due to increased cell-plating density

Figure 1. Human cytomegalovirus (HCMV) focal infections on normal human dermal fibroblast (NHDF) monolayers. NHDF monolayers were plated at the

indicated cell densities and focally infected with 2� 104 plaque forming units of HCMV-IE2-EGFP in the presence or absence of serum. A: Representative

micrographs of the infections at 43 days post infection. Radial expansion over time was measured in the (B) absence of serum or (C) presence of serum. The rate

of spread for each infection was determined by a linear regression using data points from 12 to 43 days.


DOI 10.1002/bit

was significant beyond 95% while the effect mediated by

serum addition was at 95%. The greater increase in infection

spread rate, of 30 mm/day, observed in monolayers with

5� 104 plating density suggest that there is a threshold of cell

density where infection spread is optimal. This optimal cell

density was achieved in these monolayers when they were

incubated with serum. Correspondingly, infection spread

rates did not increase as dramatically in monolayers plated at

2.5� 104 and 1� 105 cells/well since these monolayers

either did not reach or were already beyond this optimal cell

density. Slower rates of spread in low-density monolayers

were not due to a lack of neighboring cells to support virus

propagation. All monolayers were observed to be confluent

by 15 days post infection (results not shown). The cell counts

by all focally infected monolayers, including those plated at

low density and incubated without serum, at 15 days post-

infection ranged from 3.1� 105 to 7.4� 105 cells/well. Cell

densities were determined by quantifying the number of

DAPI labeled cell nuclei in fixed monolayers. Phalloidin

labeled cells in Figure 5B and D show confluent monolayers

at, respectively, 4� 105 and 8.2� 105 cells/well.

Anti-Interferon-b Antibody Alters Viral GeneExpression Patterns But Does Not AffectHCMV Spread Rates

Interferon-b (IFN-b) restricts viral replication by repressing

numerous cellular functions necessary for efficient viral

infection (Stark et al., 1998). Similar to other viruses, HCMV

robustly induces IFN-b expression upon infection. To assess

the effect of the IFN-b response on HCMV spread, an IFN-bneutralizing antibody was added to the inoculum of focal

infection samples. Inhibition of IFN-b activity had negligible

effects on the spread rate of HCMV (Fig. 2). Interestingly, a

slight decrease in the spread rate (8 mm/day) was observed in

monolayers plated at 1� 105 cells/well, both in the presence

and absence of serum. However, these differences were not

statistically significant.

Although addition of the IFN-b neutralizing antibody

minimally affected HCMV spread rates, significant differ-

ences in viral gene expression patterns were observed under

serum-free infections (Fig. 3A–F), but not in cultures

incubated with serum (Fig. 3G–L). Expression of viral

envelope glycoprotein B (gB), a late-stage gene product, was

monitored in combination with IE2-EGFP to assess the

effects of different culture conditions on genes with different

temporal expression patterns. In control cultures minimal

expression of viral IE2 gene expression was observed at

1� 104 and 5� 104 cells/well (Fig. 3A and B). At high cell

density, 1� 105 cells/well, greater expansion of viral gene

expression was observed (Fig. 3C), IE2-EGFP and gB were

broadly distributed and overlapped significantly. In contrast

to the control cultures, expansion of the viral gene expression

front was increased in monolayers treated with the anti-IFN-

b antibody (Fig. 3D–E). Further, IE2-EGFP expression was

localized at the leading edge of the infection. Lagging behind

the IE2-EGFP front was a uniformly distributed ring of gB

expression. This pattern of expression is consistent with the

tiered gene expression program utilized by HCMV.

The larger foci observed in the presence of the antibody

may be attributable to an increase in the efficiency of

inoculation. Although antibody addition had no effect on the

rate of infection spread, neutralization of IFN-b activity

facilitated HCMV infection of cells at the time of focal

inoculation. Extrapolation from data obtained between 11

and 35 days post-infection, reveals that the initial infection

radii in control cultures are smaller than those in the presence

Figure 2. Rates of HCMV infection spread are increased at higher cell-

plating densities and in the presence of serum. NHDF monolayers were

plated at 2.5� 104 (white bar), 5� 104 (hatched bar), and 1� 105 cells (black

bar) per well and inoculated with 2� 104 plaque forming units of HCMV-

IE2-EGFP in the presence or absence of anti-interferon antibody. Infected

monolayers were either incubated with or without serum. Error bars

represent the standard error of the mean for three replicate samples except for

data points highlighted with *where there were only two replicates.

**Increase in mean spread rate with respect to cell-plating density and

serum incubation was significant beyond 95% (using the paired t-test). *The

difference in these two spread rates was only significant to 69%.

Table I. Rates of HCMV infection spread in NHDF cell monolayers.

Monolayer plating density (cells/well)

Spread rate (mm/day)

2.5� 104 5� 104a 1� 105a

No AIFN

No serum 19� 3.1 26� 4.3 59� 9.4

Serumb 28� 1.1 55� 6.4 76� 7.0

AIFNc

No serum 20� 1.3 30� 3.6 51� 8.8

Serumb 26� 3.9 55� 7.9 67� 6.1

aSpread rates were compared to those observed in monolayers plated at2.5� 104 cells/well, P< 0.05.

bSpread rates were compared to those observed in monolayers incubatedwith serum, P¼ 0.05.

cDifferences in spread rate due to antibody addition were not significant.



of the anti-IFN-b antibody (Table II). Given comparable rates

of spread, infections with larger initial size would produce

larger sized infections at all times post infection.

Spatial Spread of HCMV in MonolayersIncubated Without and With Serum ProducedDistinct Patterns of IE2-EGFP Expression

In general, viral IE2-EGFP expression was more uniform

and symmetric in monolayers incubated without serum.

As described above, IE2-EGFP expression was visible

as a narrow band at the leading edge of the infection

followed by extensive expression of viral glycoprotein B

(Fig. 3D–F). In contrast, in monolayers incubated with

serum IE2-EGFP expression was less spatially uniform

and appeared as individual arms of fluorescence localized

to specific sections of the infections (Fig. 3G–L). How-

ever, viral glycoprotein B (gB) expression was uniformly

distributed in the radial direction within these focal

infections.

Figure 3. Spatial patterns of viral IE2-EGFP and glycoprotein B expression on focally infected monolayers incubated without (A–F) and with serum (G–L).

Micrographs show representative HCMV focal infections at 35 days post infection. Distribution of IE2-EGFP, viral glycoprotein B, and cell nuclei in the images

were color-encoded as green, red, and blue, respectively. Apparent colors of yellow, magenta, and cyan in the images are due to overlapping green/red, red/blue,

and blue/green colors, respectively. HCMV gB was detected with an anti-gB primary antibody (monoclonal antibody 27–78) and a Cy3 conjugated secondary

antibody. Cell nuclei were labeled with DAPI. Cell density of the monolayers was approximately 4.4� 105 cells/well in monolayers incubated without serum

(A–F) and 8.5� 105 cells/well in monolayers incubated with serum (G–L). The temporal spread of IE2-EGFP expression in samples 3E and 3K over the course

of the experiment is presented in Figure 4.


DOI 10.1002/bit

In the absence of serum, IE2-EGFP expression was visible

as rings offluorescence that spread radially from 17 to 35 days

post infection (Fig. 4A, corresponds to Fig. 3E). Earlier

regions of IE2-EGFP expression generally correlated with

later regions of gB expression. By contrast, IE2-EGFP

expression in monolayers incubated with serum appeared to

spread with an angular rather than radial component (Fig. 4B,

corresponds to the infection in Fig. 3K). Of particular interest

is the right arm of the focal infection where IE2-EGFP

expression spreads toward the lower right hand corner of the

images between 17 and 21 days (Fig. 4B, arrow 1). Between

25 and 28 days, the IE2-EGFP expression spreads in the

downward direction (Fig. 4B, arrow 2), and by 32 and 35 days

spread was toward the lower left (Fig. 4B, arrow 3). The

observed oriented spread of IE2-EGFP expression and

expansive gaps between areas of IE2-EGFP fluorescence

suggests that the infections did not spread in these areas.

However, the gaps in IE2-EGFP expression stained positive

for viral gB expression (Fig. 3E, infection at 5� 104 cells

with anti-IFN-b antibody), suggesting that in monolayers

incubated with serum, HCMV infections spread more

uniformly than the expression of IE2-EGFP would indicate.

Currently we cannot rule out the possibility that the

expression of IE2-EGFP within these gap areas are below

our detection limit.

Staining of the cellular actin cytoskeleton with phalloidin

revealed that cells in monolayers incubated with serum, at all

cell plating densities, formed large regions of relatively

oriented cells as compared with monolayers incubated

without serum (representative samples are shown in Fig. 5).

In monolayers incubated without serum, the infections

spread through confluent monolayers of cells having

relatively random orientations (Fig. 5A and B). Alignment

in cell orientation was limited to a few neighboring cells and

was observed only in parts of the monolayers. In monolayers

incubated with serum, however, cells were more densely

packed and formed large sections of oriented cells that were

aligned in the same direction (Fig. 5C and D). Further, the

directional expression of IE2-EGFP noted above (Fig. 4B,

arrows 1–3) appears to correlate with the alignment of cells

in the monolayer (Fig. 5C and D). Initially, the outward

spread of IE2-EGFP expression, from below point 1 right-

ward, was directed downwards by the vertically aligned cells

located below point 2 (Fig. 5D). Then the diagonally aligned

cells below point 3 gradually limited the rightward expres-

sion of IE2-EGFP and instead redirected it left and downward

(Fig. 5A, arrow 3).

DISCUSSION

Several factors likely contribute mechanistically to the

observation that higher cell densities achieved either by

plating monolayers at high cell densities or including serum

in the agar medium, enable infections to spread at higher

rates. These effects are, to some extent, coupled since the

presence of serum can promote cell growth and thereby

increase cell density in the monolayers. Incubation of cells

with serum also facilitates their transition from G1 to S phase.

Cell entry into S phase induces the intracellular expression of

factors that are favorable to viral replication, a process that is

promoted in HCMV infected cells by the viral IE2 protein

(Castillo et al., 2000; Wiebusch et al., 2003a,b).

Serum-independent effects linked to higher cell densities

may also contribute to viral spread. As the cell density in

monolayers increases we observed reduced cell spreading,

increased cell–cell contact, and larger regions of aligned cell

populations (Fig. 5B and D). Such changes to cell

morphology and interactions are likely coupled to extensive

changes to the cytoskeleton such as altered fibronectin

splicing (Masur et al., 1996), increased collagen VI

expression (Hatamochi et al., 1989), and increased micro-

tubule formation (Ostlund et al., 1980). Moreover, these

changes can affect cell proliferation and influence the

clustering and distribution of integrins on the cell surface

(Giancotti and Ruoslahti, 1999). Integrins are cell-

surface receptors that mediate cell adhesion and induce

intracellular signaling cascades through their association

with signaling adapter proteins and the cytoskeleton at focal

adhesions (Giancotti and Ruoslahti, 1999).

HCMV infection and replication is intimately tied to the

cytoskeleton of the host. At the time of infection, HCMV

utilizes integrins for entry into cells (Feire et al., 2004). Upon

entry, the viral capsid is transported along cellular micro-

tubules toward the cell nucleus (Ogawa-Goto et al.,

2003). Within hours following infection, the virus depoly-

merizes and reorganizes the cell’s microtubules (Pfeiffer

et al., 1983) and actin microfilaments (Losse et al., 1982).

Depolymerization of cellular microtubules enhances the

initiation of HCMV DNA synthesis (Carney et al., 1986)

while depolymerization of the actin cytoskeleton stimulates

progeny production (Jones et al., 1986). Late in the infection

cycle, by 72 h post infection, the newly reformed cytoske-

leton facilitates the nuclear localization of HCMV capsid and

matrix proteins to facilitate the assembly of progeny virions

(Yamauchi et al., 1985). The efficacy of microtubule-

mediated capsid transport is believed to dictate the

endothelial-cell tropism of certain strains of HCMV (Sinzger

et al., 2000). These observations suggest that the correlation

between cell density and HCMV propagation from our in

Table II. Inoculation radii of samples shown in Figure 3a.

Plating density

(cells/well) Control (mm)

Anti-interferon

(mm)

No serum

1.0Eþ 04 0.14 (Fig. 3A) 0.58 (Fig. 3D)5.0Eþ 04 0.84 (Fig. 3B) 1.0 (Fig. 3E)1.0Eþ 05 0.74 (Fig. 3C) 1.0 (Fig. 3F)

With Serum

1.0Eþ 04 0.47 (Fig. 3G) 0.64 (Fig. 3J)5.0Eþ 04 0.77 (Fig. 3H) 1.0 (Fig. 3K)1.0Eþ 05 0.71 (Fig. 3I) 1.25 (Fig. 3L)

aRadii were extrapolated from infection spread data obtained between 11and 35 days post infection.



vitro experiments may be intimately coupled with cell

morphology, cell–cell communication, and interactions

between the cell and the underlying material substrate.

New methods are being developed to define, control and

characterize at the molecular-level how these factors may

influence viral spread (Endler et al., 2003, 2005).

Higher cell densities may also facilitate infection spread

by suppressing intracellular antiviral responses. Increased

cell density is known to suppress the activation of the stress-

activated transcription factor c-jun (Lallemand et al., 1998)

and the lateral mobility of immune proteins such as the

major histocompatibility antigens within the cell membrane

(Wier and Edidin, 1986). A reduction in transcription activity

and transport of surface receptors may interfere with in-

tracellular signal transduction and thus the expression of

antiviral genes. Further, HCMV infection can induce the

production of interferon-b (Boehme et al., 2004; Zhu

et al., 1998), and interferon signaling can inhibit HCMV

Figure 4. Distinctive patterns of HCMV IE2-EGFP expression in monolayers incubated without and with serum. Time-lapsed images of IE2-EGFP

expression in representative focally infected monolayers incubated without (A) and with serum (B). The sample shown in A corresponds to the color encoded

micrograph in Figure 3E with plating density of 5� 104 cells/well and infected with anti-interferon-b antibody. The sample shown in B corresponds to the color

encoded micrograph in Figure 3K with plating density of 5� 104 cells/well and infected with anti-interferon-b antibody. Numbers and arrows indicate the

apparent directions of IE2-EGFP spread over time. All scale bars represent 1 mm.


DOI 10.1002/bit

replication (Nakamura et al., 1988). We hypothesized that

inhibition of interferon-b activity by the inclusion of an

interferon-b neutralizing antibody with the inoculum would

enhance HCMV infection and spread. Interestingly we found

that while the antibody did facilitate HCMV infection at the

time of inoculation, it had minimal effects on the rate

of infection spread. This result suggests that at the time of

focal inoculation, inhibition of cellular interferon signaling

suppressed cellular antiviral responses and facilitated

HCMV infection and replication. However, once the in-

fection was established the effects of interferon signaling

on virus propagation were negligible. This is consistent

with the ability of HCMV to negatively modulate interferon

and interferon-stimulated gene expression during infection

(Browne et al., 2001; Zhu et al., 1998). HCMV utilizes a

multifaceted approach to inhibiting host interferon responses

indicating that inhibition of host antiviral responses is of

paramount importance to the virus. The HCMV TRS1 and

IRS1 gene products can overcome protein kinase R-mediated

inhibition of protein synthesis (Cassady, 2005; Hakki and

Geballe, 2005). A structural component of HCMV, pp65,

contributes to the global down-regulation of interferon-

stimulated gene expression (Abate et al., 2004; Browne and

Shenk, 2003). Most recently, the IE2 gene product was shown

to inhibit interferon-b gene transcription (Taylor and

Bresnahan, 2005). We hypothesize that inhibition of the

interferon pathway by HCMV masked the interferon-bneutralizing activity of the antibody and thus no differences

in the rates of infection spread when the antibody was

added to the focal inoculations. Additionally, induction of a

refractory state in cells to interferon signaling may also

contribute to the antibody independent propagation of

HCMV infections. Interferon stimulation is known to cause

a refractory state in cells, which includes a reduction in the

expression level of interferon receptors and interferon-

stimulated genes for up to 4 days (Dupont et al., 2002). A

loss of cellular response to interferon stimulation may disrupt

the perpetuation of interferon signaling and thus the antiviral

state of the monolayer.

Serum incubation and high cell density may also influence

the spatial expression pattern of viral genes. In monolayers

incubated without serum, where cell density is low and cell

Figure 5. Oriented spread of HCMV IE2-EGFP expression in monolayers incubated with serum correlates with cell alignment.A, C: Color encoded images

of IE2-EGFP, viral glycoprotein B, and cell nuclei at 35 days post infection. Both A and C samples were plated at 5� 104 cells/well and focally inoculated in the

presence of anti-IFN b antibody. Awas incubated without serum while C was incubated with serum.B,D: Magnified views of the areas highlighted by the white

boxes in A and C, respectively. In B and D, the micrographs show the fluorescence signal from both IE2-EGFP and cellular cytoskeleton labeled with Alexa 488

conjugated phalloidin. Numbers in D highlight regions of different cell orientation in the monolayer.



orientation in the monolayer was randomly distributed, IE2-

EGFP spread radially over 35 days, and its expression was

enriched at the leading edge of the infections. This spatial

expression pattern was not surprising because susceptible

cells at the outermost periphery of the focal infection are the

most recently infected cells, and IE2 is expressed at the

earliest stage in the HCMVinfection cycle. A related method,

using viral spatial spread to deduce the relative timing of

intracellular events, has been elegantly employed in the study

of herpes simplex virus type 1 (Pomeranz and Blaho, 1999).

In monolayers incubated with serum where cell density

was higher, it was evident that IE2-EGFP expression spread

preferentially along the major axes of locally oriented cells

(Fig. 5). The direction of IE2-EGFP expression was visibly

altered several times over the course of the experiment, and

the regions of directional change correlated with observed

changes in the orientation of cells within the monolayer. We

cannot yet explain the visible gaps in IE2-EGFP expression in

the infections. The most likely explanation is that IE2-EGFP

expression peaked prior to 17 days post-infection, waned

after that, and was no longer visible at the times of

fluorescence imaging. Another possibility is that the loss of

IE2-EGFP expression in the gap areas may be due to muta-

tions or deletions in the viral genome that allowed mutants to

be locally enriched during spread. There is precedent for

such spatially resolved mutation-selection processes in

spreading infections (Lee and Yin, 1996a; Yin, 1993).

Alternatively, the expression of IE2-EGFP in cells could be

influenced by a bi-stable gene expression network (Gardner

et al., 2000; Lai et al., 2004), whose state is mediated by

coupling to specific viral or cellular factors. Subtle interac-

tions between each cell and the propagating virus might favor

one IE2-EGFP expression pattern over the other. Finally, we

cannot yet rule out the possibility that the expression level of

IE2-EGFP in these gap areas was simply below our detection

limit.

In summary, we have applied focal infections to visualize

and quantify HCMV infection spread and assessed how the

rate of spread can be affected by different tissue-culture

conditions. Our results suggest that cell shape, cell spread,

and cell orientation may individually or collectively

influence spread of an infection. Improved understanding

of how HCMV infection spread and gene expression is

mediated by such factors will enrich our fundamental

understanding of HCMV-host interactions and enable the

development of new antiviral strategies.

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spatial patterns of protein expression in focal infections of human cytomegalovirus

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