functional and structural defects in hiv type 1 nef genes derived from...
TRANSCRIPT
AIDS RESEARCH AND HUMAN RETROVIRUSESVolume 16, Number 17, 2000, pp. 1855–1868Mary Ann Liebert, Inc.
Functional and Structural Defects in HIV Type 1 nef GenesDerived from Pediatric Long-Term Survivors
REBECA GEFFIN,1 DIETLINDE WOLF,2 RÜDIGER MÜLLER,2 MARTIN D. HILL,3,4
ELISABETH STELLWAG,2 MARTINA FREITAG,2 GABI SASS,2 GWENDOLYN B. SCOTT,1
and ANDREAS S. BAUR2
ABSTRACT
DNA sequences and three distinct in vitro functions of Nef were evaluated in a group of seven perinatally infected children. nef gene sequences obtained before and after virus culture showed that one of the five non-/slow progressors harbored a virus with large deletions. nef genes from the remaining four children werefull length but contained discrete changes at a higher frequency than the rapid progressors. In functionalstudies, 40 of 44 Nef proteins derived from the whole study group were capable of binding the cellular ser-ine kinase p62, indicating that this function is well conserved among naturally occurring viruses. In contrast,representative Nef proteins derived from the long-term non-/slow progressors were found to be defective orfar less capable of enhancing viral replication and/or viral infectivity in herpesvirus saimiri-transformed hu-man T cells and peripheral blood mononuclear cells. On reversion of highly prevalent point mutations inthe defective proteins, viral replication could be restored to wild-type levels. Our results suggest that nefgenes derived from pediatric long-term nonprogressors have gross deletions in isolated cases but a higherprevalence of discrete changes that may impair Nef function in primary T cell assays, but not all functionsreported for Nef.
1855
INTRODUCTION
LONG-TERM NONPROGRESSORS (LTNPs) with HIV-1 infectioncarry the secret of a successful status quo with the virus,
which allows them to survive disease free for extended periodsof time. Studies have shown that some LTNPs carry viruses withgross deletions in their nef genes.1–4 These studies comple-mented and supported earlier findings indicating that rhesusmacaques infected with nef-deleted simian immunodeficiencyvirus (SIV) do not develop high viral loads or progress to AIDS.5
To understand the role played by Nef in the pathogenesis ofHIV/SIV, extensive efforts have been undertaken to analyze themolecular functions of Nef in vitro. At present, at least threeNef-associated activities have been described: (1) downregula-tion of CD4 and MHC class I,6–9 (2) enhancement of infectiv-ity and replication,10–12 and (3) modulation/activation of T cellsignaling pathways.13–15 For reviews see Trono16 and Harris.17
However, a clear correlation between viral pathogenesis in vivoand the reported functions of Nef in vitro has not been estab-lished. Sawai and collaborators have described a link betweenthe binding of Nef to a serine kinase (p62 or NAK for Nef-associated kinase) and pathogenesis in the SIV model18; how-ever, these results have been disputed by others.19
The number of LTNP individuals exceeds by far the num-ber of individuals infected with nef-deleted HIV strains, sug-gesting that host factors or defects in other viral genes may con-tribute significantly to their long-term survival.20–25 On theother hand, Hanna et al.,26 using a transgenic mouse model,have provided evidence that in this system, only the nef genecontains a major determinant for pathogenicity. In view of theimportant role of Nef in disease progression, one may specu-late that nef genes in LTNPs, which do not contain extendeddeletions, may bear discrete changes/mutations rendering theprotein dysfunctional. The latter situation may have clinical
1Department of Pediatrics, University of Miami School of Medicine, Miami, Florida 33136.2Institut für Klinische and Molekulare Virologie, Universität Erlangen/Nürnberg, 91054 Erlangen, Germany.3Nova Southeastern University, Ft. Lauderdale, Florida 33328.4Present address: Department of Pharmacology and Toxicology, Ponce School of Medicine, Ponce, Puerto Rico 00732.
consequences similar to those resulting from a deleted nef gene,at least until the changes revert to wild type-like sequences.
In this study, we have investigated the occurrence of nef dele-tions and Nef dysfunction at the protein level in HIV-1-infectedchildren with different disease courses. We have first deter-mined the sequences of Nef proteins derived from five perina-tally infected LTNP/slow progressors (SPs) and two rapid pro-gressors (RPs). Second, we have analyzed the function ofrepresentative proteins by three different functional assays, in-cluding a replication assay in herpesvirus saimiri-transformedT cells. Our study found gross deletions in nef in a single caseof pediatric LTNP, which supports similar findings inadults.1,2,4 In addition, we found that nef genes sequenced frompediatric LNTPs/SPs may have discrete changes and mutationsat a higher frequency than in RPs. In representative nef genes,which were investigated in more detail, these changes renderedNef proteins dysfunctional in viral replication and infectivity.Thus, functionally impaired Nef proteins appear to be moreprevalent in pediatric LTNPs than anticipated from the sequenceexamination, possibly reflecting their clinical status.
MATERIALS AND METHODS
Study population
Seven children with perinatal infection were enrolled in thisstudy from a cohort of infected children monitored at the Uni-versity of Miami/Jackson Memorial Hospital (Miami, FL). Ofthe seven children, four were long-term nonprogressors (iden-tified as LTNP 001, 002, 003, and 011), one was a slow pro-gressor (identified as SP 007), and two were rapid progressors(RP 004 and 006) at entry into the study. The LTNPs were olderthan 8 years of age, had CD41 numbers above 900/mm3, wereasymptomatic or minimally symptomatic (classified in stagesN1 or A1 according to the Centers for Disease Control [CDC]classification for children),27 and had not received antiretrovi-ral therapy. The SP was also older than 8 years of age, with aCD41 lymphocyte count above 650/mm3, but was moderatelysymptomatic and had received zidovudine (ZDV) therapy. TheRPs were younger than 2 years of age, symptomatic, and hadCD41 lymphocyte numbers below the fifth percentile for age.28
Informed consent was obtained from all study participantsand/or their guardians.
Collection and processing of blood specimens
Blood was collected in acid citrate tubes and processedwithin 6 hr. Peripheral blood mononuclear cells (PBMCs) wereseparated using lymphocyte separation medium (OrganonTeknika, Westchester, PA). After separation, an aliquot of4–5 3 106 cells was saved for polymerase chain reaction (PCR)of genomic nef sequences, and one aliquot of at least 5 3 106
cells was mixed with an equal number of lymphocytes from anuninfected donor for HIV-1 lymphocyte culture. To monitorvirus production in the culture, a supernatant sample was re-trieved every 3–4 days to test for virus production, using a p24antigen capture enzyme-linked immunosorbent assay (ELISA)(Beckman Coulter, Miami, FL). The cultures were kept for aperiod between 16 and 21 days, after which time the cells werefrozen in aliquots for PCR amplification.
DNA separation and PCR amplification of nefsequences
DNA was extracted from purified PBMCs by standard meth-ods. nef genes were amplified from genomic DNA by nestedPCR, using the following primers: 59-GCAGTAGCTGAG-GGGACAGATAGG-3 9 and 59-CTGGTCTAACCAGAGA-GACCCAGTAC-39, corresponding to nucleotides 8675–8698and 9549–9524 of NL43 (outer primers). nef sequences weresubsequently amplified with the following nested primers: 59-GCCACATACCTAGAAGAATAA-G ACAGG-39 and 59-CCACGCCTCCCTGGAAAGTC CC-39, corresponding to nu-cleotides 8736–8762 and 9460–9439, respectively. For the firstround of amplification, 2 pmol of each primer was added to astandard reaction containing approximately 2 mg of patientDNA. Amplification was performed first by denaturation at94°C for 5 min followed by 10 cycles of 94°C (1 min), 45°C(1 min), and 72°C (1 min). Subsequently, 20 pmol of eachprimer was added and amplified under the same conditions for30 additional cycles. The second round of amplification wasperformed with the same PCR parameters (30 cycles), using a5% volume of the first reaction.
Cloning and sequencing of nef genes
PCR products from the second amplification were directlycloned into the pCR3 vector (InVitrogen, San Diego, CA). Be-tween four and nine colonies were selected for sequencing ofthe nef gene, using the T7 sequencing kit (Pharmacia Biotech,Piscataway, NJ), or an automated DNA sequencer (Perkin-Elmer/Applied Biosystems [Foster City, CA], model 373Strech). CD8-Nef constructs containing patient nef genes weregenerated by a two-step PCR procedure and cloned into thepRcCMV expression vector as described previously.29 To gen-erate heterologous viruses containing patient nef genes, an MluIrestriction site was introduced between env and nef open read-ing frames of the NL4-3 proviral clone by PCR mutagenesis.In addition, a ClaI site was introduced at the 39 end of the nefopen reading frame (ORF) by changing a single nucleotide atposition 9409 from ATC GAG to ATC GAT. The patient nefgenes were amplified by PCR and cloned directly into the provi-ral clone, using the MluI/ClaI restriction sites. All constructsand mutations were verified by sequencing.
Generation of infectious viral supernatants
Infectious molecular clones were transfected into 293T cellsby LipofectAMINE according to the manufacturer instructions(GIBCO-BRL, Gaithersburg, MD). Forty-eight hours aftertransfection the supernatants were harvested, clarified at 400 3g for 10 min, and filtered through a 0.2-mm pore size filter. Su-pernatants were subsequently assayed for reverse transcriptase(RT) activity and p24 antigen contents, using a p24 antigen cap-ture ELISA (Beckman Coulter). For infection, viral super-natants were then normalized for p24. In addition, aliquots ofthe virus stocks were stored in liquid nitrogen for repeated useand confirmation of results.
Infection of quiescent and prestimulated PBMCs
Two million freshly isolated, unstimulated PBMCs were in-fected with various virus concentration ranging from 1.25 to 20
GEFFIN ET AL.1856
ng. The infections were carried out in 2-ml volumes in 24-wellplates (Costar, Cambridge, MA). Infection was allowed to oc-cur overnight, after which the virus was removed by three sub-sequent washes. Forty-eight hours after the wash step, the cellswere stimulated with anti-CD3 antibodies (20 ng/ml; BectonDickinson, San Jose, CA). Aliquots of 1.0 ml were obtainedevery 2–4 days to monitor virus production. Infectivity assaysusing stimulated PBMCs were performed with the same con-centration of virus stocks and the same number of cells, exceptthat the PBMCs had been stimulated for 24 hr prior to the in-fection with anti-CD3 antibodies (20 ng/ml). All experimentswere repeated several times with PBMCs from different donors.
Infection of herpesvirus saimiri-transformed human T cells
Eighteen human T cell clones transformed with herpesvirussaimiri were analyzed for their ability to discriminate betweenthe replication of viruses containing or lacking the nef gene.Two cell clones, U4 and N, were selected. In the absence of in-terleukin 2 (IL-2), only HIV strains with a functional nef genereplicated in U4 cells, whereas in the N cell clone, HIV chimerascontaining or lacking nef replicated at comparable levels. Thislatter clone was used as a control to ensure the overall capa-bility of the different recombinant HIV strains to replicate in vitro. The cells were grown in RPMI 1640 supplementedwith 20% fetal bovine serum, and IL-2 (5 U/ml). For a givenexperiment, only viral stocks generated at the same time by thesame transfection were used. For transfections, only DNA gen-erated by the same method (Qiagen [Valencia, CA] columns)and at the same time was used. Viral stocks were used onlywhen the p24 or RT values were comparable within all sam-ples generated at a given transfection (a 30–50% deviation wasaccepted). For infections, the IL-2 was washed out of themedium 3 days in advance. NL43 viruses containing heterolo-gous nef genes from the patient population were normalized forp24 contents as described above. The equivalent of 5 ng of p24was used to infect 3 3 105 cells. Every 2 days, half of themedium was retrieved for RT activity measurements, and re-placed with fresh medium. All assays were repeated severaltimes.
Protein expression assays and antibodies
COS-7 cells were transfected as described above for 293Tcells, with 5–10 mg of the various pRcCMV-CD8-Nef con-structs. Constructs, immunoprecipitations (using anti-CD8 an-tibodies), and in vitro kinase assays were performed as de-scribed previously.30 Anti-Nef Western blots were performedon cytoplasmic lysates from 293T cells transfected with provi-ral constructs. The sheep anti-Nef serum was kindly providedby M. Harris (University of Leeds, Leeds, UK).
RESULTS
Characteristics of the study population
The clinical characteristics and viral and immunological pa-rameters of the study participants at enrollment and at the endof the study are summarized in Table 1. Six children in the co-
hort were black, five of them belonging to the LTNP or SPgroups and one to the RP group. The second RP was Hispanic.Five children were females. At entry, all the LTNPs had viralloads below 7000 RNA copies/ml, and two children, (LTNP002 and LTNP 003) had viral loads that were below 400 RNAcopies/ml. The slow progressor, child SP 007, had CD41 lym-phocyte numbers of 659/mm3, but had two episodes of herpeszoster and herpes simplex virus (HSV). Child RP 004, a rapidprogressor, had an AIDS-defining illness and died at the age of18.5 months, while RP 006 has had multiple symptoms and anearly and rapid decline of CD41 lymphocyte numbers. The tworapid progressors (RP 004 and RP 006) had significantly highervirus loads (.700,000 RNA copies/ml) and their CD41 lym-phocyte numbers were below the fifth percentile for age.28 Bythe end of the study (January 1999), child LTNP 001 had be-come symptomatic, had experienced a decline in CD41 lym-phocyte numbers, and was treated with antiretroviral drugs. Asa consequence, viral loads declined to undetectable levels. Chil-dren LTNP 002 and 003 had no significant changes in theirclinical characteristics or viral loads. Their CD41 lymphocytenumbers had diminished, but only slightly.
Analysis of nef sequences
Nested PCR amplification of nef sequences from the sevenstudy participants was performed from genomic DNA obtainedfrom uncultured and cultured PBMCs. nef sequences were ob-tained from cultured PBMCs for three reasons: first, to inves-tigate whether specific sequences/amino acid changes areprevalent in uncultured and cultured cells and thus are repre-sentative for nef genes found in the particular individual; sec-ond, to determine whether specific sequences/amino acids areselected through culture; third, to provide validation for the au-thenticity of the uncultured as well as cultured sequencesthrough phylogenetic analysis.
PCR amplification yielded products of the expected size inall but one child (LTNP 002), whose nef sequences had reducedmolecular weight (data not shown). A total of 69 nucleotide se-quences were obtained from the study population. On se-quencing, 11 of 11 nef genes derived from cultured PBMCsfrom LTNP 002 revealed deletions that encompassed approxi-mately 69% of the nucleotides (see consensus sequence in Fig.1). Attempts to obtain sequences from uncultured PBMCs wereunsuccessful at the time. The deletions found in LTNP 002started at the thymidine in the ATG initiation codon for Nefand continued up to amino acid 84 (methionine in some clones),or amino acid 90 in the majority of the other clones. Eventhough some clones encode a methionine at the beginning ofthe reading frame, it is followed by a termination codon. In allclones sequenced the polypurine tract and the U3 terminal se-quences overlapping the long terminal repeat (LTR) and nef,deemed necessary for viral replication, were present. Despitethe deletions, the virus could be cultured and isolated in vitro;however, it grew with low efficiency (data not shown). At thepresent time, it is unknown whether a truncated Nef protein isbeing translated from these sequences. Studies by Greenway etal. have shown that ELISAs using defined Nef peptides wereable to discern sera from individuals who were infected withnef-deleted strains from sera of individuals who were infectedwith viruses containing intact nef.31
DEFECTIVE Nef PROTEINS IN PEDIATRIC LTNPs 1857
TA
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Age
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Age
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fica
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NP
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16,
988
No
18.9
yr
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Neg
ativ
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P 0
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yr
925
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5 yr
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9A
1,
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No
13.2
5 yr
975
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442
No
LT
NP
011
9.5
yr99
7N
12,
982
No
11.3
yr
916
A1
816
No
SP
007
8.5
yr65
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114
,211
Yes
12 y
r16
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330
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RP
004
7 m
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31,
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612
No
Exp
ired
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RP
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371
3,08
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224
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1858
The remaining children in the study harbored viruses withfull-length nef sequences. The consensus sequences for eachchild are shown in Fig. 1. To determine whether they containedsignificant changes on the amino acid level, they were com-pared with the consensus sequence published by Shugars et al.32
and sequences published in the Los Alamos database.33 Achange was noted as such when an amino acid in a given po-sition was different from the consensus defined by Shugars etal.,32 in half or more of the clones from one LTNP/SP childand was not found in the control group (i.e., the RP sequences),or vice versa. This analysis indicated that sequences obtainedfrom LTNPs/SPs had about three times more changes than thoseobtained from the RPs (on average six vs. two per child). Thesechanges (Fig. 2) included small deletions, changes in net chargeand/or hydrophobic properties of the amino acids that clusteredin particular between amino acids 45–60 and 155–181. Most ofthese changes were found in a minority of nef genes listed inthe Los Alamos database33 belonging to clade B and other HIV-
1 clades. Some of the changes, however, were rare (see belowand data not shown).
In children LTNP 001 and LTNP 011, a deletion of two aminoacids at positions 53 and 54 was found. This small deletion wasalso described for an LTNP from a different study.34 Substitu-tion of asparagine for glycine at amino acid position 3, found infive of six Nef proteins from child SP 007, is found only in afew sequences submitted to the Los Alamos database, but wasalso found in the LTNP patient LM from the study by Huang etal.35 The changes described above for LTNP 001 and 011 andfor SP 007 were also found in all their cultured sequences (Fig.7 and data not presented) and must therefore be considered asrepresentative changes. Thus, these amino acid changes were in-vestigated in more detail in functional assays (see below). Thedomains involved in the binding of Nef to the SH3 domain oftyrosine kinases are mostly conserved. However, R76 in the(PxxP)4 motif, which forms a bond with Tyr-137 of SH3,36 ischanged to T in five of nine clones from child LTNP 003. This
DEFECTIVE Nef PROTEINS IN PEDIATRIC LTNPs 1859
FIG. 1. Amino acid sequence alignment of consensus Nef proteins from the study population.Sequences were aligned by hand. Nucleotide gaps (.) were assigned after amino acid conversionto maintain translational integrity whenever possible. Sequences were aligned according to SF2Nef. Known domains of Nef are depicted on top of the corresponding sequences. (?) No con-sensus sequence could be determined; (*) termination codon; (X) one or two nucleotides present,no amino acid could be inferred. Each sequence has been identified as follows: first the child’sidentification number, followed by U (from uncultured cells) or C (from cultured cells), and bycon (consensus).
introduces a hydrophobic amino acid in place of a positivelycharged one. Interestingly, all five T variants formed a separatesequence cluster (see Fig. 3) and one representative allele of thiscluster was therefore investigated in more detail (see below).
Intrapatient and interpatient sequence variation andphylogenetic relationship of nef sequences
The sequence variability within the study population was in-vestigated by determining the intra- and interpatient nucleotidedistances (Table 2). Mean intrapatient nucleotide distancesranged from 1.74% (RP 004) to 4.63% (LTNP 003), with a ten-dency to be higher in LTNPs/SP than in RPs. These distancesare similar to those already published by Huang et al. (between0.04 and 3.54%) and by Mariani et al. (0–4.5%).35,37 Mean in-terpatient nucleotide distances were much higher, ranging be-tween 6.84 and 19.3%. Mariani et al.37 also found higher in-terpatient distances of between 10 and 16%. In general, theintrapatient variability in sequences derived from cultured cellswas less than in uncultured cells. For example, cultured se-quences from LTNP 003 showed a mean variability of only0.98% while uncultured sequences varied by 5.61%. Eventhough the differences in mean distances between cultured anduncultured cells were not different between the LTNP/SP andthe RP group, the deviations from the mean were different (datanot shown). Together with the phylogenetic analysis (see be-low) and functional data (see Figs. 5A and 6A), these resultspointed to the selection of Nef forms on culture.
Phylogenetic trees of nucleotide sequences were constructedby the neighbor-joining method after calculating maximumlikelihood distances. This analysis indicated that each child hada cluster of unique sequences that could be differentiated fromthose of the other children and the laboratory strains SF2 andNL43 (Fig. 3). nef clones derived from uncultured and culturedcells clustered together with the exception of LTNP 003, whohad a separate cluster of uncultured cells. In addition, SP 007harbored two distinguishable forms, separating cultured and un-cultured sequences, both of which cluster together in the tree.In summary, these results indicated that the sequences obtainedwere free from contamination and pointed to a selection of sub-variants in virus cultures of LTNP 003 and SP 007.
Nef functions in vitro: Association with the cellularserine kinase p62 (NAK)
The ability of Nef proteins from our study population to bindthe cellular serine kinase p62/NAK was investigated. For thispurpose, CD8-Nef chimeric constructs were generated, trans-fected into 293T cells, and assayed for p62/NAK binding by invitro kinase assays. This method has been successfully used inthe past30 to consistently detect the association of p62 with Nef,because it provides the advantage of easy precipitation of thecomplex with anti-CD8 antibodies, and avoids using anti-Nefantibodies, which vary in their ability to recognize primary Nefproteins. Using this system, a total of 44 Nef proteins weretested. Twenty-three proteins were derived from three LTNPs
GEFFIN ET AL.1860
FIG. 2. Amino acid changes in Nef proteins derived from the study population. Amino acid sequences in Nef proteins derivedfrom uncultured cells were compared with the consensus amino acid sequence obtained by Shugars et al.32 Nef sequences weredivided into two groups: LTNPs plus SP, and RP. The number of clones analyzed for each patient is enclosed in parentheses,e.g., (6) for LTNP 001. All amino acids that were prevalent in half or more of the clones sequenced for one child, but differentin the consensus sequence and in Nef proteins of the control group (e.g., RPs), are depicted together with the number of clonesin which the amino acid is present. Amino acid changes that were investigated in functional assays (N3 in SP 007; deletion 53/54in LTNP 001; T76 in LTNP 003) are shown in boldface.
(from child 001, 5 proteins were tested from uncultured cellsand 4 proteins from cultured cells; from child 003, 4 proteinsfrom uncultured cells and 6 proteins from cultured cells; andfrom child 011, 4 proteins from uncultured cells). Five proteinswere tested from uncultured cells of SP 007 and 16 proteinsfrom the two RPs (from child 004, 4 proteins from unculturedand 5 from cultured cells, and from child 006, 4 Nef proteinswere tested from uncultured cells and 3 proteins from culturedcells). Only 9% of the Nef proteins (4 of the 44; 3 from LTNP001 and 1 from LTNP 003) did not associate with p62 (Fig. 4A,lanes 3 and 7, representing 001.U.09 and 003.C.05, respec-tively), even though these proteins were expressed at compara-ble levels in transfected cells (Fig. 4B). Some of the Nef pro-teins bound p62 strongly, such as 004.U.16 (Fig. 4A, lane 8),and some others less strongly (exemplified in Fig. 4A, lanes4–6). Quantification of differences in p62 binding was not per-formed, and so we have considered binding to p62 to be posi-tive if a band was observed in the gel, even if it was less intensethan that of the reference protein, SF2 Nef. The sequences ofNef proteins not binding p62 are shown in Fig. 4C. A compar-ison of these sequences and those of Nef proteins binding NAKdid not reveal apparent changes that could account for the dif-ference in phenotype (data not shown). In summary, these re-sults indicated that although binding of the serine kinase is aconserved function of Nef, it did not correlate with clinical pro-gression.
Nef functions in vitro: Enhancement of viral replication
To test whether Nef proteins derived from our study popu-lation differed in their ability to enhance viral replication invitro, virus chimeras were constructed containing the backboneof NL43, and a representative nef gene derived from PBMCsof patients LTNP 001 and 003, SP 007, and RPs 004 and 006.An NL43 proviral construct containing SF2 nef or a nef dele-tion (nef-negative) were used as controls. Virus stocks of theseconstructs were generated in 293T cells and tested for their abil-ity to replicate in herpesvirus saimiri-transformed primary hu-man T cells (see Materials and Methods for details). In a sim-
DEFECTIVE Nef PROTEINS IN PEDIATRIC LTNPs 1861
FIG. 3. Phylogenetic relationship of nef genes derived fromLTNPs, SP, and RPs. Analysis of nucleotide sequences wasperformed with the program PHYLIP, version 3.5c (Felsen-stein J: PHYLIP, version 3.5c. Copyright 1993, Felsenstein andthe University of Washington). Sequences have been labeledaccording to patient number, and according to the source: u,obtained from uncultured PBMCs; c, obtained from culturedPBMCs.
TABLE 2. INTRA- AND INTERPATIENT NUCLEOTIDE DISTANCESa
Intrapatient distance
Mean, Mean, Interpatient distanceChild Mean, all clones uncultured cultured (range)
LTNP 001 3.18 3.22 3.04 6.84–15.16LTNP 002 2.93 — 2.93 15.09–19.30LTNP 003 4.63 5.61 0.98 6.84–15.09LTNP 011 2.49 2.49 — 7.75–16.46
SP 007 3.91 4.17 3.17 11.03–19.30
RP 004 1.74 2.14 1.04 10.05–16.77
RP 006 2.56 2.31 2.77 8.96–14.34
aIntrapatient nucleotide distances were calculated with sequences obtained both from cultured and uncultured cells, separately.The mean of all clones has been obtained using both. For interpatient distances, clones from both cultured and uncultured cells,if available, were used.
GEFFIN ET AL.1862
C
FIG. 4. Association of Nef with p62/NAK is conserved in most Nef proteins derived from pediatric patients. (A) In vitro ki-nase assays after transient transfection of CD8-Nef constructs into COS cells (see Materials and Methods for details). The arrowpoints to the migration of p62. Two Nef proteins (001.U.09, lane 3; and 003.C.05, lane 7) did not bind p62 despite being wellexpressed in COS cells [see (B)]. Nef protein 004.U.16 represents a strong binder (lane 8), and Nef proteins 001.U.01, 001.U.15,and 003.U.24 (lanes 4–6, respectively) bound p62 moderately. (B) 35S metabolic labeling of COS cells transiently transfectedwith Nef proteins not binding p62. Lanes 3–6 represent the Nef proteins 001.C.09, 001.C.22, 001.C.24, and 003.C.05 respec-tively; see (C) for sequences. Transfection with SF2 nef (lane 2) was used as a control. The arrow points to the migration of theCN chimeras. (C) Amino acid sequence alignment of Nef proteins not binding to p62/NAK. Sequence alignments were performedas described for Fig. 1. Consensus amino acid sequences represent the entire cohort and are listed at the top. They were obtainedby determining amino acids present in at least 50% of the clones after weighting for multiple sequences from the same patient.
FIG. 5. Infection of herpesvirus saimiri-transformed human T cells with NL43 containing nef genes derived from LTNPs, SP,and RPs. Symbols indicate the patients from whom the genes were derived (e.g., 003), followed by the source (U, unculturedPBMCs; C, cultured PBMCs; and clone number). (A) Infection of control (N) cells allowing HIV replication independent of thepresence of the nef gene. (B) Infection of U4 cells, where HIV replication requires a functional nef gene. (C) Anti-Nef Westernblot of 293T cell lysates transfected with chimeric viruses (provirus) or a SF2 expression construct, showing that the function-ally defective Nef proteins were expressed in vitro.
ilar cell system (transformed monkey PBMCs), SIV replicationwas shown to be Nef dependent under certain circumstances.15
To establish a comparable system for HIV, we analyzed 18transformed human cell clones for Nef-dependent HIV repli-
cation (data not shown). In one cell clone (U4), a striking dif-ference in replication between nef-positive and nef-negativeviruses was observed (Fig. 5B, upper panel) and was subse-quently used for this study. A second cell clone (N) was iden-
DEFECTIVE Nef PROTEINS IN PEDIATRIC LTNPs 1863
tified in which no difference in replication was observed be-tween viruses containing and lacking Nef. This latter cell clonewas used as control to verify comparable replication of the different constructs used (Fig. 5A, upper panel). In addition tobeing able to discriminate between Nef-deleted and Nef-con-taining viruses, clone U4 was a slower growing clone. The re-lationship between these two phenotypes is unknown.
Using this cell system, NL43 with nef genes derived fromLTNPs 001 and 003 (001.C.24 and 003.U.15) and SP 007(007.U.17) replicated poorly in U4 cells, whereas no differencewas observed in the control N cells (Fig. 5). Conversely, virusescontaining nef genes from RP 004 (clone 004.C.04) and RP 006(006.U.06) had growth characteristics similar to those of theSF2 positive control. Next, we asked whether some of the se-quence differences observed in LTNP 001 and 003 and SP 007(Fig. 2 and also described above) had any effect on viral repli-cation. In LTNP 001, the two-amino acid deletion at aminoacids 53–54 was filled with the wild-type amino acids TA. InLTNP 003 the threonine in the PxxP motif was changed to argi-nine, and in SP 007 the asparagine at position 3 was replacedwith the conserved amino acid glycine. In all cases, the rever-sions improved the replication kinetics of the recombinantviruses (Fig. 5). In Nef proteins from LTNP 003 and SP 007,the difference was particularly striking, changing from slowreplication to SF2-like replication. In line with this result, wenoted that all Nef proteins sequenced from cultured PBMCsfrom LTNP 003 contained an arginine in the (PxxP)4 domain,while the sequences obtained from uncultured cells were mixed,containing T and R. Assuming that this selection in culturewould render the Nef protein fully competent, we tested one ofthese alleles (003.C.05) which contains an R at position 76, andindeed the replication competence was comparable to the lev-els seen in the RP and SF2 controls. Terwilliger and co-work-ers38 found that the LAI nef allele, which is identical to theNL43 allele, failed to replicate in primary PBMCs. Interest-ingly, NL43, like LTNP 003 Nef, has a threonine at position76.
To exclude that the lack of replication in 003.U.15 and007.U.17 was due to poor expression of Nef proteins in vitro,Western blots of cells infected with the chimeric viruses wereperformed. The blots, shown in Fig. 5C, indicate that all de-fective Nef proteins were being produced in the infected cells,
GEFFIN ET AL.1864
FIG. 5. Continued.
C
FIG. 6. Infection of resting and prestimulated PBMCs withNL43 containing nef genes derived from LTNPs, SP, and RPs(same as in Fig. 5). (A) Infection of unstimulated PBMCs withvirus containing 5 ng of p24. (B) Infection of stimulated PBMCswith virus containing 2.5 ng of p24. For details see Materialsand Methods.
and that lack of function was not due to lack of Nef expression.Although it may appear that the Nef proteins from LTNP 003and SP 007 are expressed in lower amounts than SF2 Nef, thisdifference may be due to a lower binding of the antibodies tothe primary Nef proteins. In addition, Nef 007G appears to bepartly degraded. (see lower band in Fig. 5C). This apparentdegradation is not sufficient to affect function, since Nef 007Gwas significantly better at enhancing virus replication than the007N counterpart. As is shown below, 001.C.24 had infectiv-ity enhancement characteristics similar to those of wild type,and was therefore considered to be expressed in vitro.
Nef functions in vitro: Enhancement of viral infectivity
To further investigate possible defects in nef alleles from ourstudy population, Nef-mediated enhancement of infectivity wasinvestigated. The proviral constructs described above weretested for their ability to infect unstimulated PBMCs (see Ma-terials and Methods for details). Nef proteins derived from un-cultured PBMCs from LTNP 003 and SP 007 rendered com-parable growth characteristics to the Nef-negative construct,thus being defective in enhancing viral infectivity (Fig. 6A).Conversely, Nef from LTNP 001 and RPs 004 and 006 en-hanced infectivity in a manner somewhat similar to the SF2positive control. Next, we asked whether the sequence differ-ences observed in LTNPs 001 and 003 and SP 007 (Figs. 2 and5) had any effect on viral infectivity. However, none of these“reversions” significantly affected the infectivity enhancementproperties of the viruses tested (Fig. 6A). Since the revertantsshowed no effect, LTNP 003 and SP 007 Nefs derived fromcultured PBMCs were tested. The cultured nef allele of SP 007
had infectivity enhancement properties similar to those of SF2,whereas the LTNP 003 allele was still defective. In the partic-ular case of SP 007, the amino acid differences are concentratedin the amino terminus of Nef (Fig. 7). Notably, a threonine ischanged to an isoleucine at position 49, an aspargine to a ser-ine at position 56, and a cysteine to a serine at position 60 inthe inactive Nef.
As control for these experiments, similar infections were per-formed using prestimulated PBMCs. In these cells, the differ-ences between nef-containing and nef-deleted viruses are lessdramatic. Although chimeric viruses replicated well, those con-taining the functionally defective Nef proteins replicated at aslower rate that was similar to that of the nef-deleted counter-part (Fig. 6B). These experiments again indicated that the Nefproteins from LTNP 003 and SP 007 were defective in en-hancing viral infectivity.
This study, as well as those of Luo et al.,39 have concludedthat association of Nef with p62/NAK is independent of itsinfectivity enhancement properties. Nef proteins 003.U.15 and007.U.17 associate with p62/NAK but are unable to enhanceinfectivity and replication. Conversely, Nef protein 001.C.24does not associate with p62 but effectively enhances viral in-fectivity.
DISCUSSION
This study found structurally and functionally defective nefgenes/proteins in all five pediatric patients studied (four LTNPsand one SP). Our results differ from those obtained in previousstudies of Nef proteins from LTNPs,3,40 which raises a number
DEFECTIVE Nef PROTEINS IN PEDIATRIC LTNPs 1865
FIG. 7. Amino acid sequence alignment of Nef proteins tested for en-hancement of infectivity/replication (Figs. 5 and 6). Alignments were per-formed as described for Figs. 1 and 4. Sequences are labeled according to thepatient, source (cultured, C; or uncultured, U), and clone number.
of questions: (1) Are the different results due to a different se-lection of the study population (pediatric versus adult LTNPs)?(2) Are the described changes and functional defects due toPCR errors and thus represent artifacts? (3) Are the observedfunctional data truly representative? And finally, (4) are the dif-ferent results, in particular those obtained using the functionalassays, due to the different assay systems used?
In newborns, viral infections are severe and usually progressrapidly because their immune systems are still immature andhave an overall higher level of immunological activation. InHIV infection, most of the perinatally infected children nottreated with antiretroviral drugs develop HIV-associated symp-toms within the first 2 years of life, and pediatric LTNPs olderthan 10 years are rare.41–44 Immaturity of the immune systemat the time of perinatal HIV exposure may more easily allowthe establishment of infection by defective HIV strains, whilein adults, defective stains may be selected out by a robust im-mune reaction. In line with these hypotheses, we found that oneof the five children studied had a grossly deleted nef gene andthe other four children had a 3-fold higher number of signifi-cant sequence changes as compared with the RP population. In-terestingly, LTNPs 001 and 011 had the same two-amino aciddeletion at positions 53–54 in all of their clones. In summary,it is possible that the different results obtained in our study areat least in part due to the particular population we selected.
PCR amplification, in particular nested PCR, is prone to er-rors and contamination. Thus, validation of the sequences ob-tained is of paramount importance. The intra- as well as inter-patient sequence variability and the phylogenetic data analysiswere well within the expected range and distance.35,37 In addi-tion, the cultured sequences clustered in all but one case withthe uncultured sequences. These findings indicate that a cont-amination of the sequences is unlikely. The observed aminoacid changes clustered more predominantly in areas (aminoacids 45–60; amino acids 155–181) where others also have ob-served a higher variability.3,35 More importantly, these changesare not completely unique. All of them have been observed be-fore in sequences compiled by the Los Alamos database (datanot shown), albeit some in only a few sequences and/or in adifferent HIV subtype. Taken together, these facts gave littlereason to believe that the observed amino acid changes werethe result of PCR errors. On the contrary, one could argue thatthe frequency and location of the observed amino acid changesare all but unusual in view of other LTNP-derived sequences.However, it is difficult to assess the occurrence of a combina-tion of different changes, which together, but not by themselves,may have significant effects on Nef function. The latter fact isa clear limitation of the sequence analysis approach to detectpotential Nef defects.
Genetic variability of HIV in a given host is vast and PCRamplification may select only certain subvariants at a given timepoint. In addition, when viral loads are as different as betweenthe LTNP and the RP children in this study, the relative fre-quencies of variants cannot be estimated accurately. This hasseveral important implications: (1) We cannot rule out the pos-sibility that defective Nef variants exist in the RP population,but cannot be identified because they constitute a minority in apopulation that is mostly functional; (2) we cannot exclude theexistence in the LTNP population of Nef proteins not bearingthe amino acid changes that abolish function. This is a clear
technical limitation in assessing the function of Nef proteinsderived from an infected individual, and additional informationcould be obtained by sequencing nef genes from later timepoints. Thus, in view of these limitations, we focused on rep-resentative sequences/amino acid changes prevalent in both un-cultured and cultured PBMCs. In LTNP 001 and SP 007, theamino acid changes investigated in detail were conserved innearly all sequences. Moreover, the two-amino acid deletion inLTNP 001 was also found in LTNP 011 and in an adult LTNPfrom a different study.34 Thus, the amino acid mutations stud-ied were considered as representative in viruses from these in-dividuals. In LTNP 003, the T 76 change was prevalent in allsequences of a distinct subcluster but not in all uncultured se-quences. Moreover, T 76 did not appear in any of the culturedsequences. Thus T 76 was at least representative of a major sub-cluster.
Studies of Nef function in LTNPs have focused on the CD4downregulation capacity of representative alleles3,37 and in onestudy on the ability of Nef proteins to increase viral infectiv-ity.40 In this study, we have assessed the ability of Nef proteinsto bind p62/NAK and to increase viral replication in a primaryT cell system. In addition, like Huang and co-workers, we havealso investigated Nef-mediated increase of viral infectivity inprimary PBMCs. We believe that the replication assay in pri-mary cells reveals a truly important function of Nef, which asshown here may not correlate with the other known functions,such as NAK binding or increasing viral infectivity. Even moresignificantly, the replication data correlated with certain aminoacid mutations. In summary, our study used a more sophisti-cated approach to assess Nef function and it may be for thatreason, in addition to reasons discussed above, that our studycame to different conclusions.
Binding of Nef to p62/NAK was conserved in 91% (40 of44) of Nef proteins tested. All four nonbinding alleles were de-rived from LTNPs 001 and 003, perhaps reflecting the greatervariability in sequences of LTNPs. Although the importance ofthis interaction has been disputed, at least in the animal model,19
it appears to be a conserved property of Nef and did not cor-relate with long-term nonprogression. However, since the truefunctional importance of p62/NAK remains obscure, it is diffi-cult to speculate on this result or, for example, on the relativeability of a Nef variant to bind p62/NAK.
To assess Nef-dependent replication of HIV, we used twoherpesvirus saimiri-transformed human T cells clones. Such acellular system was originally described by Alexander and co-workers for SIV replication and suggested that Nef increasedviral growth through the activation of T cells.15 The results re-vealed significant replication defects of the recombinantviruses, which improved on reversion of the described aminoacid changes. Thus, it appears that the Nef-dependent increasein replication is a mutation-sensitive property of Nef. The lat-ter was also observed by others when analyzing different nefalleles and nef mutations in primary cell systems.45,46 Like thesestudies, we suggest that the overall secondary structure of Nefmay be important for the replicative function of Nef. In viewof these data, it is therefore possible that nef alleles describedin other studies as functional3,40 would reveal defects in pri-mary T cell replication assays.
In two LTNPs we also discovered defects in the Nef-medi-ated increase in viral infectivity/replication. Defects in infec-
GEFFIN ET AL.1866
tivity enhancement, however, may be difficult to differentiatefrom defects in replication since both Nef effects may overlapin this particular primary T cell assay. Nevertheless, it appearsthat they are distinct functions, since in contrast to the replica-tion data, Nef proteins from LTNP 001 behaved like the wild-type protein, and none of the reversions could restore the Nefinfectivity function. In this assay we used anti-CD3 instead ofphytohemagglutinin (PHA)/IL-2 to stimulate the cells 2 daysafter infection. In our hands, PHA/IL-2 stimulation rendered re-sults that were inconsistent and erratic, including results ob-tained after infections with the Nef-negative construct. Haughnet al. reported that IL-2 uncouples the T cell receptor (TCR)from CD3 signaling, leaving inactive Lck at the plasma mem-brane.47 This process could affect Nef function, in particular inprimary cells, and may help explain the differences observedbetween this study and that by Huang and co-workers.40
In summary, our data imply that some but not all Nef func-tions may be defective in nef alleles from pediatric LTNPs. De-spite looking at representative changes characteristic of Nef pro-teins in individual children, our results could be coincidentaland statistically nonsignificant. However, it is possible that, infact, they reflect the functional capacity of viral quasispeciesfound in an individual, and thus are related to their disease pro-gression.
ACKNOWLEDGMENTS
Authors R.G. and D.W. contributed equally to this work.This work was supported by grants PAF 50586-18-PG and50655-21-PG from the Pediatric AIDS Foundation.
The authors are indebted to Ana Garcia and her team of so-cial workers for their dedication and their assistance with thepatients in the study. We thank Celia Hutto for a patient’s spec-imen, and Walter A. Scott for helpful discussions.
SEQUENCE DATA
Nef sequences obtained for this study were submitted to theGenBank. They were given accession numbers AF252897–AF252918 and AF252919–AF252966.
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Address reprint requests to:Andreas S. Baur
Department of DermatologyHartmannstr. 14
91052 Erlangen, Germany
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