scholarly journals In vivo serial passaging of human-simian immunodeficiency virus clones identifies viral characteristics that enhance persistent viral replication

2021 ◽  
Author(s):  
Rajesh Thippeshappa ◽  
Patricia Polacino ◽  
Shaswath S Chandrasekar ◽  
Khanghy Truong ◽  
Anisha Misra ◽  
...  
Keyword(s):  
Viral Replication ◽  
Simian Immunodeficiency Virus ◽  
Peak Plasma ◽  
Viral Fitness ◽  
Replication Capacity ◽  
Viral Loads ◽  
Immunodeficiency Virus ◽  
Replication Fitness ◽  
Hiv 1

We previously reported that a human immunodeficiency virus type 1 with a simian immunodeficiency virus vif substitution (HSIV-vif-NL4-3) could replicate in pigtailed macaques (PTMs), demonstrating that Vif is a species-specific tropism factor of primate lentiviruses. However, infections did not result in high peak viremia or setpoint plasma viral loads, as observed during SIV infection of PTMs. Here, we characterized variants isolated from one of the original infected animals with CD4 depletion after nearly four years of infection to identify determinants of increased replication fitness. In our studies, we found that the HSIV-vif clones did not express the HIV-1 Vpr protein due to interference from the vpx open reading frame in singly spliced vpr mRNA. To examine whether these viral genes contribute to persistent viral replication, we generated infectious HSIV-vif clones expressing either the HIV-1 Vpr or SIV Vpx protein. And then to determine viral fitness determinants of HSIV-vif, we conducted three rounds of serial in vivo passaging in PTMs, starting with an initial inoculum containing a mixture of CXCR4-tropic (Vpr- HSIV-vif-NL4-3 isolated at 196 (C/196) and 200 (C/200) weeks post-infection from a PTM with depressed CD4 counts) and CCR5-tropic HSIV (Vpr+ HSIV-vif derivatives based NL-AD8 and Bru-Yu2 and a Vpx expressing HSIV-vif-Yu2). Interestingly, all infected PTMs showed peak plasma viremia close to or above 105 copies/ml and persistent viral replication for more than 20 weeks. The passage 3 PTM showed peak viral loads greater than 105 viral RNA copies/ml. Infectious molecular clones (IMCs) recovered from the passage 3 PTM (HSIV-P3 IMCs) included mutations required for HIV-1 Vpr expression and those mutations encoded by the CXCR4-tropic HSIV-vifNL4-3 isolates C/196 and C/200. The data indicate that the biological isolates selected during long-term infection acquired HIV-1 Vpr expression to enhance their replication fitness in PTMs. Further passaging of HSIV-P3 IMCs in vivo may generate pathogenic variants with higher replication capacity, which will be a valuable resource as challenge virus in vaccine and cure studies.

scholarly journals In vivo Serial Passaging of Human–Simian Immunodeficiency Virus Clones Identifies Characteristics for Persistent Viral Replication

2021 ◽  
Vol 12 ◽  
Author(s):  
Rajesh Thippeshappa ◽  
Patricia Polacino ◽  
Shaswath S. Chandrasekar ◽  
Khanghy Truong ◽  
Anisha Misra ◽  
...  
Keyword(s):  
Viral Replication ◽  
Simian Immunodeficiency Virus ◽  
Peak Plasma ◽  
Viral Fitness ◽  
Replication Capacity ◽  
Challenge Virus ◽  
Initial Inoculum ◽  
Immunodeficiency Virus ◽  
Hiv 1

We previously reported that a human immunodeficiency virus type 1 with a simian immunodeficiency virus vif substitution (HSIV-vifNL4-3) could replicate in pigtailed macaques (PTMs), demonstrating that Vif is a species-specific tropism factor of primate lentiviruses. However, infections did not result in high-peak viremia or setpoint plasma viral loads, as observed during simian immunodeficiency virus (SIV) infection of PTMs. Here, we characterized variants isolated from one of the original infected animals with CD4 depletion after nearly 4years of infection to identify determinants of increased replication fitness. In our studies, we found that the HSIV-vif clones did not express the HIV-1 Vpr protein due to interference from the vpx open reading frame (ORF) in singly spliced vpr mRNA. To examine whether these viral genes contribute to persistent viral replication, we generated infectious HSIV-vif clones expressing either the HIV-1 Vpr or SIV Vpx protein. And then to determine viral fitness determinants of HSIV-vif, we conducted three rounds of serial in vivo passaging in PTMs, starting with an initial inoculum containing a mixture of CXCR4-tropic [Vpr-HSIV-vifNL4-3 isolated at 196 (C/196) and 200 (C/200) weeks post-infection from a PTM with depressed CD4 counts] and CCR5-tropic HSIV (Vpr+ HSIV-vif derivatives based NL-AD8 and Bru-Yu2 and a Vpx expressing HSIV-vifYu2). Interestingly, all infected PTMs showed peak plasma viremia close to or above 105 copies/ml and persistent viral replication for more than 20weeks. Infectious molecular clones (IMCs) recovered from the passage 3 PTM (HSIV-P3 IMCs) included mutations required for HIV-1 Vpr expression and those mutations encoded by the CXCR4-tropic HSIV-vifNL4-3 isolate C/196. The data indicate that the viruses selected during long-term infection acquired HIV-1 Vpr expression, suggesting the importance of Vpr for in vivo pathogenesis. Further passaging of HSIV-P3 IMCs in vivo may generate pathogenic variants with higher replication capacity, which will be a valuable resource as challenge virus in vaccine and cure studies.

scholarly journals The Human Immunodeficiency Virus Type 1nef Gene Can to a Large Extent Replace Simian Immunodeficiency Virus nef In Vivo

1999 ◽  
Vol 73 (10) ◽  
pp. 8371-8383 ◽  
Author(s):  
Frank Kirchhoff ◽  
Jan Münch ◽  
Silke Carl ◽  
Nicole Stolte ◽  
Kerstin Mätz-Rensing ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Viral Replication ◽  
Simian Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Loads ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Chimeric Viruses

ABSTRACT The nef gene of the pathogenic simian immunodeficiency virus (SIV) 239 clone was replaced with primary human immunodeficiency virus type 1 (HIV-1) nef alleles to investigate whether HIV-1 Nef can substitute for SIV Nef in vivo. Initially, two rhesus macaques were infected with the chimeric viruses (Nef-SHIVs). Most of the nef alleles obtained from both animals predicted intact open reading frames. Furthermore, forms containing upstream nucleotide substitutions that enhanced expression of the inserted gene became predominant. One animal maintained high viral loads and slowly progressed to immunodeficiency. nef long terminal repeat sequences amplified from this animal were used to generate a second generation of Nef-SHIVs. Two macaques, which were subsequently infected with a mixture of cloned chimeric viruses, showed high viral loads and progressed to fatal immunodeficiency. Five macaques received a single molecular clone, named SHIV-40K6. The SHIV-40K6 nef allele was active in CD4 and class I major histocompatibility complex downregulation and enhanced viral infectivity and replication. Notably, all of the macaques inoculated with SHIV-40K6 showed high levels of viral replication early in infection. During later stages, however, the course of infection was variable. Three animals maintained high viral loads and developed immunodeficiency. Of the remaining two macaques, which showed decreasing viral loads after the acute phase of infection, only one efficiently controlled viral replication and remained asymptomatic during 1.5 years of follow-up. The other animal showed an increasing viral load and developed signs of progressive infection during later stages. Our data demonstrate that HIV-1 nefcan, to a large extent, functionally replace SIVmac nef in vivo.

scholarly journals Gag-Specific Cellular Immunity DeterminesIn VitroViral Inhibition andIn VivoVirologic Control following Simian Immunodeficiency Virus Challenges of Vaccinated Rhesus Monkeys

2012 ◽  
Vol 86 (18) ◽  
pp. 9583-9589 ◽  
Author(s):  
Kathryn E. Stephenson ◽  
Hualin Li ◽  
Bruce D. Walker ◽  
Nelson L. Michael ◽  
Dan H. Barouch
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Rhesus Monkeys ◽  
Viral Inhibition ◽  
Viral Loads ◽  
Immunodeficiency Virus ◽  
Virologic Control ◽  
Cellular Immune ◽  
Hiv 1

A comprehensive vaccine for human immunodeficiency virus type 1 (HIV-1) would block HIV-1 acquisition as well as durably control viral replication in breakthrough infections. Recent studies have demonstrated that Env is required for a vaccine to protect against acquisition of simian immunodeficiency virus (SIV) in vaccinated rhesus monkeys, but the antigen requirements for virologic control remain unclear. Here, we investigate whether CD8+T lymphocytes from vaccinated rhesus monkeys mediate viral inhibitionin vitroand whether these responses predict virologic control following SIV challenge. We observed that CD8+lymphocytes from 23 vaccinated rhesus monkeys inhibited replication of SIVin vitro. Moreover, the magnitude of inhibition prior to challenge was inversely correlated with set point SIV plasma viral loads after challenge. In addition, CD8 cell-mediated viral inhibition in vaccinated rhesus monkeys correlated significantly with Gag-specific, but not Pol- or Env-specific, CD4+and CD8+T lymphocyte responses. These findings demonstrate thatin vitroviral inhibition following vaccination largely reflects Gag-specific cellular immune responses and correlates within vivovirologic control following infection. These data suggest the importance of including Gag in an HIV-1 vaccine in which virologic control is desired.

scholarly journals Impact of Primary Elvitegravir Resistance-Associated Mutations in HIV-1 Integrase on Drug Susceptibility and Viral Replication Fitness

2013 ◽  
Vol 57 (6) ◽  
pp. 2654-2663 ◽  
Author(s):  
Michael E. Abram ◽  
Rebecca M. Hluhanich ◽  
Derrick D. Goodman ◽  
Kristen N. Andreatta ◽  
Nicolas A. Margot ◽  
...  
Keyword(s):  
Viral Replication ◽  
Drug Susceptibility ◽  
Viral Fitness ◽  
Cross Resistance ◽  
Replication Capacity ◽  
Strand Transfer ◽  
Recombinant Viruses ◽  
Transfer Inhibitor ◽  
Replication Fitness ◽  
Hiv 1

ABSTRACTElvitegravir (EVG) is an effective HIV-1 integrase (IN) strand transfer inhibitor (INSTI) in advanced clinical development. Primary INSTI resistance-associated mutations (RAMs) at six IN positions have been identified in HIV-1-infected patients failing EVG-containing regimens in clinical studies: T66I/A/K, E92Q/G, T97A, S147G, Q148R/H/K, and N155H. In this study, the effect of these primary IN mutations, alone and in combination, on susceptibility to the INSTIs EVG, raltegravir (RAL), and dolutegravir (DTG); IN enzyme activities; and viral replication fitness was characterized. Recombinant viruses containing the six most common mutations exhibited a range of reduced EVG susceptibility: 92-fold for Q148R, 30-fold for N155H, 26-fold for E92Q, 10-fold for T66I, 4-fold for S147G, and 2-fold for T97A. Less commonly observed primary IN mutations also showed a range of reduced EVG susceptibilities: 40- to 94-fold for T66K and Q148K and 5- to 10-fold for T66A, E92G, and Q148H. Some primary IN mutations exhibited broad cross-resistance between EVG and RAL (T66K, E92Q, Q148R/H/K, and N155H), while others retained susceptibility to RAL (T66I/A, E92G, T97A, and S147G). Dual combinations of primary IN mutations further reduced INSTI susceptibility, replication capacity, and viral fitness relative to either mutation alone. Susceptibility to DTG was retained by single primary IN mutations but reduced by dual mutation combinations with Q148R. Primary EVG RAMs also diminished IN enzymatic activities, concordant with their structural proximity to the active site. Greater reductions in viral fitness of dual mutation combinations may explain why some primary INSTI RAMs do not readily coexist on the same HIV-1 genome but rather establish independent pathways of resistance to EVG.

scholarly journals Viral Characteristics Associated with the Clinical Nonprogressor Phenotype Are Inherited by Viruses from a Cluster of HIV-1 Elite Controllers

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Concepción Casado ◽  
Sara Marrero-Hernández ◽  
Daniel Márquez-Arce ◽  
María Pernas ◽  
Sílvia Marfil ◽  
...  
Keyword(s):  
Viral Replication ◽  
Clinical Phenotype ◽  
Functional Characterization ◽  
Viral Fitness ◽  
Elite Controller ◽  
Replication Capacity ◽  
Viral Fusion ◽  
Elite Controllers ◽  
Hiv 1

ABSTRACTA small group of HIV-1-infected individuals, called long-term nonprogressors (LTNPs), and in particular a subgroup of LTNPs, elite controllers (LTNP-ECs), display permanent control of viral replication and lack of clinical progression. This control is the result of a complex interaction of host, immune, and viral factors. We identified, by phylogenetic analysis, a cluster of LTNP-ECs infected with very similar low-replication HIV-1 viruses, suggesting the contribution of common viral features to the clinical LTNP-EC phenotype. HIV-1 envelope (Env) glycoprotein mediates signaling and promotes HIV-1 fusion, entry, and infection, being a key factor of viral fitnessin vitro, cytopathicity, and infection progressionin vivo. Therefore, we isolated full-lengthenvgenes from viruses of these patients and from chronically infected control individuals. Functional characterization of the initial events of the viral infection showed that Envs from the LTNP-ECs were ineffective in the binding to CD4 and in the key triggering of actin/tubulin-cytoskeleton modifications compared to Envs from chronic patients. The viral properties of the cluster viruses result in a defective viral fusion, entry, and infection, and these properties were inherited by every virus of the cluster. Therefore, inefficient HIV-1 Env functions and signaling defects may contribute to the low viral replication capacity and transmissibility of the cluster viruses, suggesting a direct role in the LTNP-EC phenotype of these individuals. These results highlight the important role of viral characteristics in the LTNP-EC clinical phenotype. These Env viral properties were common to all the cluster viruses and thus support the heritability of the viral characteristics.IMPORTANCEHIV-1 long-term nonprogressor elite controller patients, due to their permanent control of viral replication, have been the object of numerous studies to identify the factors responsible for this clinical phenotype. In this work, we analyzed the viral characteristics of the envelopes of viruses from a phylogenetic cluster of LTNP-EC patients. These envelopes showed ineffective binding to CD4 and the subsequent signaling activity to modify actin/tubulin cytoskeletons, which result in low fusion and deficient entry and infection capacities. These Env viral characteristics could explain the nonprogressor clinical phenotype of these patients. In addition, these inefficientenvviral properties were present in all viruses of the cluster, supporting the heritability of the viral phenotype.

scholarly journals HLA-B57/B*5801 Human Immunodeficiency Virus Type 1 Elite Controllers Select for Rare Gag Variants Associated with Reduced Viral Replication Capacity and Strong Cytotoxic T-Lymphotye Recognition

2008 ◽  
Vol 83 (6) ◽  
pp. 2743-2755 ◽  
Author(s):  
Toshiyuki Miura ◽  
Mark A. Brockman ◽  
Arne Schneidewind ◽  
Michael Lobritz ◽  
Florencia Pereyra ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Viral Replication ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Fitness ◽  
Replication Capacity ◽  
Elite Controllers ◽  
Immunodeficiency Virus ◽  
Viral Replication Capacity

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) elite controllers (EC) maintain viremia below the limit of commercial assay detection (<50 RNA copies/ml) in the absence of antiviral therapy, but the mechanisms of control remain unclear. HLA-B57 and the closely related allele B*5801 are particularly associated with enhanced control and recognize the same Gag240-249 TW10 epitope. The typical escape mutation (T242N) within this epitope diminishes viral replication capacity in chronically infected persons; however, little is known about TW10 epitope sequences in residual replicating viruses in B57/B*5801 EC and the extent to which mutations within this epitope may influence steady-state viremia. Here we analyzed TW10 in a total of 50 B57/B*5801-positive subjects (23 EC and 27 viremic subjects). Autologous plasma viral sequences from both EC and viremic subjects frequently harbored the typical cytotoxic T-lymphocyte (CTL)-selected mutation T242N (15/23 sequences [65.2%] versus 23/27 sequences [85.1%], respectively; P = 0.18). However, other unique mutants were identified in HIV controllers, both within and flanking TW10, that were associated with an even greater reduction in viral replication capacity in vitro. In addition, strong CTL responses to many of these unique TW10 variants were detected by gamma interferon-specific enzyme-linked immunospot assay. These data suggest a dual mechanism for durable control of HIV replication, consisting of viral fitness loss resulting from CTL escape mutations together with strong CD8 T-cell immune responses to the arising variant epitopes.

scholarly journals Direct Evidence of Lower Viral Replication Rates In Vivo in Human Immunodeficiency Virus Type 2 (HIV-2) Infection than in HIV-1 Infection

2007 ◽  
Vol 81 (10) ◽  
pp. 5325-5330 ◽  
Author(s):  
Adam MacNeil ◽  
Abdoulaye Dieng Sarr ◽  
Jean-Louis Sankalé ◽  
Seema Thakore Meloni ◽  
Souleymane Mboup ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Mrna ◽  
Viral Dna ◽  
Viral Loads ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Studies have shown that human immunodeficiency virus type 2 (HIV-2) is less pathogenic than HIV-1, with a lower rate of disease progression. Similarly, plasma viral loads are lower in HIV-2 infection, suggesting that HIV-2 replication is restricted in vivo in comparison to that of HIV-1. However, to date, in vivo studies characterizing replication intermediates in the viral life cycle of HIV-2 have been limited. In order to test the hypothesis that HIV-2 has a lower replication rate in vivo than HIV-1 does, we quantified total viral DNA, integrated proviral DNA, cell-associated viral mRNA, and plasma viral loads in peripheral blood samples from groups of therapy-naïve HIV-1-infected (n = 21) and HIV-2-infected (n = 18) individuals from Dakar, Senegal, with CD4+ T-cell counts of >200/μl. Consistent with our previous findings, total viral DNA loads were similar between HIV-1 and HIV-2 and plasma viral loads were higher among HIV-1-infected individuals. Proportions of DNA in the integrated form were also similar between these viruses. In contrast, levels of viral mRNA were lower in HIV-2 infection. Our study indicates that HIV-2 is able to establish a stable, integrated proviral infection in vivo, but that accumulation of viral mRNA is attenuated in HIV-2 infection relative to that in HIV-1 infection. The differences in viral mRNA are consistent with the differences in plasma viral loads between HIV-1 and HIV-2 and suggest that lower plasma viral loads, and possibly the attenuated pathogenesis of HIV-2, can be explained by lower rates of viral replication in vivo.

scholarly journals Simian Immunodeficiency Virus SIVrcm, a Unique CCR2-Tropic Virus, Selectively Depletes Memory CD4+ T Cells in Pigtailed Macaques through Expanded Coreceptor Usage In Vivo

2009 ◽  
Vol 83 (16) ◽  
pp. 7894-7908 ◽  
Author(s):  
Rajeev Gautam ◽  
Thaidra Gaufin ◽  
Isolde Butler ◽  
Aarti Gautam ◽  
Mary Barnes ◽  
...  
Keyword(s):  
T Cells ◽  
Simian Immunodeficiency Virus ◽  
Effector Memory ◽  
Coreceptor Usage ◽  
New Host ◽  
Viral Loads ◽  
Immunodeficiency Virus ◽  
Memory Cd4 T Cells

ABSTRACT Simian immunodeficiency virus SIVrcm, which naturally infects red-capped mangabeys (RCMs), is the only SIV that uses CCR2 as its main coreceptor due to the high frequency of a CCR5 deletion in RCMs. We investigated the dynamics of SIVrcm infection to identify specific pathogenic mechanisms associated with this major difference in SIV biology. Four pigtailed macaques (PTMs) were infected with SIVrcm, and infection was monitored for over 2 years. The dynamics of in vivo SIVrcm replication in PTMs was similar to that of other pathogenic and nonpathogenic lymphotropic SIVs. Plasma viral loads (VLs) peaked at 107 to 109 SIVrcm RNA copies/ml by day 10 postinoculation (p.i.). A viral set point was established by day 42 p.i. at 103 to 105 SIVrcm RNA copies/ml and lasted up to day 180 p.i., when plasma VLs decreased below the threshold of detection, with blips of viral replication during the follow-up. Intestinal SIVrcm replication paralleled that of plasma VLs. Up to 80% of the CD4+ T cells were depleted by day 28 p.i. in the gut. The most significant depletion (>90%) involved memory CD4+ T cells. Partial CD4+ T-cell restoration was observed in the intestine at later time points. Effector memory CD4+ T cells were the least restored. SIVrcm strains isolated from acutely infected PTMs used CCR2 coreceptor, as reported, but expansion of coreceptor usage to CCR4 was also observed. Selective depletion of effector memory CD4+ T cells is in contrast with predicted in vitro tropism of SIVrcm for macrophages and is probably due to expansion of coreceptor usage. Taken together, these findings emphasize the importance of understanding the selective forces driving viral adaptation to a new host.

scholarly journals Polyubiquitination of APOBEC3G Is Essential for Its Degradation by HIV-1 Vif

2010 ◽  
Vol 84 (9) ◽  
pp. 4840-4844 ◽  
Author(s):  
Qiujia Shao ◽  
Yudi Wang ◽  
James E. K. Hildreth ◽  
Bindong Liu
Keyword(s):  
Human Immunodeficiency Virus ◽  
Simian Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Proteasomal Degradation ◽  
Human Cells ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Proteasomal degradation of APOBEC3G is a critical step for human immunodeficiency virus type 1 (HIV-1) replication. However, the necessity for polyubiquitination of APOBEC3G in this process is still controversial. In this study, we showed that although macaque simian immunodeficiency virus (SIVmac) Vif is more stable than HIV-1 Vif in human cells, SIVmac Vif induces degradation of APBOEC3G as efficiently as HIV-1 Vif. Overexpression of APOBEC3G or lysine-free APOBEC3G stabilized HIV-1 Vif, indicating that APOBEC3G degradation is independent of the degradation of Vif. Furthermore, an in vivo polyubiquitination assay showed that lysine-free APOBEC3G was also polyubiquitinated. These data suggest that polyubiquitination of APOBEC3G, not that of HIV-1 Vif, is crucial for APOBEC3G degradation.

scholarly journals The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages.

1994 ◽  
Vol 179 (1) ◽  
pp. 101-113 ◽  
Author(s):  
M D Miller ◽  
M T Warmerdam ◽  
I Gaston ◽  
W C Greene ◽  
M B Feinberg
Keyword(s):  
Human Immunodeficiency Virus ◽  
Tissue Culture ◽  
Viral Replication ◽  
Gene Product ◽  
Mononuclear Cells ◽  
Positive Effects ◽  
Viral Spread ◽  
Immunodeficiency Virus ◽  
Hiv 1

Considerable controversy and uncertainty have surrounded the biological function of the Human Immunodeficiency Virus (HIV)-1 nef gene product. Initial studies suggested that this early, nonstructural viral protein functioned as a negative regulatory factor; thus, it was proposed to play a role in establishing or maintaining viral latency. In contrast, studies in Simian Immunodeficiency Virus (SIV)mac-infected rhesus monkeys have suggested that Nef is not a negative factor but rather plays a central role in promoting high-level viral replication and is required for viral pathogenesis in vivo. We sought to define a tissue culture system that would approximate the in vivo setting for virus infection in order to assess the role of HIV-1 Nef in viral replication. We show that infection of mitogen-activated peripheral blood mononuclear cells (PBMC) with Nef+ HIV results in enhanced replication as evidenced by earlier gag p24 expression when compared with infections performed with nef mutant viruses. Moreover, when unstimulated freshly isolated PBMC are infected with Nef+ and Nef- viruses and then subsequently activated with mitogen, the Nef-induced difference in viral replication kinetics is even more pronounced, with the Nef- viruses requiring much more time in culture for appreciable growth. A positive effect of Nef on viral replication was also observed in primary macrophages infected with a recombinant of YU-2, a patient-derived molecular clone with macrophage tropism. These positive effects of Nef on viral replication are dependent on the initial multiplicity of infection (MOI), in that infections of unstimulated PBMC at low MOI are most dependent upon intact nef for subsequent viral growth. We now provide evidence that the Nef+ HIV is more infectious than Nef- HIV from both a tissue culture infectious dose analysis, and a single-cell HIV infection assay. In the latter case, we demonstrate that infection with equivalent doses of HIV based on virion-associated gag p24 yields five- to sixfold more infected cells if Nef+ viral stocks were used. Furthermore, we find that the differential infectivity is not dependent on CD4 down-regulation as Nef+ virus produced from transfected COS cells lacking CD4 is also more infectious. However, normalization of PBMC infections to equivalent infectivity between that of the Nef+ and Nef- viruses continues to reveal delayed viral replication in the absence of Nef, suggesting that secondary viral spread in PBMC is also enhanced in Nef+ infections. We demonstrate this directly by showing a 13-15-fold increase in infectivity of PBMC-derived Nef+ HIC.(ABSTRACT TRUNCATED AT 400 WORDS)

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