Macaque-Tropic HIV-1 Derivatives: A Novel Experimental Approach to Understand Viral Replication and Evolution in Vivo

ABSTRACT There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZα reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 × 10−5 mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro. Although the mutation rate was not affected by the orientation of lacZα, the sites favored for mutations (hot spots) in lacZα depended on which strand of lacZα was present in the viral RNA. The pattern of hot spots seen in lacZα in vivo did not match any of the published data obtained when purified RT was used to copy lacZα in vitro.

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

Journal of Experimental Medicine ◽

10.1084/jem.179.1.101 ◽

1994 ◽

Vol 179 (1) ◽

pp. 101-113 ◽

Cited By ~ 369

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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HIV-1 Vpr Accelerates Viral Replication during Acute Infection by Exploitation of Proliferating CD4+ T Cells In Vivo

PLoS Pathogens ◽

10.1371/journal.ppat.1003812 ◽

2013 ◽

Vol 9 (12) ◽

pp. e1003812 ◽

Cited By ~ 32

Author(s):

Kei Sato ◽

Naoko Misawa ◽

Shingo Iwami ◽

Yorifumi Satou ◽

Masao Matsuoka ◽

...

Keyword(s):

T Cells ◽

Viral Replication ◽

Cd4 T Cells ◽

Acute Infection ◽

Hiv 1

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Differential Ability of Primary HIV-1 Nef Isolates To Downregulate HIV-1 Entry Receptors

Journal of Virology ◽

10.1128/jvi.01548-15 ◽

2015 ◽

Vol 89 (18) ◽

pp. 9639-9652 ◽

Cited By ~ 18

Author(s):

Mako Toyoda ◽

Yoko Ogata ◽

Macdonald Mahiti ◽

Yosuke Maeda ◽

Xiaomei T. Kuang ◽

...

Keyword(s):

Viral Replication ◽

Viral Entry ◽

Immune Escape ◽

Elite Controllers ◽

Functional Impairments ◽

Disease Phenotypes ◽

Infected Cells ◽

Entry Receptor ◽

Hiv 1

ABSTRACTHIV-1 Nef downregulates the viral entry receptor CD4 as well as the coreceptors CCR5 and CXCR4 from the surface of HIV-infected cells, and this leads to promotion of viral replication through superinfection resistance and other mechanisms. Nef sequence motifs that modulate these functions have been identified viain vitromutagenesis with laboratory HIV-1 strains. However, it remains unclear whether the same motifs contribute to Nef activity in patient-derived sequences and whether these motifs may differ in Nef sequences isolated at different infection stages and/or from patients with different disease phenotypes. Here,nefclones from 45 elite controllers (EC), 46 chronic progressors (CP), and 43 acute progressors (AP) were examined for their CD4, CCR5, and CXCR4 downregulation functions. Nef clones from EC exhibited statistically significantly impaired CD4 and CCR5 downregulation ability and modestly impaired CXCR4 downregulation activity compared to those from CP and AP. Nef's ability to downregulate CD4 and CCR5 correlated positively in all cohorts, suggesting that they are functionally linkedin vivo. Moreover, impairments in Nef's receptor downregulation functions increased the susceptibility of Nef-expressing cells to HIV-1 infection. Mutagenesis studies on three functionally impaired EC Nef clones revealed that multiple residues, including those at novel sites, were involved in the alteration of Nef functions and steady-state protein levels. Specifically, polymorphisms at highly conserved tryptophan residues (e.g., Trp-57 and Trp-183) and immune escape-associated sites were responsible for reduced Nef functions in these clones. Our results suggest that the functional modulation of primary Nef sequences is mediated by complex polymorphism networks.IMPORTANCEHIV-1 Nef, a key factor for viral pathogenesis, downregulates functionally important molecules from the surface of infected cells, including the viral entry receptor CD4 and coreceptors CCR5 and CXCR4. This activity enhances viral replication by protecting infected cells from cytotoxicity associated with superinfection and may also serve as an immune evasion strategy. However, how these activities are maintained under selective pressurein vivoremains elusive. We addressed this question by analyzing functions of primary Nef clones isolated from patients at various infection stages and with different disease phenotypes, including elite controllers, who spontaneously control HIV-1 viremia to undetectable levels. The results indicated that downregulation of HIV-1 entry receptors, particularly CCR5, is impaired in Nef clones from elite controllers. These functional impairments were driven by rare Nef polymorphisms and adaptations associated with cellular immune responses, underscoring the complex molecular pathways responsible for maintaining and attenuating viral protein functionin vivo.

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Effect of Antibody to HIV-1 Tat Protein on Viral Replication in Vitro and Progression of HIV-1 Disease in Vivo

Journal of Acquired Immune Deficiency Syndromes & Human Retrovirology ◽

10.1097/00042560-199512000-00003 ◽

1995 ◽

Vol 10 (4) ◽

pp. 408-416 ◽

Cited By ~ 78

Author(s):

M C Re ◽

G Furlini ◽

M Vignoli ◽

E Ramazzotti ◽

G Roderigo ◽

...

Keyword(s):

Viral Replication ◽

Tat Protein ◽

Hiv 1

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A Novel Motif in HIV-1 Nef That Regulates MIP-1β Chemokine Release in Macrophages

Journal of Virology ◽

10.1128/jvi.00741-10 ◽

2010 ◽

Vol 84 (16) ◽

pp. 8327-8331 ◽

Cited By ~ 11

Author(s):

Lue Dai ◽

Mario Stevenson

Keyword(s):

Major Histocompatibility Complex ◽

Viral Replication ◽

Accessory Protein ◽

Major Histocompatibility ◽

Histocompatibility Complex ◽

Virus Dissemination ◽

Primate Lentiviruses ◽

Hiv 1

ABSTRACT All primate lentiviruses encode Nef, an accessory protein that is important for viral pathogenicity in vivo. Lentiviral Nef proteins regulate the release of chemokines (MIP-1 α/β) from infected macrophages, thereby enhancing virus dissemination (S. Swingler, A. Mann, J. Jacque, B. Brichacek, V. G. Sasseville, K. Williams, A. A. Lackner, E. N. Janoff, R. Wang, D. Fisher, and M. Stevenson, Nat. Med. 5:997-1003, 1999). In the current study, we have identified a novel domain within Nef (K92EK) that is required for Nef-dependent MIP-1β production by infected macrophages. Mutations in this domain abrogated MIP-1β induction but did not affect other Nef-ascribed activities, such as CD4 or major histocompatibility complex (MHC) class Ι downregulation. This further underscores Nef as a modular protein with genetically separable activities that may contribute to its role in viral replication and pathogenicity.

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In vitro and in vivo cleavage of HIV-1 RNA by new SOFA-HDV ribozymes and their potential to inhibit viral replication

RNA Biology ◽

10.4161/rna.8.2.15200 ◽

2011 ◽

Vol 8 (2) ◽

pp. 343-353 ◽

Cited By ~ 11

Author(s):

Sébastien Lainé ◽

Robert J. Scarborough ◽

Dominique Lévesque ◽

Ludovic Didierlaurent ◽

Kaitlin J. Soye ◽

...

Keyword(s):

Viral Replication ◽

Hiv 1

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

Journal of Virology ◽

10.1128/jvi.73.10.8371-8383.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8371-8383 ◽

Cited By ~ 35

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.

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Simian-Human Immunodeficiency Virus SHIV.CH505 Infection of Rhesus Macaques Results in Persistent Viral Replication and Induces Intestinal Immunopathology

Journal of Virology ◽

10.1128/jvi.00372-19 ◽

2019 ◽

Vol 93 (18) ◽

Cited By ~ 7

Author(s):

Katharine J. Bar ◽

Ernesto Coronado ◽

Tiffany Hensley-McBain ◽

Megan A. O’Connor ◽

Jessica M. Osborn ◽

...

Keyword(s):

T Cells ◽

Hiv Infection ◽

Viral Replication ◽

Cell Function ◽

Immune Cell ◽

Rhesus Macaques ◽

Subtype C ◽

Viral Kinetics ◽

Hiv 1

ABSTRACTSimian-human immunodeficiency viruses (SHIVs) have been utilized to test vaccine efficacy and characterize mechanisms of viral transmission and pathogenesis. However, the majority of SHIVs currently available have significant limitations in that they were developed using sequences from chronically HIV-infected individuals or uncommon HIV subtypes or were optimized for the macaque model by serially passaging the engineered virusin vitroorin vivo. Recently, a newly developed SHIV, SHIV.C.CH505.375H.dCT (SHIV.CH505), which incorporates vpu-env (gp140) sequences from a transmitted/founder HIV-1 subtype C strain, was shown to retain attributes of primary HIV-1 strains. However, a comprehensive analysis of the immunopathology that results from infection with this virus, especially in critical tissue compartments like the intestinal mucosa, has not been completed. In this study, we evaluated the viral dynamics and immunopathology of SHIV.CH505 in rhesus macaques. In line with previous findings, we found that SHIV.CH505 is capable of infecting and replicating efficiently in rhesus macaques, resulting in peripheral viral kinetics similar to that seen in pathogenic SIV and HIV infection. Furthermore, we observed significant and persistent depletions of CCR5+and CCR6+CD4+T cells in mucosal tissues, decreases in CD4+T cells producing Th17 cell-associated cytokines, CD8+T cell dysfunction, and alterations of B cell and innate immune cell function, indicating that SHIV.CH505 elicits intestinal immunopathology typical of SIV/HIV infection. These findings suggest that SHIV.CH505 recapitulates the early viral replication dynamics and immunopathogenesis of HIV-1 infection of humans and thus can serve as a new model for HIV-1 pathogenesis, treatment, and prevention research.IMPORTANCEThe development of chimeric SHIVs has been instrumental in advancing our understanding of HIV-host interactions and allowing forin vivotesting of novel treatments. However, many of the currently available SHIVs have distinct drawbacks and are unable to fully reflect the features characteristic of primary SIV and HIV strains. Here, we utilize rhesus macaques to define the immunopathogenesis of the recently developed SHIV.CH505, which was designed without many of the limitations of previous SHIVs. We observed that infection with SHIV.CH505 leads to peripheral viral kinetics and mucosal immunopathogenesis comparable with those caused by pathogenic SIV and HIV. Overall, these data provide evidence of the value of SHIV.CH505 as an effective model of SIV/HIV infection and an important tool that can be used in future studies, including preclinical testing of new therapies or prevention strategies.

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Lentiviral Nef Proteins Manipulate T Cells in a Subset-Specific Manner

Journal of Virology ◽

10.1128/jvi.03104-14 ◽

2014 ◽

Vol 89 (4) ◽

pp. 1986-2001 ◽

Cited By ~ 8

Author(s):

Hangxing Yu ◽

Mohammad Khalid ◽

Anke Heigele ◽

Jan Schmökel ◽

Shariq M. Usmani ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Viral Replication ◽

Cell Subset ◽

T Cell Subsets ◽

Immunodeficiency Virus ◽

Primate Lentiviruses ◽

Selective Preservation ◽

Hiv 1

ABSTRACTThe role of the accessory viral Nef protein as a multifunctional manipulator of the host cell that is required for effective replication of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV)in vivois well established. It is unknown, however, whether Nef manipulates all or just specific subsets of CD4+T cells, which are the main targets of virus infection and differ substantially in their state of activation and importance for a functional immune system. Here, we analyzed the effect of Nef proteins differing in their T cell receptor (TCR)-CD3 downmodulation function in HIV-infected human lymphoid aggregate cultures and peripheral blood mononuclear cells. We found that Nef efficiently downmodulates TCR-CD3 in naive and memory CD4+T cells and protects the latter against apoptosis. In contrast, highly proliferative CD45RA+CD45RO+CD4+T cells were main producers of infectious virus but largely refractory to TCR-CD3 downmodulation. Such T cell subset-specific differences were also observed for Nef-mediated modulation of CD4 but not for enhancement of virion infectivity. Our results indicate that Nef predominantly modulates surface receptors on CD4+T cell subsets that are not already fully permissive for viral replication. As a consequence, Nef-mediated downmodulation of TCR-CD3, which distinguishes most primate lentiviruses from HIV type 1 (HIV-1) and itsvpu-containing simian precursors, may promote a selective preservation of central memory CD4+T cells, which are critical for the maintenance of a functional immune system.IMPORTANCEThe Nef proteins of human and simian immunodeficiency viruses manipulate infected CD4+T cells in multiple ways to promote viral replication and immune evasionin vivo. Here, we show that some effects of Nef are subset specific. Downmodulation of CD4 and TCR-CD3 is highly effective in central memory CD4+T cells, and the latter Nef function protects this T cell subset against apoptosis. In contrast, highly activated/proliferating CD4+T cells are largely refractory to receptor downmodulation but are main producers of infectious HIV-1. Nef-mediated enhancement of virion infectivity, however, was observed in all T cell subsets examined. Our results provide new insights into how primate lentiviruses manipulate their target cells and suggest that the TCR-CD3 downmodulation function of Nef may promote a selective preservation of memory CD4+T cells, which are critical for immune function, but has little effect on activated/proliferating CD4+T cells, which are the main targets for viral replication.

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