PSGL-1 restricts HIV-1 infectivity by blocking virus particle attachment to target cells.

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-like, 120-kDa glycoprotein that binds to P-, E-, and L-selectins. PSGL-1 is expressed primarily on the surface of lymphoid and myeloid cells and is up-regulated during inflammation to mediate leukocyte tethering and rolling on the surface of endothelium for migration into inflamed tissues. Although it has been reported that PSGL-1 expression inhibits HIV-1 replication, the mechanism of PSGL-1-mediated anti-HIV activity remains to be elucidated. Here we report that PSGL-1 in virions blocks the infectivity of HIV-1 particles by preventing the binding of particles to target cells. This inhibitory activity is independent of the viral glycoprotein present on the virus particle; the binding of particles bearing the HIV-1 envelope glycoprotein or vesicular stomatitis virus G glycoprotein or even lacking a viral glycoprotein is impaired by PSGL-1. Mapping studies show that the extracellular N-terminal domain of PSGL-1 is necessary for its anti-HIV-1 activity, and that the PSGL-1 cytoplasmic tail contributes to inhibition. In addition, we demonstrate that the PSGL-1-related monomeric E-selectin-binding glycoprotein CD43 also effectively blocks HIV-1 infectivity. HIV-1 infection, or expression of either Vpu or Nef, down-regulates PSGL-1 from the cell surface; expression of Vpu appears to be primarily responsible for enabling the virus to partially escape PSGL-1-mediated restriction. Finally, we show that PSGL-1 inhibits the infectivity of other viruses, such as murine leukemia virus and influenza A virus. These findings demonstrate that PSGL-1 is a broad-spectrum antiviral host factor with a unique mechanism of action.

Potent Inhibition of HIV-1 Replication in Resting CD4 T Cells by Resveratrol and Pterostilbene.

HIV-1 infection of resting CD4 T cells plays a crucial and numerically dominant role during virus transmission at mucosal sites and during subsequent acute replication and T cell depletion. Resveratrol and pterostilbene are plant stilbenoids associated with several health-promoting benefits. Resveratrol has been shown to inhibit the replication of several viruses, including herpes simplex viruses 1 and 2, papillomaviruses, severe acute respiratory syndrome virus, and influenza virus. Alone, resveratrol does not inhibit HIV-1 infection of activated T cells, but it does synergize with nucleoside reverse transcriptase inhibitors in these cells to inhibit reverse transcription. Here, we demonstrate that resveratrol and pterostilbene completely block HIV-1 infection at a low micromolar dose in resting CD4 T cells, primarily at the reverse transcription step. The anti-HIV effect was fully reversed by exogenous deoxynucleosides and Vpx, an HIV-1 and simian immunodeficiency virus protein that increases deoxynucleoside triphosphate (dNTP) levels. These findings are consistent with the reported ability of resveratrol to inhibit ribonucleotide reductase and to lower dNTP levels in cells. This study supports the potential use of resveratrol, pterostilbene, or related compounds as adjuvants in anti-HIV preexposure prophylaxis (PrEP) formulations.

Germinal Center T Follicular Helper Cells Are Highly Permissive to HIV-1 and Alter Their Phenotype during Virus Replication.

HIV-1 replication is concentrated within CD4(+) T cells in B cell follicles of secondary lymphoid tissues during asymptomatic disease. Limited data suggest that a subset of T follicular helper cells (TFH) within germinal centers (GC) is highly permissive to HIV-1. Whether GC TFH are the major HIV-1 virus-producing cells in vivo has not been established. In this study, we investigated TFH permissivity to HIV-1 ex vivo by spinoculating and culturing tonsil cells with HIV-1 GFP reporter viruses. Using flow cytometry, higher percentages of GC TFH (CXCR5(high)PD-1(high)) and CXCR5(+)programmed cell death-1 (PD-1)(low) cells were GFP(+) than non-GC TFH (CXCR5(+)PD-1(intermediate)) or extrafollicular (EF) (CXCR5(-)) cells. When sorted prior to spinoculation, however, GC TFH were substantially more permissive than CXCR5(+)PD-1(low) or EF cells, suggesting that many GC TFH transition to a CXCR5(+)PD-1(low) phenotype during productive infection. In situ hybridization on inguinal lymph node sections from untreated HIV-1-infected individuals without AIDS revealed higher frequencies of HIV-1 RNA(+) cells in GC than non-GC regions of follicle or EF regions. Superinfection of HIV-1-infected individuals' lymph node cells with GFP reporter virus confirmed the permissivity of follicular cells ex vivo. Lymph node immunostaining revealed 96% of CXCR5(+)CD4(+) cells were located in follicles. Within sorted lymph node cells from four HIV-infected individuals, CXCR5(+) subsets harbored 11-66-fold more HIV-1 RNA than CXCR5(-) subsets, as determined by RT PCR. Thus, GC TFH are highly permissive to HIV-1, but downregulate PD-1 and, to a lesser extent, CXCR5 during HIV-1 replication. These data further implicate GC TFH as the major HIV-1-producing cells in chronic asymptomatic HIV-1 infection.

Distinct patterns of Bcl-2 expression occur in R5- and X4-tropic HIV-1-producing lymphoid tissue cells infected ex vivo.

Most HIV-1 replication occurs in secondary lymphoid tissues in T cells within B cell follicles. Mechanisms underlying the accumulation of HIV-1-producing cells at these sites are not understood. Antiapoptotic proteins such as Bcl-2 could promote follicular CD4(+) T cell survival, contributing to sustained virus production. Tonsils obtained from subjects without known HIV infection were disaggregated and analyzed for Bcl-2 expression in follicular (CXCR5(+)) and extrafollicular (CXCR5(-)) CD3(+)CD4(+) cells by flow cytometry. Additional tonsil cells were cultured with phytohemagglutinin (PHA) and interleukin-2 (IL-2) for 2 days, infected with either CCR5(R5) or CXCR4-tropic (X4) GFP reporter viruses, and analyzed for Bcl-2 expression. In freshly disaggregated CD3(+)CD4(+) tonsil cells, mean florescence intensity (MFI) for Bcl-2 was higher in CXCR5(+) (median, 292) compared to CXCR5(-) cells (median, 194; p=0.001). Following in vitro stimulation with PHA and IL-2, Bcl-2 MFI was higher in both CXCR5(+) cells (median, 757; p=0.03) and CXCR5(-) cells (median, 884; p=0.002) in uninfected cultures compared to freshly isolated tonsil cells. Bcl-2 MFI was higher in GFP(+)CD3(+)CD8(-) R5-producing cells (median, 554) than in X4-producing cells (median, 393; p=0.02). Bcl-2 MFI was higher in R5-producing CXCR5(+) cells (median, 840) compared to all other subsets including R5-producing CXCR5(-) cells (median, 524; p=0.04), X4-producing CXCR5(+) cells (median, 401; p=0.02), and X4-producing CXCR5(-) cells (median, 332; p=0.008). Bcl-2 expression is elevated in R5 HIV-1-producing CXCR5(+) T cells in vitro, which may contribute to propagation of R5 virus in B cell follicles in vivo.

Suppression of Foxo1 activity and down-modulation of CD62L (L-selectin) in HIV-1 infected resting CD4 T cells.

HIV-1 hijacks and disrupts many processes in the cells it infects in order to suppress antiviral immunity and to facilitate its replication. Resting CD4 T cells are important early targets of HIV-1 infection in which HIV-1 must overcome intrinsic barriers to viral replication. Although resting CD4 T cells are refractory to infection in vitro, local environmental factors within lymphoid and mucosal tissues such as cytokines facilitate viral replication while maintaining the resting state. These factors can be utilized in vitro to study HIV-1 replication in resting CD4 T cells. In vivo, the migration of resting naïve and central memory T cells into lymphoid tissues is dependent upon expression of CD62L (L-selectin), a receptor that is subsequently down-modulated following T cell activation. CD62L gene transcription is maintained in resting T cells by Foxo1 and KLF2, transcription factors that maintain T cell quiescence and which regulate additional cellular processes including survival, migration, and differentiation. Here we report that HIV-1 down-modulates CD62L in productively infected naïve and memory resting CD4 T cells while suppressing Foxo1 activity and the expression of KLF2 mRNA. Partial T cell activation was further evident as an increase in CD69 expression. Several other Foxo1- and KLF2-regulated mRNA were increased or decreased in productively infected CD4 T cells, including IL-7rα, Myc, CCR5, Fam65b, S1P1 (EDG1), CD52, Cyclin D2 and p21CIP1, indicating a profound reprogramming of these cells. The Foxo1 inhibitor AS1842856 accelerated de novo viral gene expression and the sequella of infection, supporting the notion that HIV-1 suppression of Foxo1 activity may be a strategy to promote replication in resting CD4 T cells. As Foxo1 is an investigative cancer therapy target, the development of Foxo1 interventions may assist the quest to specifically suppress or activate HIV-1 replication in vivo.

Adenosine deaminase acting on RNA-1 (ADAR1) inhibits HIV-1 replication in human alveolar macrophages.

While exploring the effects of aerosol IFN-γ treatment in HIV-1/tuberculosis co-infected patients, we observed A to G mutations in HIV-1 envelope sequences derived from bronchoalveolar lavage (BAL) of aerosol IFN-γ-treated patients and induction of adenosine deaminase acting on RNA 1 (ADAR1) in the BAL cells. IFN-γ induced ADAR1 expression in monocyte-derived macrophages (MDM) but not T cells. ADAR1 siRNA knockdown induced HIV-1 expression in BAL cells of four HIV-1 infected patients on antiretroviral therapy. Similar results were obtained in MDM that were HIV-1 infected in vitro. Over-expression of ADAR1 in transformed macrophages inhibited HIV-1 viral replication but not viral transcription measured by nuclear run-on, suggesting that ADAR1 acts post-transcriptionally. The A to G hyper-mutation pattern observed in ADAR1 over-expressing cells in vitro was similar to that found in the lungs of HIV-1 infected patients treated with aerosol IFN-γ suggesting the model accurately represented alveolar macrophages. Together, these results indicate that ADAR1 restricts HIV-1 replication post-transcriptionally in macrophages harboring HIV-1 provirus. ADAR1 may therefore contribute to viral latency in macrophages.

Increased burst size in multiply infected cells can alter basic virus dynamics.

BACKGROUND: The dynamics of viral infections have been studied extensively in a variety of settings, both experimentally and with mathematical models. The majority of mathematical models assumes that only one virus can infect a given cell at a time. It is, however, clear that especially in the context of high viral load, cells can become infected with multiple copies of a virus, a process called coinfection. This has been best demonstrated experimentally for human immunodeficiency virus (HIV), although it is thought to be equally relevant for a number of other viral infections. In a previously explored mathematical model, the viral output from an infected cell does not depend on the number of viruses that reside in the cell, i.e. viral replication is limited by cellular rather than viral factors. In this case, basic virus dynamics properties are not altered by coinfection. RESULTS: Here, we explore the alternative assumption that multiply infected cells are characterized by an increased burst size and find that this can fundamentally alter model predictions. Under this scenario, establishment of infection may not be solely determined by the basic reproductive ratio of the virus, but can depend on the initial virus load. Upon infection, the virus population need not follow straight exponential growth. Instead, the exponential rate of growth can increase over time as virus load becomes larger. Moreover, the model suggests that the ability of anti-viral drugs to suppress the virus population can depend on the virus load upon initiation of therapy. This is because more coinfected cells, which produce more virus, are present at higher virus loads. Hence, the degree of drug resistance is not only determined by the viral genotype, but also by the prevalence of coinfected cells. CONCLUSIONS: Our work shows how an increased burst size in multiply infected cells can alter basic infection dynamics. This forms the basis for future experimental testing of model assumptions and predictions that can distinguish between the different scenarios.

HLA-DR+ CD38+ CD4+ T lymphocytes have elevated CCR5 expression and produce the majority of R5-tropic HIV-1 RNA in vivo.

Percentages of activated T cells correlate with HIV-1 disease progression, but the underlying mechanisms are not fully understood. We hypothesized that HLA-DR(+) CD38(+) (DR(+) 38(+)) CD4(+) T cells produce the majority of HIV-1 due to elevated expression of CCR5 and CXCR4. In phytohemagglutinin (PHA)-stimulated CD8-depleted peripheral blood mononuclear cells (PBMC) infected with HIV-1 green fluorescent protein (GFP) reporter viruses, DR(-) 38(+) T cells constituted the majority of CCR5 (R5)-tropic (median, 62%) and CXCR4 (X4)-tropic HIV-1-producing cells (median, 61%), although cell surface CCR5 and CXCR4 were not elevated in this subset of cells. In lymph nodes from untreated individuals infected with R5-tropic HIV-1, percentages of CCR5(+) cells were elevated in DR(+) 38(+) CD4(+) T cells (median, 36.4%) compared to other CD4(+) T-cell subsets (median values of 5.7% for DR(-) 38(-) cells, 19.4% for DR(+) 38(-) cells, and 7.6% for DR(-) 38(+) cells; n = 18; P < 0.001). In sorted CD8(-) lymph node T cells, median HIV-1 RNA copies/10(5) cells was higher for DR(+) 38(+) cells (1.8 × 10(6)) than for DR(-) 38(-) (0.007 × 10(6)), DR(-) 38(+) (0.064 × 10(6)), and DR(+) 38(-) (0.18 × 10(6)) subsets (n = 8; P < 0.001 for all). After adjusting for percentages of subsets, a median of 87% of viral RNA was harbored by DR(+) 38(+) cells. Percentages of CCR5(+) CD4(+) T cells and concentrations of CCR5 molecules among subsets predicted HIV-1 RNA levels among CD8(-) DR/38 subsets (P < 0.001 for both). Median HIV-1 DNA copies/10(5) cells was higher in DR(+) 38(+) cells (5,360) than in the DR(-) 38(-) (906), DR(-) 38(+) (814), and DR(+) 38(-) (1,984) subsets (n = 7; P ≤ 0.031). Thus, DR(+) 38(+) CD4(+) T cells in lymph nodes have elevated CCR5 expression, are highly susceptible to infection with R5-tropic virus, and produce the majority of R5-tropic HIV-1. PBMC assays failed to recapitulate in vivo findings, suggesting limited utility. Strategies to reduce numbers of DR(+) 38(+) CD4(+) T cells may substantially inhibit HIV-1 replication.

Multiploid inheritance of HIV-1 during cell-to-cell infection.

During cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1), many viral particles can be simultaneously transferred from infected to uninfected CD4 T cells through structures called virological synapses (VS). Here we directly examine how cell-free and cell-to-cell infections differ from infections initiated with cell-free virus in the number of genetic copies that are transmitted from one generation to the next, i.e., the genetic inheritance. Following exposure to HIV-1-expressing cells, we show that target cells with high viral uptake are much more likely to become infected. Using T cells that coexpress distinct fluorescent HIV-1 variants, we show that multiple copies of HIV-1 can be cotransmitted across a single VS. In contrast to cell-free HIV-1 infection, which titrates with Poisson statistics, the titration of cell-associated HIV-1 to low rates of overall infection generates a constant fraction of the newly infected cells that are cofluorescent. Triple infection was also readily detected when cells expressing three fluorescent viruses were used as donor cells. A computational model and a statistical model are presented to estimate the degree to which cofluorescence underestimates coinfection frequency. Lastly, direct detection of HIV-1 proviruses using fluorescence in situ hybridization confirmed that significantly more HIV-1 DNA copies are found in primary T cells infected with cell-associated virus than in those infected with cell-free virus. Together, the data suggest that multiploid inheritance is common during cell-to-cell HIV-1 infection. From this study, we suggest that cell-to-cell infection may explain the high copy numbers of proviruses found in infected cells in vivo and may provide a mechanism through which HIV preserves sequence heterogeneity in viral quasispecies through genetic complementation.

CD8 T cell response and evolutionary pressure to HIV-1 cryptic epitopes derived from antisense transcription.

Retroviruses pack multiple genes into relatively small genomes by encoding several genes in the same genomic region with overlapping reading frames. Both sense and antisense HIV-1 transcripts contain open reading frames for known functional proteins as well as numerous alternative reading frames (ARFs). At least some ARFs have the potential to encode proteins of unknown function, and their antigenic properties can be considered as cryptic epitopes (CEs). To examine the extent of active immune response to virally encoded CEs, we analyzed human leukocyte antigen class I-associated polymorphisms in HIV-1 gag, pol, and nef genes from a large cohort of South Africans with chronic infection. In all, 391 CEs and 168 conventional epitopes were predicted, with the majority (307; 79%) of CEs derived from antisense transcripts. In further evaluation of CD8 T cell responses to a subset of the predicted CEs in patients with primary or chronic infection, both sense- and antisense-encoded CEs were immunogenic at both stages of infection. In addition, CEs often mutated during the first year of infection, which was consistent with immune selection for escape variants. These findings indicate that the HIV-1 genome might encode and deploy a large potential repertoire of unconventional epitopes to enhance vaccine-induced antiviral immunity.

Human immunodeficiency virus evolution towards reduced replicative fitness in vivo and the development of AIDS.

Human immunodeficiency virus (HIV) infection progresses to AIDS following an asymptomatic period during which the virus is thought to evolve towards increased fitness and pathogenicity. We show mathematically that progression to the strongest HIV-induced pathology requires evolution of the virus towards reduced replicative fitness in vivo. This counter-intuitive outcome can happen if multiple viruses co-infect the same cell frequently, which has been shown to occur in recent experiments. According to our model, in the absence of frequent co-infection, the less fit AIDS-inducing strains might never emerge. The frequency of co-infection can correlate with virus load, which in turn is determined by immune responses. Thus, at the beginning of infection when immunity is strong and virus load is low, co-infection is rare and pathogenic virus variants with reduced replicative fitness go extinct. At later stages of infection when immunity is less efficient and virus load is higher, co-infection occurs more frequently and pathogenic virus variants with reduced replicative fitness can emerge, resulting in T-cell depletion. In support of these notions, recent data indicate that pathogenic simian immunodeficiency virus (SIV) strains occurring late in the infection are less fit in specific in vitro experiments than those isolated at earlier stages. If co-infection is blocked, the model predicts the absence of any disease even if virus loads are high. We hypothesize that non-pathogenic SIV infection within its natural hosts, which is characterized by the absence of disease even in the presence of high virus loads, could be explained by a reduced occurrence of co-infection in this system.

Bis-anthracycline antibiotics inhibit human immunodeficiency virus type 1 transcription.

The increasing numbers of human immunodeficiency virus type 1 (HIV-1) strains that exhibit resistance to antiretroviral agents used at present require the development of new effective antiretroviral compounds. Tat transactivation was recognized early on as an attractive target for drug interference. To screen for and analyze the effects of compounds that interfere with Tat transactivation, we developed several cell-based reporter systems in which enhanced green fluorescence protein is a direct and quantitative marker of HIV-1 expression or Tat-dependent long terminal repeat activity. Using these reporter cell lines, we found that the bis-anthracycline WP631, a recently developed DNA intercalator, efficiently inhibits HIV-1 expression at subcytotoxic concentrations. WP631 also abrogated acute HIV-1 replication in peripheral blood mononuclear cells infected with various primary virus isolates. We demonstrate that WP631-mediated HIV-1 inhibition is caused by the inhibition of Tat transactivation. The data presented suggest that WP631 could serve as a lead compound for a new type of HIV-1 inhibitor.

Dynamics of HIV-1 recombination in its natural target cells.

Genetic recombination is believed to assist HIV-1 diversification and escape from host immunity and antiviral therapies, yet this process remains largely unexamined within the natural target-cell populations. We developed a method for measuring HIV-1 recombination directly that employs reporter viruses bearing functional enhanced yellow fluorescent protein (YFP) and enhanced cyan fluorescent protein (CFP) genes in which recombination produces a modified GFP gene and GFP fluorescence in the infected cells. These reporter viruses allow simultaneous quantification of the dynamics of HIV-1 infection, coinfection, and recombination in cell culture and in animal models by flow-cytometric analysis. Multiround infection assays revealed that productive cellular coinfection was subject to little functional inhibition. As a result, generation of recombinants proceeded according to the square of the infection rate during HIV-1 replication in T lymphocytes and within human thymic grafts in severe combined immunodeficient (SCID)-hu (Thy/Liv) mice. These results suggest that increases in viral load may confer a compounding risk of virus escape by means of recombinational diversification. A single round of replication in T lymphocytes in culture generated an average of nine recombination events per virus, and infection of macrophages led to approximately 30 crossover events, making HIV-1 up to an order of magnitude more recombinogenic than recognized previously and demonstrating that the infected cell exerts a profound influence on the frequency of recombination.

CD154-CD40-induced reactivation of latent HIV-1 infection.

Reservoirs of latent HIV-1 in T cells and macrophages pose one of the major obstacles that hamper final eradication of HIV-1 from infected patients. Targeting costimulatory molecules expressed on cell types harboring latent HIV-1 to achieve reactivation may provide a new approach to overcome this problem. One such molecule is CD40, a member of the tumor necrosis factor (TNF)-receptor family. Using THP89GFP cells as a model for latently infected macrophages, we demonstrate that trimeric forms of recombinant CD154 allow for the direct reactivation of latent HIV-1 infection. Reactivation is augmented by the release of TNF-alpha. The presence of TNF-alpha is also crucial for the expression of late structural genes such as p24 Gag. In addition, levels of secreted TNF-alpha are sufficiently high to reactivate latent HIV-1 in a latently HIV-1-infected T-cell line (J89GFP). Taken together, our results demonstrate that costimulatory molecules may be attractive targets to reactivate latent HIV-1 in infected patients.

Direct and quantitative single-cell analysis of human immunodeficiency virus type 1 reactivation from latency.

The ability of human immunodeficiency virus type 1 (HIV-1) to establish latent infections in cells has received renewed attention owing to the failure of highly active antiretroviral therapy to eradicate HIV-1 in vivo. Despite much study, the molecular bases of HIV-1 latency and reactivation are incompletely understood. Research on HIV-1 latency would benefit from a model system that is amenable to rapid and efficient analysis and through which compounds capable of regulating HIV-1 reactivation may be conveniently screened. We describe a novel reporter system that has several advantages over existing in vitro systems, which require elaborate, expensive, and time-consuming techniques to measure virus production. Two HIV-1 molecular clones (NL4-3 and 89.6) were engineered to express enhanced green fluorescent protein (EGFP) under the control of the viral long terminal repeat without removing any viral sequences. By using these replication-competent viruses, latently infected T-cell (Jurkat) and monocyte/macrophage (THP-1) lines in which EGFP fluorescence and virus expression are tightly coupled were generated. Following reactivation with agents such as tumor necrosis factor alpha, virus expression and EGFP fluorescence peaked after 4 days and over the next 3 weeks each declined in a synchronized manner, recapitulating the establishment of latency. Using fluorescence microscopy, flow cytometry, or plate-based fluorometry, this system allows immediate, direct, and quantitative real-time analysis of these processes within single cells or in bulk populations of cells. Exploiting the single-cell analysis abilities of this system, we demonstrate that cellular activation and virus reactivation following stimulation with proinflammatory cytokines can be uncoupled.