Productive entry of HIV-1 during cell-to-cell transmission via dynamin-dependent endocytosis.

HIV-1 can be transmitted as cell-free virus or via cell-to-cell contacts. Cell-to-cell transmission between CD4(+) T cells is the more efficient mode of transmission and is predominant in lymphoid tissue, where the majority of virus resides. Yet the cellular mechanisms underlying productive cell-to-cell transmission in uninfected target cells are unclear. Although it has been demonstrated that target cells can take up virus via endocytosis, definitive links between this process and productive infection remain undefined, and this route of transmission has been proposed to be nonproductive. Here, we report that productive cell-to-cell transmission can occur via endocytosis in a dynamin-dependent manner and is sensitive to clathrin-associated antagonists. These data were obtained in a number of CD4(+) T-cell lines and in primary CD4(+) T cells, using both CXCR4- and CCR5-tropic virus. However, we also found that HIV-1 demonstrated flexibility in its use of such endocytic pathways as certain allogeneic transmissions were seen to occur in a dynamin-dependent manner but were insensitive to clathrin-associated antagonists. Also, depleting cells of the clathrin accessory protein AP180 led to a viral uptake defect associated with enhanced infection. Collectively, these data demonstrate that endosomal uptake of HIV-1 during cell-to-cell transmission leads to productive infection, but they are also indicative of a flexible model of viral entry during cell-to-cell transmission, in which the virus can alter its entry route according to the pressures that it encounters.

Expansion of HIV-specific T follicular helper cells in chronic HIV infection.

HIV targets CD4 T cells, which are required for the induction of high-affinity antibody responses and the formation of long-lived B cell memory. The depletion of antigen-specific CD4 T cells during HIV infection is therefore believed to impede the development of protective B cell immunity. Although several different HIV-related B cell dysfunctions have been described, the role of CD4 T follicular helper (TFH) cells in HIV infection remains unknown. Here, we assessed HIV-specific TFH responses in the lymph nodes of treatment-naive and antiretroviral-treated HIV-infected individuals. Strikingly, both the bulk TFH and HIV-specific TFH cell populations were significantly expanded in chronic HIV infection and were highly associated with viremia. In particular, GAG-specific TFH cells were detected at significantly higher levels in the lymph nodes compared with those of GP120-specific TFH cells and showed preferential secretion of the helper cytokine IL-21. In addition, TFH cell expansion was associated with an increase of germinal center B cells and plasma cells as well as IgG1 hypersecretion. Thus, our study suggests that high levels of HIV viremia drive the expansion of TFH cells, which in turn leads to perturbations of B cell differentiation, resulting in dysregulated antibody production.

The viral protein Tat can inhibit the establishment of HIV-1 latency.

The establishment of HIV-1 latency can result from limiting levels of transcription initiation or elongation factors, restrictive chromatin modifications, transcriptional interference, and insufficient Tat activity. Since the viral protein Tat can counteract many of these factors, we hypothesized that the presence of exogenous Tat during infection might inhibit the establishment of latency. This was explored using a Jurkat model of latency establishment and reactivation. PCR and reverse transcriptase PCR (RT-PCR) confirmed the latent state in this model and showed evidence of transcriptional interference. To address our hypothesis, cells undergoing infection were first exposed to either purified recombinant Tat or a transactivation-negative mutant. Only the former resulted in a modest inhibition of the establishment of latency. Next, Jurkat cells stably expressing intracellular Tat were used in our latency model to avoid limitations of Tat delivery. Experiments confirmed that intracellular Tat expression did not affect the susceptibility of these cells to viral infection. Eight weeks after infection, Jurkat cells expressing Tat harbored up to 1,700-fold fewer (P < 0.01) latent viruses than Jurkat cells that did not express Tat. Additionally, Tat delivered by a second virus was sufficient to reactivate most of the latent population. Our results suggest that inhibition of the establishment of latent infection is theoretically possible. In a hypothetical scenario of therapy that induces viral gene expression during acute infection, activation of viruses which would otherwise have entered latency could occur while concurrent highly active antiretroviral therapy (HAART) would prevent further viral spread, potentially decreasing the size of the established latent reservoir.

The HIV-1 Vpu viroporin inhibitor BIT225 does not affect Vpu-mediated tetherin antagonism.

Among its many roles, the HIV-1 accessory protein Vpu performs a viroporin function and also antagonizes the host cell restriction factor tetherin through its transmembrane domain. BIT225 is a small molecule inhibitor that specifically targets the Vpu viroporin function, which, in macrophages, resulted in late stage inhibition of virus release and decreased infectivity of released virus, a phenotype similar to tetherin-mediated restriction. Here, we investigated whether BIT225 might mediate its antiviral function, at least in part, via inhibition of Vpu-mediated tetherin antagonism. Using T-cell lines inducible for tetherin expression, we found that BIT225 does not exert its antiviral function by inhibiting Vpu-mediated tetherin downmodulation from the cell surface, the main site of action of tetherin activity. In addition, results from a bioluminescence resonance energy transfer (BRET) assay showed that the Vpu-tetherin interaction was not affected by BIT225. Our data provide support for the concept that tetherin antagonism and viroporin function are separable on the Vpu transmembrane and that viroporin function might be cell-type dependent. Further, this work contributes to the characterization of BIT225 as an inhibitor that specifically targets the viroporin function of Vpu.

Vpu-mediated tetherin antagonism of ongoing HIV-1 infection in CD4(+) T-cells is not directly related to the extent of tetherin cell surface downmodulation.

Tetherin is a host cell restriction factor that acts against HIV-1 and other enveloped viruses. The antiviral activity of tetherin is antagonized by the HIV-1 protein Vpu, that downregulates tetherin from the cell surface. Here, we report the specific detection of cell surface tetherin levels in primary activated CD4(+) T-cells and in CD4(+) T-cell lines. Differences were observed regarding tetherin cell surface expression, Vpu-mediated tetherin downmodulation and promotion of virus release. However, Vpu expression in all T-cell lines resulted in a 2-fold increase in numbers of infected cells after three days. This implies a Vpu-mediated effect in ongoing infection and possibly in cell-to-cell viral spread that is independent of the extent of Vpu-mediated tetherin cell surface downmodulation. Endogenous cell surface tetherin levels in T-cell lines were also downmodulated following infection with Vpu-deleted virus, suggesting an additional Vpu-independent mechanism of tetherin cell surface downmodulation following HIV-1 infection in T-cell lines.

Tetherin and its viral antagonists.

Restriction factors comprise an important layer of host defense to fight against viral infection. Some restriction factors are constitutively expressed whereas the majority is induced by interferon to elicit innate immunity. In addition to a number of well-characterized interferon-inducible antiviral factors such as RNaseL/OAS, ISG15, Mx, PKR, and ADAR, tetherin (BST-2/CD317/HM1.24) was recently discovered to block the release of enveloped viruses from the cell surface, which is regarded as a novel antiviral mechanism induced by interferon. Here, we briefly review the history of tetherin discovery, discuss how tetherin blocks virus production, and highlight the viral countermeasures to evade tetherin restriction.

Transcription of preintegrated HIV-1 cDNA modulates cell surface expression of major histocompatibility complex class I via Nef.

Although transcription from unintegrated human immunodeficiency virus type 1 (HIV-1) DNA can occur inside infected cells, yielding all classes of viral mRNA transcripts, the translation of viral proteins is very limited. One of the proteins made is Nef, but it is unclear whether Nef produced in this way is able to play a role in immune evasion as occurs with integrated virus. We therefore asked whether transcription from preintegrated HIV-1 cDNAs could result in Nef-mediated modulation of cell surface major histocompatibility complex class I (MHC-I) expression. We infected a Rev-CEM green fluorescent protein (GFP) reporter cell line with virus and blocked integration though use of either an inactive integrase or the integrase inhibitor raltegravir. Infected cells were assayed by flow cytometry for cell surface expression of the HLA-A, HLA-B, and HLA-C allotypes (HLA-ABC), HLA-A31, and HLA-E. Viral RNA and DNA products were assayed via quantitative PCR (qPCR). The prevention of integration had no effect, relative to productively infected cells, on levels of expression of multiply spliced viral mRNA transcripts and Nef protein. Downregulation of HLA-ABC and HLA-A31 also occurred at levels similar to those seen in cells in which integration had occurred. Parallel experiments assaying cell surface HLA-ABC expression in infected activated primary CD4(+) T cells produced a similar pattern of results. Hence, the capacity of HIV-1 to modulate MHC-I is not linked to its ability to integrate. Thus, Nef-mediated evasion of host immune responsiveness might be attributable, in part at least, to transcription from unintegrated viral DNA.

Tetherin restricts direct cell-to-cell infection of HIV-1.

BACKGROUND: Tetherin (BST-2/CD317/HM1.24) is an interferon (IFN)-inducible factor of the innate immune system, recently shown to exert antiviral activity against HIV-1 and other enveloped viruses by tethering nascent viral particles to the cell surface, thereby inhibiting viral release. In HIV-1 infection, the viral protein U (Vpu) counteracts this antiviral action by down-modulating tetherin from the cell surface. Viral dissemination between T-cells can occur via cell-free transmission or the more efficient direct cell-to-cell route through lipid raft-rich virological synapses, to which tetherin localizes. RESULTS: We established a flow cytometry-based co-culture assay to distinguish viral transfer from viral transmission and investigated the influence of tetherin on cell-to-cell spread of HIV-1. Sup-T1 cells inducible for tetherin expression were used to examine the impact of effector and target cell tetherin expression on virus transfer and transmission. Using this assay, we showed that tetherin inhibits direct cell-to-cell virus transfer and transmission. Viral Vpu promoted viral transmission from tetherin-expressing cells by down-modulating tetherin from the effector cell surface. Further, we showed that tetherin on the target cell promotes viral transfer and transmission. Viral infectivity in itself was not affected by tetherin. CONCLUSION: In addition to inhibiting viral release, tetherin also inhibits direct cell-to-cell spread. Viral protein Vpu counteracts this restriction, outweighing its possible cost of fitness in cell-to-cell transmission. The differential role of tetherin in effector and target cells suggest a role for tetherin in cell-cell contacts and virological synapses.

HIV-1 subtype B and C integrase enzymes exhibit differential patterns of resistance to integrase inhibitors in biochemical assays.

BACKGROUND: Because of high intersubtype HIV-1 genetic variability, it has been shown that subtype-specific patterns of resistance to antiretroviral drugs exist. We wished to ascertain whether this might be true for integrase inhibitors. METHODS: We compared the susceptibility of subtype B and C HIV-1 integrase enzymes, harboring the previously reported resistance mutations E92Q, N155H, and E92Q/N155H, to clinically relevant integrase inhibitors. This was performed biochemically using a microtiter plate system. RESULTS: Subtype C integrase enzymes bearing the resistance mutations E92Q/N155H were approximately 10-fold more susceptible to each of two integrase inhibitors, raltegravir and elvitegravir, than were subtype B recombinant integrase containing the same mutations. CONCLUSION: Polymorphic differences within the subtype B and C integrase genes likely cause variations in the contribution of N155H alone or in combination with E92Q to drug resistance. It is possible that different viral subtypes may favor different mutational pathways, potentially leading to varying levels of drug resistance among different subtypes.

Identification of novel mutations responsible for resistance to MK-2048, a second-generation HIV-1 integrase inhibitor.

MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to first-generation INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to approximately 13% of wt levels and increased resistance to MK-2048 to approximately 8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer "off" rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg(2+) binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.

Expression of Nef from unintegrated HIV-1 DNA downregulates cell surface CXCR4 and CCR5 on T-lymphocytes.

BACKGROUND: Transcription of HIV-1 cDNA prior to, or in the absence of, integration leads to synthesis of all classes of viral RNA transcripts. Yet only a limited range of viral proteins, including Nef, are translated in this context. Nef expression from unintegrated HIV-1 DNA has been shown to reduce cell surface CD4 levels in T-cells. We wished to determine whether Nef expressed from unintegrated DNA was also able to downregulate the chemokine coreceptors CXCR4 and CCR5.Viral integration was blocked through use of an inactive integrase or by using the integrase inhibitor raltegravir. Infected cells bearing unintegrated DNA were assayed by flow cytometry in the GFP reporter cell line, Rev-CEM, for cell surface levels of CD4, CXCR4 and CCR5. RESULTS: In cells bearing only unintegrated HIV-1 DNA, we found that surface levels of CXCR4 were significantly reduced, while levels of CCR5 were also diminished, but not to the extent of CXCR4. We also confirmed the downregulation of CD4. Similar patterns of results were obtained with both integrase-deficient virus or with wild-type infections of cells treated with raltegravir. The Alu-HIV qPCR assay that we used for detection of proviral DNA did not detect any integrated viral DNA. CONCLUSIONS: Our results demonstrate that Nef can be expressed from unintegrated DNA at functionally relevant levels and suggest a role for Nef in downregulation of CXCR4 and CCR5. These findings may help to explain how downregulation of CXCR4, CCR5 and CD4 might restrict superinfection and/or prevent signal transduction involving HIV-1 infected cells.

Stage-dependent inhibition of HIV-1 replication by antiretroviral drugs in cell culture.

Recent clinical trials have shown that the use of the HIV-1 integrase (IN) inhibitor raltegravir (RAL) results in drops in the viral load that are more rapid than those achieved by use of the reverse transcriptase (RT) inhibitor efavirenz. Previously, mathematical modeling of viral load decay that takes into account the stage of viral replication targeted by a drug has yielded data that closely approximate the clinical trial results. This model predicts greater inhibition of viral replication by drugs that act later in the viral replication cycle. In the present study, we have added drugs that target entry, reverse transcription, integration, or proteolytic processing to acutely infected cells and have shown modest viral inhibition by entry inhibitors, intermediate levels of inhibition by RT and IN inhibitors, and high levels of inhibition by protease inhibitors relative to the levels of growth for the no-drug controls. When dual or triple combinations of these drugs were added to acutely infected cells, we found that the levels of inhibition achieved by any given combination were comparable to those achieved by the latest-acting drug in the combination. In single-round infections in which the kinetics of reverse transcription and integration had been determined by quantitative PCR, addition of IN inhibitors at various times postinfection resulted in levels of inhibition equal to or greater than those achieved by addition of RT inhibitors. Collectively, our data provide in vitro evidence of the stage-dependent inhibition of HIV-1 by clinically relevant drugs. We discuss how stage-dependent inhibition helps to explain the unique viral load decay dynamics observed clinically with RAL.

Comparative biochemical analysis of HIV-1 subtype B and C integrase enzymes.

BACKGROUND: Integrase inhibitors are currently being incorporated into highly active antiretroviral therapy (HAART). Due to high HIV variability, integrase inhibitor efficacy must be evaluated against a range of integrase enzymes from different subtypes. METHODS: This study compares the enzymatic activities of HIV-1 integrase from subtypes B and C as well as susceptibility to various integrase inhibitors in vitro. The catalytic activities of both enzymes were analyzed in regard to each of 3' processing and strand transfer activities both in the presence and absence of the integrase inhibitors raltegravir (RAL), elvitegravir (EVG), and MK-2048. RESULTS: Our results show that integrase function is similar with enzymes of either subtype and that the various integrase strand transfer inhibitors (INSTIs) that were employed possessed similar inhibitory activity against both enzymes. CONCLUSION: This suggests that the use of integrase inhibitors against HIV-1 subtype C will result in comparable outcomes to those obtained against subtype B infections.