The innate immune factor RPRD2/REAF and its role in the Lv2 restriction of HIV.

Intracellular innate immunity involves co-evolved antiviral restriction factors that specifically inhibit infecting viruses. Studying these restrictions has increased our understanding of viral replication, host-pathogen interactions, and pathogenesis, and represent potential targets for novel antiviral therapies. Lentiviral restriction 2 (Lv2) was identified as an unmapped early-phase restriction of HIV-2 and later shown to also restrict HIV-1 and simian immunodeficiency virus. The viral determinants of Lv2 susceptibility have been mapped to the envelope and capsid proteins in both HIV-1 and HIV-2, and also viral protein R (Vpr) in HIV-1, and appears dependent on cellular entry mechanism. A genome-wide screen identified several likely contributing host factors including members of the polymerase-associated factor 1 (PAF1) and human silencing hub (HUSH) complexes, and the newly characterized regulation of nuclear pre-mRNA domain containing 2 (RPRD2). Subsequently, RPRD2 (or RNA-associated early-stage antiviral factor) has been shown to be upregulated upon T cell activation, is highly expressed in myeloid cells, binds viral reverse transcripts, and potently restricts HIV-1 infection. RPRD2 is also bound by HIV-1 Vpr and targeted for degradation by the proteasome upon reverse transcription, suggesting RPRD2 impedes reverse transcription and Vpr targeting overcomes this block. RPRD2 is mainly localized to the nucleus and binds RNA, DNA, and DNA:RNA hybrids. More recently, RPRD2 has been shown to negatively regulate genome-wide transcription and interact with the HUSH and PAF1 complexes which repress HIV transcription and are implicated in maintenance of HIV latency. In this review, we examine Lv2 restriction and the antiviral role of RPRD2 and consider potential mechanism(s) of action.

IFITM proteins: Understanding their diverse roles in viral infection, cancer, and immunity.

Interferon-induced transmembrane proteins (IFITMs) are broad spectrum antiviral factors that inhibit the entry of a wide range of clinically important pathogens including influenza A virus, HIV-1, and Dengue virus. IFITMs are thought to act primarily by antagonizing virus-cell membrane fusion in this regard. However, recent work on these proteins has uncovered novel post-entry viral restriction mechanisms. IFITMs are also increasingly thought to have a role regulating immune responses, including innate antiviral and inflammatory responses as well as adaptive T-cell and B-cell responses. Further, IFITMs may have pathological activities in cancer, wherein IFITM expression can be a marker of therapeutically resistant and aggressive disease courses. In this review, we summarize the respective literatures concerning these apparently diverse functions with a view to identifying common themes and potentially yielding a more unified understanding of IFITM biology.

The Role of IFITM Proteins in Tick-Borne Encephalitis Virus Infection.

Tick-borne encephalitis virus (TBEV), of the genus Flavivirus, is a causative agent of severe encephalitis in regions of endemicity of northern Asia and central and northern Europe. Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the replication cycles of numerous viruses, including flaviviruses such as West Nile virus, dengue virus, and Zika virus. Here, we demonstrate the role of IFITM1, IFITM2, and IFITM3 in the inhibition of TBEV infection and in protection against virus-induced cell death. We show that the most significant role is that of IFITM3, including the dissection of its functional motifs by mutagenesis. Furthermore, through the use of CRISPR-Cas9-generated IFITM1/3-knockout monoclonal cell lines, we confirm the role and additive action of endogenous IFITMs in TBEV suppression. However, the results of coculture assays suggest that TBEV might partially escape interferon- and IFITM-mediated suppression during high-density coculture infection when the virus enters naive cells directly from infected donor cells. Thus, cell-to-cell spread may constitute a strategy for virus escape from innate host defenses. IMPORTANCE TBEV infection may result in encephalitis, chronic illness, or death. TBEV is endemic in northern Asia and Europe; however, due to climate change, new centers of endemicity have arisen. Although effective TBEV vaccines have been approved, vaccination coverage is low, and due to the lack of specific therapeutics, infected individuals depend on their immune responses to control the infection. IFITM proteins are components of the innate antiviral defenses that suppress cell entry of many viral pathogens. However, no studies on the role of IFITM proteins in TBEV infection have been published thus far. Understanding antiviral innate immune responses is crucial for the future development of antiviral strategies. Here, we show the important role of IFITM proteins in the inhibition of TBEV infection and virus-mediated cell death. However, our data suggest that TBEV cell-to-cell spread may be less prone to both interferon- and IFITM-mediated suppression, potentially facilitating escape from IFITM-mediated immunity.

Priming of NLRP3 inflammasome activation by Msn kinase MINK1 in macrophages.

The nucleotide-binding domain, leucine-rich-repeat containing family, pyrin domain-containing 3 (NLRP3) inflammasome is essential in inflammation and inflammatory disorders. Phosphorylation at various sites on NLRP3 differentially regulates inflammasome activation. The Ser725 phosphorylation site on NLRP3 is depicted in multiple inflammasome activation scenarios, but the importance and regulation of this site has not been clarified. The present study revealed that the phosphorylation of Ser725 was an essential step for the priming of the NLRP3 inflammasome in macrophages. We also showed that Ser725 was directly phosphorylated by misshapen (Msn)/NIK-related kinase 1 (MINK1), depending on the direct interaction between MINK1 and the NLRP3 LRR domain. MINK1 deficiency reduced NLRP3 activation and suppressed inflammatory responses in mouse models of acute sepsis and peritonitis. Reactive oxygen species (ROS) upregulated the kinase activity of MINK1 and subsequently promoted inflammasome priming via NLRP3 Ser725 phosphorylation. Eliminating ROS suppressed NLRP3 activation and reduced sepsis and peritonitis symptoms in a MINK1-dependent manner. Altogether, our study reveals a direct regulation of the NLRP3 inflammasome by Msn family kinase MINK1 and suggests that modulation of MINK1 activity is a potential intervention strategy for inflammasome-related diseases.

HIV-1 Accessory Protein Vpr Interacts with REAF/RPRD2 To Mitigate Its Antiviral Activity.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and, to a lesser extent, cycling T cells. Virion-packaged Vpr is released in target cells shortly after entry, suggesting it is required in the early phase of infection. Previously, we described REAF (RNA-associated early-stage antiviral factor; RPRD2), a constitutively expressed protein that potently restricts HIV replication at or during reverse transcription. Here, we show that a virus without an intact vpr gene is more highly restricted by REAF and, using delivery by virus-like particles (VLPs), that Vpr alone is sufficient for REAF degradation in primary macrophages. REAF is more highly expressed in macrophages than in cycling T cells, and we detected, by coimmunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts quickly during the early phase of replication and induces the degradation of REAF within 30 min of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localization and interaction with cullin 4A-DBB1 (DCAF1) E3 ubiquitin ligase are required for REAF degradation by Vpr. In response to infection, cells upregulate REAF levels. This response is curtailed in the presence of Vpr. These findings support the hypothesis that Vpr induces the degradation of a factor, REAF, that impedes HIV infection in macrophages.IMPORTANCE For at least 30 years, it has been known that HIV-1 Vpr, a protein carried in the virion, is important for efficient infection of primary macrophages. Vpr is also a determinant of the pathogenic effects of HIV-1 in vivo A number of cellular proteins that interact with Vpr have been identified. So far, it has not been possible to associate these proteins with altered viral replication in macrophages or to explain why Vpr is carried in the virus particle. Here, we show that Vpr mitigates the antiviral effects of REAF, a protein highly expressed in primary macrophages and one that inhibits virus replication during reverse transcription. REAF is degraded by Vpr within 30 min of virus entry in a manner dependent on the nuclear localization of Vpr and its interaction with the cell's protein degradation machinery.

IFITM proteins inhibit HIV-1 protein synthesis.

Interferon induced transmembrane proteins (IFITMs) inhibit the cellular entry of a broad range of viruses, but it has been suspected that for HIV-1 IFITMs may also inhibit a post-integration replicative step. We show that IFITM expression reduces HIV-1 viral protein synthesis by preferentially excluding viral mRNA transcripts from translation and thereby restricts viral production. Codon-optimization of proviral DNA rescues viral translation, implying that IFITM-mediated restriction requires recognition of viral RNA elements. In addition, we find that expression of the viral accessory protein Nef can help overcome the IFITM-mediated inhibition of virus production. Our studies identify a novel role for IFITMs in inhibiting HIV replication at the level of translation, but show that the effects can be overcome by the lentiviral protein Nef.

RNA-Associated Early-Stage Antiviral Factor Is a Major Component of Lv2 Restriction.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication in human cells is restricted at early postentry steps by host inhibitory factors. We previously described and characterized an early-phase restriction of HIV-1 and -2 replication in human cell lines, primary macrophages, and peripheral blood mononuclear cells. The restriction was termed lentiviral restriction 2 (Lv2). The viral determinants of Lv2 susceptibility mapped to the HIV-2 envelope (Env) and capsid (CA). We subsequently reported a whole-genome small interfering RNA screening for factors involved in HIV that identified RNA-associated early-stage antiviral factor (REAF). Using HIV-2 chimeras of susceptible and nonsusceptible viruses, we show here that REAF is a major component of the previously described Lv2 restriction. Further studies of the viral CA demonstrate that the CA mutation I73V (previously called I207V), a potent determinant for HIV-2, is a weak determinant of susceptibility for HIV-1. More potent CA determinants for HIV-1 REAF restriction were identified at P38A, N74D, G89V, and G94D. These results firmly establish that in HIV-1, CA is a strong determinant of susceptibility to Lv2/REAF. Similar to HIV-2, HIV-1 Env can rescue sensitive CAs from restriction. We conclude that REAF is a major component of the previously described Lv2 restriction.IMPORTANCE Measures taken by the host cell to combat infection drive the evolution of pathogens to counteract or sidestep them. The study of such virus-host conflicts can point to possible weaknesses in the arsenal of viruses and may lead to the rational design of antiviral agents. Here we describe our discovery that the host restriction factor REAF fulfills the same criteria previously used to describe lentiviral restriction (Lv2). We show that, like the HIV-2 CA, the CA of HIV-1 is a strong determinant of Lv2/REAF susceptibility. We illustrate how HIV counteracts Lv2/REAF by using an envelope with alternative routes of entry into cells.

Identification of an env-defective HIV-1 mutant capable of spontaneous reversion to a wild-type phenotype in certain T-cell lines.

BACKGROUND: Attempts to eradicate HIV from cellular reservoirs are vital but depend on a clear understanding of how viral variants are transmitted and survive in the different cell types that constitute such reservoirs. Mutations in the env gene of HIV may be able to exert a differential influence on viral transmission ability in regard to cell-free and cell-associated viral forms. METHODS: The ability of HIV containing an env G367R mutation in cell-free and cell-associated viruses to cause infection and to revert to wild-type was measured using several T cell lines. To determine factors that might potentially influence the reversion of G367R, we studied each of entry inhibitors, inhibitors of cellular endocytosis, and modulators of cell growth and activation. RESULTS: We demonstrate that an HIV-1 variant containing a G367R substitution within the CD4 binding site of gp120 was non-infectious as free virus in culture but was infectious when infected cells were co-cultured with certain T cell lines or when cells were transfected by a relevant proviral plasmid. Differences in viral infectivity by cell-associated G367R viruses were determined by the type of target cell employed, regardless which type of donor cell was used. Reversion was slowed or inhibited by entry inhibitors and by inhibitors of cellular endocytosis. Interleukin 2 was able to block G367R reversion in only one of the T cell lines studied but not in the other, while phorbol 12-myristate 13-acetate (PMA) inhibited G367R reversion in all the T cell lines. CONCLUSIONS: Env-defective HIV may have a different phenotype as cell-free versus cell-associated virus. The persistence of defective forms can potentially lead to the emergence of virulent forms. The heterogeneity of cell types that constitute the HIV reservoir can contribute to viral variability, even among similar types of cells. This is the first demonstration of a mutation in the HIV envelope, i.e. G367R, that can compromise infection by cell-free virus but less severely by cell-associated virus and that does so in a cell type-dependent manner.

Fitness impaired drug resistant HIV-1 is not compromised in cell-to-cell transmission or establishment of and reactivation from latency.

Both the presence of latently infected cells and cell-to-cell viral transmission are means whereby HIV can partially evade the inhibitory activities of antiretroviral drugs. The clinical use of a novel integrase inhibitor, dolutegravir (DTG), has established hope that this compound may limit HIV persistence, since no treatment-naïve patient treated with DTG has yet developed resistance against this drug, even though a R263K substitution in integrase confers low-level resistance to this drug in tissue culture. Here, we have studied the impact of R263K on HIV replication capacity and the ability of HIV to establish or be reactivated from latency and/or spread through cell-to-cell transmission. We affirm that DTG-resistant viruses have diminished capacity to replicate and establish infection. However, DTG-resistant viruses were efficiently transmitted via cell-to-cell contacts, and were as likely to establish and be reactivated from latent infection as wildtype viruses. Both cell-to-cell transmission of HIV and the establishment of and reemergence from latency are important for the establishment and maintenance of viral reservoirs. Since the DTG and other drug-resistant viruses studied here do not seem to have been impaired in regard to these activities, studies should be undertaken to characterize HIV reservoirs in patients who have been treated with DTG.

Biochemical analysis of the role of G118R-linked dolutegravir drug resistance substitutions in HIV-1 integrase.

Drug resistance mutations (DRMs) have been reported for all currently approved anti-HIV drugs, including the latest integrase strand transfer inhibitors (INSTIs). We previously used the new INSTI dolutegravir (DTG) to select a G118R integrase resistance substitution in tissue culture and also showed that secondary substitutions emerged at positions H51Y and E138K. Now, we have characterized the impact of the G118R substitution, alone or in combination with either H51Y or E138K, on 3' processing and integrase strand transfer activity. The results show that G118R primarily impacted the strand transfer step of integration by diminishing the ability of integrase-long terminal repeat (LTR) complexes to bind target DNA. The addition of H51Y and E138K to G118R partially restored strand transfer activity by modulating the formation of integrase-LTR complexes through increasing LTR DNA affinity and total DNA binding, respectively. This unique mechanism, in which one function of HIV integrase partially compensates for the defect in another function, has not been previously reported. The G118R substitution resulted in low-level resistance to DTG, raltegravir (RAL), and elvitegravir (EVG). The addition of either of H51Y or E138K to G118R did not enhance resistance to DTG, RAL, or EVG. Homology modeling provided insight into the mechanism of resistance conferred by G118R as well as the effects of H51Y or E138K on enzyme activity. The G118R substitution therefore represents a potential avenue for resistance to DTG, similar to that previously described for the R263K substitution. For both pathways, secondary substitutions can lead to either diminished integrase activity and/or increased INSTI susceptibility.

Latent HIV-1 can be reactivated by cellular superinfection in a Tat-dependent manner, which can lead to the emergence of multidrug-resistant recombinant viruses.

The HIV-1 latent reservoir represents an important source of genetic diversity that could contribute to viral evolution and multidrug resistance following latent virus reactivation. This could occur by superinfection of a latently infected cell. We asked whether latent viruses might be reactivated when their host cells are superinfected, and if so, whether they could contribute to the generation of recombinant viruses. Using populations of latently infected Jurkat cells, we found that latent viruses were efficiently reactivated upon superinfection. Pathways leading to latent virus reactivation via superinfection might include gp120-CD4/CXCR4-induced signaling, modulation of the cellular environment by Nef, and/or the activity of Tat produced upon superinfection. Using a range of antiviral compounds and genetic approaches, we show that gp120 and Nef are not required for latent virus reactivation by superinfection, but this process depends on production of functional Tat by the superinfecting virus. In a primary cell model of latency in unstimulated CD4 T cells, superinfection also led to latent virus reactivation. Drug-resistant latent viruses were also reactivated following superinfection in Jurkat cells and were able to undergo recombination with the superinfecting virus. Under drug-selective pressure, this generated multidrug-resistant recombinants that were identified by unique restriction digestion band patterns and by population-level sequencing. During conditions of poor drug adherence, treatment interruption or treatment failure, or in drug-impermeable sanctuary sites, reactivation of latent viruses by superinfection or other means could provide for the emergence or spread of replicatively fit viruses in the face of strong selective pressures.

HIV-1 subtype variability in Vif derived from molecular clones affects APOBEC3G-mediated host restriction.

BACKGROUND: The host protein APOBEC3G (A3G) can limit HIV-1 replication. Its protective effect is overcome by the HIV-1 'viral infectivity factor' (Vif), which targets A3G for proteosomal degradation. Although Vif is considered to be essential for HIV-1 replication, the effect of Vif variability among commonly used HIV-1 molecular clones of different genetic backgrounds on viral infectiousness and pathogenesis has not been fully determined. METHODS: We cloned the intact Vif coding regions of available molecular clones of different subtypes into expression vectors. Δvif full-length HIV-1 clonal variants were generated from corresponding subtype-specific full-length molecular clones. Replication-competent viruses were produced in 293T cells in the presence or absence of A3G, with Vif being supplied by the full-length HIV-1 clone or in trans. The extent of A3G-mediated restriction was then determined in a viral replication assay using a reporter cell line. RESULTS AND CONCLUSIONS: In the absence of A3G, Vif subtype origin did not impact viral replication. In the presence of A3G the subtype origin of Vif had a differential effect on viral replication. Vif derived from a subtype C molecular clone was less effective at overcoming A3G-mediated inhibition than Vif derived from either subtype B or CRF02_AG molecular clones.

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.

Antiviral drug resistance and the need for development of new HIV-1 reverse transcriptase inhibitors.

Highly active antiretroviral therapy (HAART) consists of a combination of drugs to achieve maximal virological response and reduce the potential for the emergence of antiviral resistance. Despite being the first antivirals described to be effective against HIV, reverse transcriptase inhibitors remain the cornerstone of HAART. There are two broad classes of reverse transcriptase inhibitor, the nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs). Since the first such compounds were developed, viral resistance to them has inevitably been described; this necessitates the continuous development of novel compounds within each class. In this review, we consider the NRTIs and NNRTIs currently in both preclinical and clinical development or approved for second-line therapy and describe the patterns of resistance associated with their use as well as the underlying mechanisms that have been described. Due to reasons of both affordability and availability, some reverse transcriptase inhibitors with a low genetic barrier are more commonly used in resource-limited settings. Their use results in the emergence of specific patterns of antiviral resistance and so may require specific actions to preserve therapeutic options for patients in such settings.

Maraviroc and other HIV-1 entry inhibitors exhibit a class-specific redistribution effect that results in increased extracellular viral load.

HIV entry inhibitors, such as maraviroc (MVC), prevent cell-free viruses from entering the cells. In clinical trials, patients who were treated with MVC often displayed viral loads that were above the limit of conventional viral load detection compared to efavirenz-based regimens. We hypothesize that viruses blocked by entry inhibitors may be redistributed to plasma, where they artificially increase viral load measurements compared to those with the use of antiretroviral drugs (ARVs) that act intracellularly. We infected PM-1 cells with CCR5-tropic HIV-1 BaL or CXCR4-tropic HIV-1 NL4-3 in the presence of inhibitory concentrations of efavirenz, raltegravir, enfuvirtide, maraviroc, and AMD3100, the latter three being entry inhibitors. Supernatant viral load, reverse transcriptase enzyme activity, and intracellular nucleic acid levels were measured at times up to 24 h postinfection. Infectivity of redistributed dual-tropic HIV-1 was assessed using TZM-bl cells. Extracellular viral load analysis revealed that entry inhibitor-treated cells had higher levels of virus in the supernatant than the cells treated with other ARVs at 8 h postinfection. By 24 h, the supernatant viral load was still higher for entry inhibitors than other ARVs. We observed a correlation between viral load and the step of entry inhibition. Dual-tropic virus infectivity was undiminished utilizing the CCR5 coreceptor following redistribution by CXCR4 entry inhibition. This in vitro model indicates that entry inhibitors exhibit a redistribution effect unseen with intracellular ARV drugs. Based on these results, the effectiveness of some entry inhibitors may be underestimated if plasma viral load is used as a sole indicator of clinical success.

Novel therapeutic strategies targeting HIV integrase.

Integration of the viral genome into host cell chromatin is a pivotal and unique step in the replication cycle of retroviruses, including HIV. Inhibiting HIV replication by specifically blocking the viral integrase enzyme that mediates this step is an obvious and attractive therapeutic strategy. After concerted efforts, the first viable integrase inhibitors were developed in the early 2000s, ultimately leading to the clinical licensure of the first integrase strand transfer inhibitor, raltegravir. Similarly structured compounds and derivative second generation integrase strand transfer inhibitors, such as elvitegravir and dolutegravir, are now in various stages of clinical development. Furthermore, other mechanisms aimed at the inhibition of viral integration are being explored in numerous preclinical studies, which include inhibition of 3' processing and chromatin targeting. The development of new clinically useful compounds will be aided by the characterization of the retroviral intasome crystal structure. This review considers the history of the clinical development of HIV integrase inhibitors, the development of antiviral drug resistance and the need for new antiviral compounds.

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.

The role of unintegrated DNA in HIV infection.

Integration of the reverse transcribed viral genome into host chromatin is the hallmark of retroviral replication. Yet, during natural HIV infection, various unintegrated viral DNA forms exist in abundance. Though linear viral cDNA is the precursor to an integrated provirus, increasing evidence suggests that transcription and translation of unintegrated DNAs prior to integration may aid productive infection through the expression of early viral genes. Additionally, unintegrated DNA has the capacity to result in preintegration latency, or to be rescued and yield productive infection and so unintegrated DNA, in some circumstances, may be considered to be a viral reservoir. Recently, there has been interest in further defining the role and function of unintegrated viral DNAs, in part because the use of anti-HIV integrase inhibitors leads to an abundance of unintegrated DNA, but also because of the potential use of non-integrating lentiviral vectors in gene therapy and vaccines. There is now increased understanding that unintegrated viral DNA can either arise from, or be degraded through, interactions with host DNA repair enzymes that may represent a form of host antiviral defence. This review focuses on the role of unintegrated DNA in HIV infection and additionally considers the potential implications for antiviral therapy.