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.

cHighly 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.

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 firstgeneration 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 Ϸ13% of wt levels and increased resistance to MK-2048 to Ϸ8-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.Selective pressure exerted by antiretroviral drugs, in conjunction with high viral mutation rates, promotes the inevitable emergence of drug-resistant HIV-1 variants. This necessitates an ongoing search for novel antiretroviral compounds that either have novel mechanisms and inhibit different stages of viral replication or inhibit targets that have acquired resistance to existing drugs. In the latter case, such newer next-generation agents should ideally display resistance profiles which are distinct and nonoverlapping with those of the first-generation drugs.Integration of viral cDNA into the host cell genome is a distinct feature of retroviral replication, and inhibitors of HIV-1 integrase have recently been added to the arsenal of clinically approved antiretroviral drugs. Raltegravir (RAL) was the first integrase strand transfer inhibitor (INSTI) to be approved by the U.S. Food and Drug Administration (FDA) after clinical trials showed that this drug promoted a rapid and sustained antiviral effect (13). Elvitegravir (EVG), another integrase inhibitor, is currently in phase III clinical trials (27). Resistance mutations common to both of these first-generation integrase inhibitors have been reported and can result in high levels of drug resistance (26). Mutations which engender crossresistance between RAL and EVG have been reported in clinical trials, cell culture studies, and biochemical assays (9,26). This has prompted the search for second-generation integrase inhibitors that might display novel patterns of resistance, allowing their use in patients who have failed therapy with RAL or E...

BackgroundTetherin (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.ResultsWe 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.ConclusionIn 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.

Canada dHIV-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.H IV-1 can be transmitted as free virus or directly between cells via cell-cell contacts. Cell-to-cell transmission is a more efficient and rapid means of viral spread and is the predominant mode of HIV-1 transmission in lymphoid tissue (1, 2). Given that the vast majority of virus within an infected individual is found in lymphoid tissue and in CD4ϩ T cells, cell-to-cell transmission between CD4 ϩ T cells likely represents the most common mode of HIV-1 spread.Improved understanding of the direct and coordinated interactions between T cells and antigen-presenting cells, termed immunological synapses (3), ultimately led to the first description of coordinated retroviral transmission between T cells. Human Tlymphotropic virus type I (HTLV-I) is transmitted via a polarized T-cell structure termed the virological synapse that is analogous to the immunological synapse (4). Subsequent studies revealed that HIV-1 could also be transmitted via virological synapses between CD4 ϩ T cells (5) and that infected cells could even form polysynapses, thereby allowing simultaneous cell-to-cell transmissions from a single infected cell to multiple uninfected target cells (6). Cell-to-cell transmission between infected macrophages and uninfected CD4ϩ T cells has also been described (7). Further, a less common mode of transmission between CD4 ϩ T cells was shown to exist in which HIV-1 can be transmitted by long membrane nanotubes that are formed after cell division (8). A vis...

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 Despite the success of highly active antiretroviral therapy (HAART) for the treatment of HIV/AIDS, the presence of latently infected resting memory CD4 ϩ T cells represents a major barrier to eradication of HIV-1 from an infected individual (45, 55). The latent reservoir is established during acute infection (10), and represents an archive of both wild-type (wt) and drugresistant viruses (26). Since latent proviruses do not express viral gene products, they are shielded from both antiretroviral drugs and the host immune response, and the long-lived, latently infected host cell is not exposed to viral cytopathic effect. Reactivation of latently infected cells likely serves as the major source of viral rebound upon treatment interruption or failure (31). Thus, HIV-1 latency provides for lifelong persistence of infection and is the target of intense research effort. Several approaches attempting to reactivate the latent reservoir have been used in clinical trials, with the goal of rendering latently infected cells susceptible to immune clearance and/or viral cytopathicity. No clear, longterm benefits have yet been observed from this approach (reviewed in references 6, 22, and 59), although further trials are under way (12). Additional strategies concerning the latent reservoir are clearly needed.HIV-1 gene expression is dependent upon the viral protein Tat. In the absence of Tat, transcription initiates normally at the 5= long terminal repeat (LTR) but results in short, abortive viral transcripts due to RNA polymerase II (RNAPII) pausing shortly after promoter clearance. Tat is initially expressed from rare full-length transcripts that are multiply spliced, and it functions as a powerful activator of viral transcription. In contrast to classic DNA-binding transcription factors that control the initiation of transcription, Tat controls transcription at the level of RNAPII elongation through interaction with the TAR RNA (the first 59 nucleotides of each viral transcript) and the positive transcription elongation factor b (P-TEFb, composed of Cyclin T1 [CycT1] and cyclindependent k...

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.

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. The HIV integrase (IN) enzyme catalyzes the insertion of viral DNA into host DNA, a process known as integration (1). In a reaction termed 3= processing, integrase recognizes and cleaves off a dinucleotide GT downstream of a conserved dinucleotide CA signal, located within the last 15 bp of the long terminal repeats (LTR) that flank the viral DNA, and this effectively creates new 3= hydroxyl ends (2). The second step in integration, termed strand transfer, is the integrase-mediated insertion of the processed viral DNA into host DNA by a 5-bp staggered cleavage of target DNA. The exposed 3= hydroxyl groups on the viral insert interact with exposed 5= phosphates on the host DNA. Integration, which occurs primarily in highly expressed genes (3), causes the host machinery to transcribe viral genes and leads to successful propagation of viral particles. Integration is essential for productive infection and the establishment of viral persistence; therefore, integration was an early choice for the development of inhibitory compounds (4).The development of in vitro microtiter plate-based biochemical assays for the measurement of the various biochemical activities of integrase facilitated compound screening and identification of viable integrase inhibitors (5). The first specific integrase inhibitors, identified in 2000 (5), posses...