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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 antagonising 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 summarise 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 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.

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.

The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and in cycling T cells to a lesser extent. Virion packaged Vpr is released in target cells shortly after entry, suggesting its requirement 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 intactvpris more highly restricted by REAF and, using delivery by 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 detect, by co-immunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts very quickly during the early phase of replication and induces the degradation of REAF within 30 minutes of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localisation and interaction with cullin4A-DBB1 (DCAF1) E3 ubiquitin ligase is 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, which impedes HIV infection in macrophages.ImportanceFor 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-1in 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 which inhibits virus replication early during reverse transcription. REAF is degraded by Vpr within 30 minutes of virus entry, in a manner dependent on the nuclear localization of Vpr and its interaction with the cell’s protein degradation machinery.

Phosphatase PP2A expression levels are positively correlated to the clinical severity of systemic lupus erythematosus (SLE) and IL17A cytokine overproduction, indicating a potential role of PP2A in controlling T H 17 differentiation and inflammation. By generating a mouse strain with ablation of the catalytic subunit α of PP2A in peripheral mature T cells (PP2A cKO), we demonstrate that the PP2A complex is essential for T H 17 differentiation. These PP2A cKO mice had reduced T H 17 cell numbers and less severe disease in an experimental autoimmune encephalomyelitis (EAE) model. PP2A deficiency also ablated C-terminal phosphorylation of SMAD2 but increased C-terminal phosphorylation of SMAD3. By regulating the activity of RORγt via binding, the changes in the phosphorylation status of these R-SMADs reduced Il17a gene transcription. Finally, PP2A inhibitors showed similar effects on T H 17 cells as were observed in PP2A cKO mice, i.e., decreased T H 17 differentiation and relative protection of mice from EAE. Taken together, these data demonstrate that phosphatase PP2A is essential for T H 17 differentiation and that inhibition of PP2A could be a possible therapeutic approach to controlling T H 17-driven autoimmune diseases.

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.

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.

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.

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.

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.

Retroviruses are defined by the integration of their reverse-transcribed genome into host cell chromatin. This process enables transcription and translation of viral genes by host cells, ultimately resulting in new viral progeny. However, human immunodeficiency virus type 1 (HIV-1) gene transcription and translation can also occur prior to, or even in the absence of, viral integration (8, 53, 57), since unintegrated, reverse-transcribed viral cDNAs can also serve as a template for transcription (22). Three species of unintegrated HIV cDNAs are found in natural infections; these are linear reverse-transcribed cDNA, which is the template for integration, and 1-long terminal repeat (LTR) and 2-LTR circular forms, which are the products of autointegration or nonhomologous recombination and nonhomologous end-joining events of linear cDNAs, respectively (15, 26, 37). The circular cDNAs were long considered to be “dead end” products, which cannot serve as templates for integration, though it is now understood that unintegrated cDNA can be complemented by superinfecting virus to yield productive infection (17, 39, 53). Transcription of preintegrated HIV-1 cDNA can yield all classes of viral RNA transcripts (25, 37, 52); however, only the accessory and regulatory proteins Nef (18, 58), Tat (2, 14, 46), and Rev (22, 29) are translated in readily detectable amounts, and the full extent of the function of these proteins needs to be further characterized. Differences in transcription between integrated and unintegrated HIV-1 may be due to the fact that unintegrated HIV-1 cDNA is organized into chromatin structures, with histone modifications typical of silenced chromatin (23). Additionally, the low levels of Rev synthesized prior to integration may also limit the translation of unspliced viral RNA transcripts and ultimate expression of late gene products (58). Studies of integrase-defective HIV-1 mutants that bear mutations in the catalytic D(64)D(116)E(152) triad of integrase have been particularly useful in the study of preintegration transcription (18, 36). Indeed, patterns of transcription and translation arising from unintegrated DNA following infection with D116N mutated HIV-1 are identical to those observed from preintegrated viral DNA and in infections of T-cell lines, activated CD4+ T cells, resting T cells, and macrophages (25, 56, 58). Transcription and translation from unintegrated cDNA following use of integrase strand transfer inhibitors (INSTIs) are also indistinguishable from those seen with preintegrated virus or integrase-defective virus (21, 58). 2-LTR circles were previously proposed as a likely transcriptional template, as their levels were found to be elevated when viral integration was inhibited (14, 21). Moreover, a novel viral transcript spanning the LTR-LTR junction was detected, demonstrating that 2-LTR circles can act as a transcriptional template (7). In contrast, a recent study calculated that there were insufficient levels of 2-LTR circles to account for the numbers of cells bearing transcriptionally active preintegrated virus (22, 54, 55). Translation of nef, tat, and rev from preintegrated templates has been linked to a number of cellular effects which aid viral infection. For example, preintegration translation of tat and nef has been shown to increase the activation state of resting T cells, making them more amenable to productive infection (58). Preintegration translation of nef has been linked to reduced cell surface expression of CD4 in primary T cells and T cell lines (18, 36), and we have recently demonstrated that the CXCR4 and CCR5 coreceptors are also affected in this manner (45). In a study of macrophages, transcription of preintegrated HIV-1 cDNA was also linked to altered patterns of cytokine expression (25). CD8+ cytotoxic T-lymphocytes (CTLs) play a central role in the adaptive immune response to control HIV-1, as they recognize viral antigens presented through major histocompatibility complex class I (MHC-I) on infected cells and can limit infection either by direct lysis (5) or through release of inhibitory factors, such as RANTES, MIP-1 alpha, or MIP-1 beta (9). Therefore, modulation of cell surface expression of MHC-I is a common viral immune evasion strategy and avoids the presentation of viral antigens to CTLs, thereby preventing lysis of the infected cell (20). In the case of HIV-1, the virus-encoded protein Nef performs this function (11, 44). Nef downregulates MHC-I by forming a complex with the cytoplasmic tail of MHC-I and the clathrin adaptor AP-1 in the trans-Golgi network (TGN). This allows it to divert normal migration of newly synthesized MHC-I to the cell surface and instead targets it for endosomal degradation (28, 41, 47, 51). However, there is also some evidence that Nef may mediate accelerated endocytosis of MHC-I from the plasma membrane (28), and it has been suggested that cell type differences might also be important (24). HIV-1 Nef can mediate downregulation of the MHC-I/human leukocyte antigen (HLA) HLA-A and HLA-B allotypes, which are recognized by CTLs. In contrast, HIV-1 does not downregulate HLA-C and HLA-E, which is advantageous since a reduction in cell surface expression of these allotypes would lead to natural killer (NK) cell-mediated lysis, as NK cells respond to reduced MHC-I levels (10). We therefore hypothesized that the preintegration translation of nef has the capacity to modulate cell surface MHC-I expression. Here we show that preintegration transcription and translation of nef can modulate cell surface MHC-I in the same manner as when integration occurs. This suggests that transcription from preintegrated viral DNA can influence viral immune evasion even prior to viral integration into the host genome.

Feline immunodeficiency virus (FIV), an immunosuppressive lentivirus found in cats worldwide, is studied to illuminate mechanisms of lentiviral pathogenesis and to identify key components of protective immunity. During replication, lentiviruses accumulate errors of nucleotide mis-incorporation due to the low-fidelity of reverse transcriptase and recombination between viral variants, resulting in the emergence of a complex viral "quasispecies". In patients infected with HIV-1, env sequences may vary by up to 10% and the detection of quasispecies with greater heterogeneity is associated with higher viral loads and reduced CD4+ T cell numbers [1], indicating that transmission of more complex quasispecies may lead to disease progression. However, little is known about how FIV evolves as disease progresses, or why some cats develop AIDS rapidly while disease progression is slow in others. The aim of this study was to determine whether disease progression may be governed by viral evolution and to examine the diversity of viral variants emerging following infection with an infectious molecular clone. The FIV env gene encoding the envelope glycoprotein (Env) was examined at early (12 weeks) and late (322 weeks) stages of FIV infection in two groups of cats infected experimentally with the FIV-GL8 molecular clone. Viral variants were detected within quasispecies in cats in the late stages of FIV infection that contained differing amino acid compositions in several variable loops of Env, some of which were identified as determinants of receptor usage and resistance to neutralization. Therefore these results indicate that the FIV env gene evolves during the course of infection, giving rise to variants that resist neutralization and likely lead to disease progression.

Background: Increasing data shows the relevance of HBV genotypes in the outcome of infection. Most studies investigating the relationship between the genotypic characteristics of hepatitis B virus (HBV) and the clinical or epidemiological aspects of HBV infection originate from studies of patients with chronic rather than acute hepatitis B. Objectives: To study a convenience sample representing ca. 5% of reported acute hepatitis B in England between 1997 and 2001 to investigate the distribution of HBV genotypes and specific HBV variants with epidemiological risk factors, thereby providing baseline data for ongoing surveillance. Study design: From 160 serum samples, PCR was carried out to amplify the first 600 bases of the HBV S gene. Amplicons were sequenced and subjected to phylogenetic analysis and risk factor analysis. Results: Fifty-seven percent of the study samples carried HBV belonging to subtype A2, 13% to subtype D2, and the rest to genotype E (8%) and subtypes C2 and D3 (each 6%), D1 and D4 (each 3%) and B4 (1%). One particular A2 isolate was dominant, accounting for 23% of the total sample set. Drug use and homosexual transmission were equally implicated as risks within genotype A2. No mutations associated with vaccine escape or resistance to antiviral therapy were identified. Conclusion: Immigration and travel likely shape the observed genotype distribution and consequent prevalence of genotypes other than A2 or D in this population. Data suggests no genetic separation of parenteral and sexually transmitted virus. These data demonstrate the value in pursuing more extensive and recent surveillance. © 2008 Elsevier B.V. All rights reserved.

Conferences on Antiviral Research present a wealth of new information on novel drug development and the 21st annual meeting, held in Montreal between 13–17 April 2008, was no exception. The sessions topics included novel targets for HIV therapy, respiratory and emerging viruses, hepatitis viruses, clinical update on antiviral drugs, retroviruses, and herpes- and poxviruses, among others. In addition, there were a number of themed poster sessions dealing with each of the above areas, as well as novel methods for antiviral analysis, topical microbicides, medicinal chemistry, animal models, veterinary viruses and assorted viral agents. Each of these sessions were well organized and provoked thoughtful discussions. The session on novel targets for HIV therapy featured cutting-edge lectures on interactions between viral proteins and cellular factors. This work has the potential to lead to novel drug discovery as does that on peptide-based approaches to prevent replication of influenza viruses and the development of novel compounds that interfere with the neuraminidase enzymes of both influenza and parainfluenza viruses. Other areas of focus included novel approaches aimed at heightening immune effector mechanisms and the development of gene therapy strategies for certain types of viral infection. Of course, the development of compounds directed at numerous hepatitis C targets also featured prominently at the meeting.

Background The hepatitis B virus (HBV) pol gene overlaps the S gene encoding surface antigen (HBsAg). It has been reported previously that drug-induced changes in HBsAg alter its binding to sera from humans immunized against HBV. We investigate here the changes to specific epitopes in the a determinant (the major target of neutralizing antibody) caused by a number of drug-resistant mutations. Methods Recombinant HBsAgs, produced by transfection of Chinese hamster ovary cells with S gene plasmids into which lamivudine, adefovir and entecavir resistance and common antibody-escape mutations had been introduced, were probed with monoclonal antibodies to epitopes in the first and second loops of the a determinant. Results The mutations rtF166L/sF158Y (lamivudine-associated, compensatory) and rtI169T/sF161L (entecavir-associated, primary) acting alone, and the mutations rtV173L/sE164D (lamivudine-associated, compensatory) and rtSilent/sD144E (antibody escape-associated) each when combined with rtM204V/sI195M (lamivudine-associated, primary) led to decreases in antibody reactivity to epitopes in the first or second loop, or in both loops. The rtM204V/ sI195M+rtV173L/sE164D mutations yielded an epitope–antibody profile similar to the rtR153Q/sG145R vaccine escape mutant. The rtM204V/sI195M mutation combined with the rtF166L/sF158Y or rtR153Q/sG145R mutation restored reactivity to second-loop epitopes previously abrogated by single mutations. Conclusions Mutations associated with resistance to nucleos(t)ide analogue therapy, singly or in combination with each other or antibody escape-associated mutations, alter HBsAg immunoreactivity through concomitant amino acid substitutions at codons within and downstream of the a determinant. The findings have implications for understanding the native structure of HBsAg, optimizing treatment of chronic hepatitis B and evaluating the success of immunization programmes.

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.

The call for submissions at the first Frontiers of Retrovirology Conference was inclusive; all fields of retrovirology research were welcome and, as of such, participants presented on a wide range of topics; from endo-retroviruses through to HIV vaccines. With this broad palette, an enjoyable, concise exchange of thoughts and perspectives between different, though connected, fields of research was achieved. In this article, the range of topics addressed at this first meeting are discussed and we consider some of the major issues presented.

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-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.

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.

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.

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 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.

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.

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.

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 ≈13% of wt levels and increased resistance to MK-2048 to ≈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.

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.

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 providein vitroevidence 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.

retroviruses, retroelements and their hosts Meet ing abstracts - A single PDF containing all abstracts in this Supplement is available here . http://www. biomedcentral.co m/content/pdf/17 42-4690-6-S2-inf o.pdf

Methods We used both Jurkat and primary cell latency models. In Jurkat cells, wt and drug-resistant latent populations were superinfected with wt or drug-resistant viruses, including nef or tat mutants, and treated with various inhibitors. Viral reporter gene expression was measured by FACS. Sequence tags on each resistant virus permitted identification of recombinants. We also performed superinfection experiments in primary cells.

Background HIV-1 infection of CD4T-cells occurs through cell free infection or through cell-cell contacts. Cell-to-cell transmission is the predominant mode of viral spread in lymphoid tissue and cell culture. Cell free infection of HIV1 has recently been described to involve clathrinand dynamin-mediated endosomal uptake of virus. There are also accounts of cell-to-cell transfer of virus involving endosomal uptake, but the precise details of this process remain unclear. Here, we asked what forms of endocytosis might be involved in such cell-to-cell transmission.

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.

Purpose of the study: As a chemokine coreceptor antagonist, maraviroc (MVC) inhibits HIV infection by preventing viral entry into the host cell. As this effect is extracellular, we hypothesized that virus might be returned to plasma upon antagonism of CCR5 by MVC and that this thereby influences measurement of plasma viral load by qRT-PCR, in contrast to other drug classes that act intracellularly. Methods: PM-1 and TZM/bl cells were infected with titrations of a CCR5 tropic reference virus in the presence of inhibitory concentrations of MVC (500nM), efavirenz (EFV) (500nm), and raltegravir (RAL) (1µM). Viral inoculation varied between 104-107 copies of viral RNA/ml. Cells were centrifuged and supernatant viral load was measured by qRT-PCR for viral RNA at various times from shortly after infection through 48 hours after infection. Summary of results: At the highest inoculum used, the amounts of viral RNA detected in culture supernatants following treatment with MVC were consistently higher by ~ 0.5 log copies HIV-1 RNA/ml than found with untreated cells despite no detectable infection in the presence of MVC as revealed by production of p24 Ag in the case of PM-1 cells and expression of luciferase in the case of TZM/bl cells. At lower levels of inoculum, the results were consistent although less striking, but there was always a significant difference in regard to the use of MVC versus other drugs. In contrast, the results obtained with EFV and RAL resembled those obtained in the absence of drug, despite no detectable infection. Conclusions: These results suggest in a tissue culture model that MVC can return virus to plasma, where it may contribute to viral load despite complete inhibition of infection. In contrast, for drugs that act at an intracellular level, cells can absorb residual virus before such virus it is inhibited by the compound. Consequently, such virus will not contribute to viral load. In view of the fact that viral production by infected cells is an ongoing process, these findings imply that the true effectiveness of MVC is currently underestimated when viral load is used as a sole indicator of clinical success. These data may also be relevant for other classes of entry inhibitors. Supplement: Abstracts of the Tenth International Congress on Drug Therapy in HIV Infection http://www.biomedcentral.com/content/pdf/1758-2652-13-S4-info.pdf Conference: Tenth International Congress on Drug Therapy in HIV Infection 7-11 November 2010 Glasgow, UK (Published: 8 November 2010) doi:10.1186/1758-2652-13-S4-O46 Cite this article as: Sloan et al.: HIV entry blocked by maraviroc can cause an overestimation of viral load. Journal of the International AIDS Society 2010 13(Suppl 4):O46. Full text: PubMed Central: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3112861/

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.

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.

Background: Tetherin (BST-2/CD317/HM1.24) is an interferon-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 transmission between T cells can occur via cell-free transmission or the more efficient direct cell-cell route through virological synapses. Virological synapses are associated with lipid raft-rich microdomains in the membrane. Tetherin is known to localize to these microdomains and is capable of modulating actin architecture, which is crucial for viral entry, assembly and budding. Methods: We established a flow cytometry-based co-culture assay to distinguish viral transfer from viral transmission and investigated the impact of tetherin on cell-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. Results: We show that tetherin, in addition to inhibiting viral release, also inhibits direct cell-cell virus transfer and transmission. Viral Vpu promotes viral transmission from tetherin-expressing cells by down-modulating tetherin from the effector cell surface, outweighing its possible cost of fitness in cell-cell transmission. Further, we have shown that tetherin on the target cell promotes viral transfer and transmission in a manner that favours Vpu-deficient virus. Conclusions: The results suggest a role for tetherin in cell-cell contacts, specifically at virological synapses. Our data further contribute to the body of knowledge that the targeting of Vpu could be a valuable strategy aimed at blocking HIV replication. Supplement: Workshop Report and Abstracts “Towards a Cure”: HIV Reservoirs and Strategies to Control Them Shirin Heidari, Marie-Capucine Penicaud, Celine Cheng, International AIDS Society http://www.biomedcentral.com/content/pdf/1758-2652-13-S3-info.pdf Conference: International AIDS Society’s Workshop “Towards a Cure”: HIV Reservoirs and Strategies to Control Them 16–17 July 2010 Vienna, Austria (Published: 4 November 2010) doi:10.1186/1758-2652-13-S3-O8 Cite this article as: Kuhl et al.: Tetherin restricts direct cell-cell viral transfer and transmission of HIV-1. Journal of the International AIDS Society 2010 13(Suppl 3):O8. Full text: PubMed Central: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2999393/