http://www.retrovirology.com The most accessed research articles published by Retrovirology 2012-05-09T00:00:00Z The enzyme APOBEC3G (A3G) mutates the human immunodeficiency virus (HIV) genome by converting deoxycytidine (dC) to deoxyuridine (dU) on minus strand viral DNA during reverse transcription. A3G restricts viral propagation by degrading or incapacitating the coding ability of the HIV genome. Thus, this enzyme has been perceived as an innate immune barrier to viral replication whilst adaptive immunity responses escalate to effective levels. The discovery of A3G less than a decade ago led to the promise of new anti-viral therapies based on manipulation of its cellular expression and/or activity. The rationale for therapeutic approaches has been solidified by demonstration of the effectiveness of A3G in diminishing viral replication in cell culture systems of HIV infection, reports of its mutational footprint in virions from patients, and recognition of its unusually robust enzymatic potential in biochemical studies in vitro. Despite its effectiveness in various experimental systems, numerous recent studies have shown that the ability of A3G to combat HIV in the physiological setting is severely limited. In fact, it has become apparent that its mutational activity may actually enhance viral fitness by accelerating HIV evolution towards the evasion of both anti-viral drugs and the immune system. This body of work suggests that the role of A3G in HIV infection is more complex than heretofore appreciated and supports the hypothesis that HIV has evolved to exploit the action of this host factor. Here, we present an overview of recent data that bring to light historical overestimation of A3G's standing as a strictly anti-viral agent. We discuss the limitations of experimental systems used to assess its activities as well as caveats in data interpretation. http://www.retrovirology.com/content/9/1/35 Mahdis Monajemi Claire Woodworth Jessica Benkaroun Michael Grant Mani Larijani Retrovirology 2012, null:35 2012-04-30T00:00:00Z doi:10.1186/1742-4690-9-35 Despite its effectiveness in various experimental systems, numerous recent studies have shown that the ability of A3G to combat HIV in the physiological setting is severely limited. In fact, it has become apparent that A3G's mutational activity may actually enhance viral fitness by accelerating HIV evolution towards the evasion of both anti-viral drugs and the immune system. Here the authors present an overview of recent data that bring to light historical overestimation of A3G's standing as a strictly anti-viral agent. /content/figures/1742-4690-9-35-toc.gif Retrovirology 1742-4690 ${item.volume} 35 2012-04-30T00:00:00Z PDF Background: The Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF) region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region. Results: The dysfunctional HIV group O8 nef allele was restored to consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2) and non-canonical (B2 and C1422) HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF->AAA) and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef's association with the cellular p21-activated kinase 2 (PAK2), the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN) were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck. Conclusion: Based on these findings, we propose that this highly conserved three amino acid VGF motif together with the acidic cluster and the proline-rich motif form a previously unrecognized amphipathic surface on Nef. This surface appears to be essential for the majority of Nef functions and thus represents a prime target for the pharmacological inhibition of Nef. http://www.retrovirology.com/content/9/1/34 Pieter Meuwissen Bettina Stolp Veronica Iannucci Jolien Vermeire Evelien Naessens Kalle Saksela Matthias Geyer Guido Vanham Kevin Arien Oliver Fackler Bruno Verhasselt Retrovirology 2012, null:34 2012-04-27T00:00:00Z doi:10.1186/1742-4690-9-34 This study describes a highly conserved three amino acid VGF motif that together with the acidic cluster and the proline-rich motif form a previously unrecognized amphipathic surface on Nef. This surface appears to be essential for the majority of Nef functions and thus represents a prime target for the pharmacological inhibition of Nef. /content/figures/1742-4690-9-34-toc.gif Retrovirology 1742-4690 ${item.volume} 34 2012-04-27T00:00:00Z PDF Background: Disassembly of the viral capsid following penetration into the cytoplasm, or uncoating, is a poorly understood stage of retrovirus infection. Based on previous studies of HIV-1 CA mutants exhibiting altered capsid stability, we concluded that formation of a capsid of optimal intrinsic stability is crucial for HIV-1 infection. Results: To further examine the connection between HIV-1 capsid stability and infectivity, we isolated second-site suppressors of HIV-1 mutants exhibiting unstable (P38A) or hyperstable (E45A) capsids. We identified the respective suppressor mutations, T216I and R132T, which restored virus replication in a human T cell line and markedly enhanced the fitness of the original mutants as revealed in single-cycle infection assays. Analysis of the corresponding purified N-terminal domain CA proteins by NMR spectroscopy demonstrated that the E45A and R132T mutations induced structural changes that are localized to the regions of the mutations, while the P38A mutation resulted in changes extending to neighboring regions in space. Unexpectedly, neither suppressor mutation corrected the intrinsic viral capsid stability defect associated with the respective original mutation. Nonetheless, the R132T mutation rescued the selective infectivity impairment exhibited by the E45A mutant in aphidicolin-arrested cells, and the double mutant regained sensitivity to the small molecule inhibitor PF74. The T216I mutation rescued the impaired ability of the P38A mutant virus to abrogate restriction by TRIMCyp and TRIM5α. Conclusions: The second-site suppressor mutations in CA that we have identified rescue virus infection without correcting the intrinsic capsid stability defects associated with the P38A and E45A mutations. The suppressors also restored wild type virus function in several cell-based assays. We propose that while proper HIV-1 uncoating in target cells is dependent on the intrinsic stability of the viral capsid, the effects of stability-altering mutations can be mitigated by additional mutations that affect interactions with host factors in target cells or the consequences of these interactions. The ability of mutations at other CA surfaces to compensate for effects at the NTD-NTD interface further indicates that uncoating in target cells is controlled by multiple intersubunit interfaces in the viral capsid. http://www.retrovirology.com/content/9/1/30 Ruifeng Yang Jiong Shi In-Ja Byeon Jinwoo Ahn Jonathan Sheehan Jens Meiler Angela Gronenborn Christopher Aiken Retrovirology 2012, null:30 2012-04-19T00:00:00Z doi:10.1186/1742-4690-9-30 The second-site suppressor mutations in CA identified here rescue virus infection without correcting the intrinsic capsid stability defects associated with the P38A and E45A mutations. The suppressors also restored wild type virus function in several cell-based assays. Thus, while proper HIV-1 uncoating in target cells is dependent on the intrinsic stability of the viral capsid, the effects of stability-altering mutations can be mitigated by additional mutations that affect interactions with host factors in target cells or the consequences of these interactions. /content/figures/1742-4690-9-30-toc.gif Retrovirology 1742-4690 ${item.volume} 30 2012-04-19T00:00:00Z XML Background: HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. Results: To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3). We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6), an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Conclusions: Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of exocyst involvement in polarized targeting for intercellular transfer of viral proteins and viruses. http://www.retrovirology.com/content/9/1/33 Joya Mukerji Kevin Olivieri Vikas Misra Kristin Agopian Dana Gabuzda Retrovirology 2012, null:33 2012-04-25T00:00:00Z doi:10.1186/1742-4690-9-33 To identify Nef binding partners involved in Pak2-association dependent Nef functions, the authors employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A74P in NL4-3). They report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6), an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. /content/figures/1742-4690-9-33-toc.gif Retrovirology 1742-4690 ${item.volume} 33 2012-04-25T00:00:00Z PDF Background: Recent evidence proposes a novel concept that mammalian natural antisense RNAs play important roles in cellular homeostasis by regulating the expression of several genes. Identification and characterization of retroviral antisense RNA would provide new insights into mechanisms of replication and pathogenicity. HIV-1 encoded-antisense RNAs have been reported, whereas the accurate structures and functions remain to be studied. Thus, we tried to identify and characterize antisense RNAs of HIV-1 and studied their function in viral infection. Results: Characterization of transcripts of HEK293T cells that were transiently transfected with an expression plasmid with HIV-1NL4-3 DNA in the antisense orientation showed that various antisense transcripts can be expressed. By screening and characterization of antisense RNAs in HIV-1NL4-3-infected cells, we defined the primary structure of a major form of HIV-1 antisense RNAs, which corresponds to a variant of previously reported ASP mRNA. This 2.6kb RNA was transcribed from the U3 region of the 3' LTR and terminated at the env region in acutely or chronically infected cell lines and acutely infected human peripheral blood mononuclear cells. Reporter assays clearly demonstrated that the HIV-1 LTR harbours promoter activity in the reverse orientation. Mutation analyses suggested involvement of NF- binding sites in the regulation of antisense transcription. The antisense RNA was localized in the nuclei of the infected cells. Expression of the antisense RNA suppressed HIV-1 replication for more than one month. Furthermore, specific knockdown of the antisense RNA resulted in an enhancement of HIV-1 gene expression and replication. Conclusions: The results of the present study identified an accurate structure of the major form of antisense RNAs expressed from the HIV-1NL4-3 provirus and demonstrated its nuclear localization. Functional studies collectively demonstrated a new role of the antisense RNA in the viral replication, thus we suggest a novel viral mechanism that regulates self-limiting replication of HIV-1, providing a new insight into viral life cycles. http://www.retrovirology.com/content/9/1/38 Mie Kobayashi-Ishihara Makoto Yamagishi Takuma Hara Yuka Matsuda Ryutaro Takahashi Ariko Miyake Kazumi Nakano Tadanori Yamochi Takaomi Ishida Toshiki Watanabe Retrovirology 2012, null:38 2012-05-08T00:00:00Z doi:10.1186/1742-4690-9-38 The present study identified an accurate structure of the major form of antisense RNAs expressed from the HIV-1NL4-3 provirus and demonstrated a new role of the antisense RNA in viral replication, suggesting a novel viral mechanism that regulates self-limited replication of HIV-1. /content/figures/1742-4690-9-38-toc.gif Retrovirology 1742-4690 ${item.volume} 38 2012-05-08T00:00:00Z PDF Background: Macrophages, which are CD4 and CCR5 positive, can sustain HIV-1 replication for long periods of time. Thus, these cells play critical roles in the transmission, dissemination and persistence of viral infection. Of note, current antiviral therapies do not target macrophages efficiently. Previously, it was demonstrated that interactions between CCR5 and gp120 stimulate PKC. However, the PKC isozymes involved were not identified. Results: In this study, we identified PKC-delta as a major cellular cofactor for HIV-1 replication in macrophages. Indeed, PKC-delta was stimulated following the interaction between the virus and its target cell. Moreover, inhibition of PKC-delta blocked the replication of R5-tropic viruses in primary human macrophages. However, this inhibition did not have significant effects on receptor and co-receptor expression or fusion. Additionally, it did not affect the formation of the early reverse transcription product containing R/U5 sequences, but did inhibit the synthesis of subsequent cDNAs. Importantly, the inhibition of PKC-delta altered the redistribution of actin, a cellular cofactor whose requirement for the completion of reverse transcription was previously established. It also prevented the association of the reverse transcription complex with the cytoskeleton. Conclusion: This work highlights the importance of PKC-delta during early steps of the replicative cycle of HIV-1 in human macrophages. http://www.retrovirology.com/content/9/1/37 Xavier Contreras Olfa Mzoughi Fabrice Gaston B Matija Peterlin Elmostafa Bahraoui Retrovirology 2012, null:37 2012-05-03T00:00:00Z doi:10.1186/1742-4690-9-37 This study identified PKC-delta as a major cellular cofactor for HIV-1 replication in macrophages. PKC-delta was stimulated following the interaction between the virus and its target cell. Inhibition of PKC-delta blocked the replication of R5-tropic viruses in primary human macrophages. /content/figures/1742-4690-9-37-toc.gif Retrovirology 1742-4690 ${item.volume} 37 2012-05-03T00:00:00Z PDF In the mid-1990's, researchers hypothesized, based on new viral load data, that HIV-1 causes CD4+ T-cell depletion by direct cytopathic effect. New data from non-human primate studies has raised doubts about this model of HIV-1 pathogenesis. Despite having high levels of viremia, most SIV infections are well tolerated by their natural hosts. Two recent studies of these models provide information, which may be useful in determining how HIV-1 causes CD4+ T-cell loss. A full understanding of pathogenesis may lead to novel therapies, which preserve the immune system without blocking virus replication. http://www.retrovirology.com/content/3/1/60 Stephen Smith Retrovirology 2006, null:60 2006-09-08T00:00:00Z doi:10.1186/1742-4690-3-60 /content/figures/1742-4690-3-60-toc.gif Retrovirology 1742-4690 ${item.volume} 60 2006-09-08T00:00:00Z XML When endogenous retroviruses (ERV) were discovered in the late 1960s, the Mendelian inheritance of retroviral genomes by their hosts was an entirely new concept. Indeed Howard M Temin's DNA provirus hypothesis enunciated in 1964 was not generally accepted, and reverse transcriptase was yet to be discovered. Nonetheless, the evidence that we accrued in the pre-molecular era has stood the test of time, and our hypothesis on ERV, which one reviewer described as 'impossible', proved to be correct. Here I recount some of the key observations in birds and mammals that led to the discovery of ERV, and comment on their evolution, cross-species dispersion, and what remains to be elucidated. http://www.retrovirology.com/content/3/1/67 Robin Weiss Retrovirology 2006, null:67 2006-10-03T00:00:00Z doi:10.1186/1742-4690-3-67 /content/figures/1742-4690-3-67-toc.gif Retrovirology 1742-4690 ${item.volume} 67 2006-10-03T00:00:00Z XML Background: Human T-cell leukemia virus type 1 (HTLV-1) and type 2 both target T lymphocytes, yet induce radically different phenotypic outcomes. HTLV-1 is a causative agent of Adult T-cell leukemia (ATL), whereas HTLV-2, highly similar to HTLV-1, causes no known overt disease. HTLV gene products are engaged in a dynamic struggle of activating and antagonistic interactions with host cells. Investigations focused on one or a few genes have identified several human factors interacting with HTLV viral proteins. Most of the available interaction data concern the highly investigated HTLV-1 Tax protein. Identifying shared and distinct host-pathogen protein interaction profiles for these two viruses would enlighten how they exploit distinctive or common strategies to subvert cellular pathways toward disease progression. Results: We employ a scalable methodology for the systematic mapping and comparison of pathogen-host protein interactions that includes stringent yeast two-hybrid screening and systematic retest, as well as two independent validations through an additional protein interaction detection method and a functional transactivation assay. The final data set contained 166 interactions between 10 viral proteins and 122 human proteins. Among the 166 interactions identified, 87 and 79 involved HTLV-1 and HTLV-2 -encoded proteins, respectively. Targets for HTLV-1 and HTLV-2 proteins implicate a diverse set of cellular processes including the ubiquitin-proteasome system, the apoptosis, different cancer pathways and the Notch signaling pathway. Conclusions: This study constitutes a first pass, with homogeneous data, at comparative analysis of host targets for HTLV-1 and -2 retroviruses, complements currently existing data for formulation of systems biology models of retroviral induced diseases and presents new insights on biological pathways involved in retroviral infection. http://www.retrovirology.com/content/9/1/26 Nicolas Simonis Jean-Francois Rual Irma Lemmens Mathieu Boxus Tomoko Hirozane-Kishikawa Jean-Stephane Gatot Amelie Dricot Tong Hao Didier Vertommen Sebastien Legros Sarah Daakour Niels Klitgord Maud Martin Jean-Francois Willaert Franck Dequiedt Vincent Navratil Michael Cusick Arsene Burny Carine Van Lint David Hill Jan Tavernier Richard Kettmann Marc Vidal Jean-Claude Twizere Retrovirology 2012, null:26 2012-03-29T00:00:00Z doi:10.1186/1742-4690-9-26 Host-pathogen interactome mapping for HTLV-1 and -2 retroviruses This work employed a scalable methodology for the systematic mapping and comparison of pathogen-host protein interactions that includes stringent yeast two-hybrid screening and systematic retest, as well as two independent validations through an additional protein interaction detection method and a functional transactivation assay. The authors found 166 interactions between 10 viral proteins and 122 human proteins. /content/figures/1742-4690-9-26-toc.gif Retrovirology 1742-4690 ${item.volume} 26 2012-03-29T00:00:00Z XML The major histocompatibility complex class I protein HLA-C plays a crucial role as a molecule capable of sending inhibitory signals to both natural killer (NK) cells and cytotoxic T lymphocytes (CTL) via binding to killer cell Ig-like receptors (KIR). Recently HLA-C has been recognized as a key molecule in the immune control of HIV-1. Expression of HLA-C is modulated by a microRNA binding site. HLA-C alleles that bear substitutions in the microRNA binding site are more expressed at the cell surface and associated with the control of HIV-1 viral load, suggesting a role of HLA-C in the presentation of antigenic peptides to CTLs. This review highlights the role of HLA-C in association with HIV-1 viral load, but also addresses the contradiction of the association between high cell surface expression of an inhibitory molecule and strong cell-mediated immunity. To explore additional mechanisms of control of HIV-1 replication by HLA-C, we address specific features of the molecule, like its tendency to be expressed as open conformer upon cell activation, which endows it with a unique capacity to associate with other cell surface molecules as well as with HIV-1 proteins. http://www.retrovirology.com/content/9/1/39 Donato Zipeto Alberto Beretta Retrovirology 2012, null:39 2012-05-09T00:00:00Z doi:10.1186/1742-4690-9-39 This review highlights the role of HLA-C in association with HIV-1 viral load, but also addresses the contradiction of the association between high cell surface expression of an inhibitory molecule and strong cell-mediated immunity. To explore additional mechanisms of control of HIV-1 replication by HLA-C, this review addresses specific features of the molecule, like its tendency to be expressed as open conformer upon cell activation, which endows it with a unique capacity to associate with other cell surface molecules as well as with HIV-1 proteins. /content/figures/1742-4690-9-39-toc.gif Retrovirology 1742-4690 ${item.volume} 39 2012-05-09T00:00:00Z PDF