http://www.retrovirology.com The latest research articles published by Retrovirology 2013-05-20T00:00:00Z Background: Human T-cell leukemia virus type-1 (HTLV-1) is the causative retrovirus of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 gene expression is maintained at low levels in vivo by unknown mechanisms. A combination therapy of interferon-alpha (IFN-alpha) and zidovudin (AZT) shows therapeutic effects in ATL patients, although its mechanism is also obscure. We previously found that viral gene expression in IL-2-dependent HTLV-1-infected T-cells (ILTs) derived from ATL patients was markedly suppressed by stromal cells through a type I IFN response. Here, we investigated the effects of IFN-alpha with or without AZT on viral gene expression and cell growth in ILTs. Results: ILTs expressed variable but lower amounts of HTLV-1 Tax protein than HTLV-1-transformed HUT102 cells. Following the addition of IFN-alpha, the amounts of HTLV-1 p19 in the supernatants of these cells decreased in three days, while HTLV-1 gene expression decreased only in ILTs but not HUT102 cells. IFN-alpha also suppressed the spontaneous HTLV-1 induction in primary ATL cells cultured for 24 h. A time course study using ILTs revealed that the levels of intracellular Tax proteins decreased in the first 24 h after addition of IFN-alpha, before the reduction in HTLV-1 mRNA levels. The initial decreases of Tax protein following IFN-alpha treatment were observed in 6 of 7 ILT lines tested, although the reduction rates varied among ILT lines. An RNA-dependent protein kinase (PKR)-inhibitor reversed IFN-mediated suppression of Tax in ILTs. IFN-alpha also induced cell cycle arrest at the G0/G1 phase and suppressed NF-kappaB activities in these cells. AZT alone did not affect HTLV-1 gene expression, cell viability or NF-kappaB activities. AZT combined with IFN-alpha markedly induced cell apoptosis associated with phosphorylation of p53 and induction of p53-responsive genes in ILTs. Conclusions: IFN-alpha suppressed HTLV-1 gene expression at least through a PKR-mediated mechanism, and also induced cell cycle arrest in ILTs. In combination with AZT, IFN-alpha further induced p53 signaling and cell apoptosis in these cells. These findings suggest that HTLV-1-infected cells at an IL-2-dependent stage retain susceptibility to type I IFN-mediated regulation of viral expression, and partly explain how AZT/IFN-alpha produces therapeutic effects in ATL. http://www.retrovirology.com/content/10/1/52 Shuichi Kinpara Mami Kijiyama Ayako Takamori Atsuhiko Hasegawa Amane Sasada Takao Masuda Yuetsu Tanaka Atae Utsunomiya Mari Kannagi Retrovirology 2013, null:52 2013-05-20T00:00:00Z doi:10.1186/1742-4690-10-52 /content/figures/1742-4690-10-52-toc.gif Retrovirology 1742-4690 ${item.volume} 52 2013-05-20T00:00:00Z PDF Background: OX40 is a member of the tumor necrosis factor receptor family that is expressed primarily on activated CD4+ T cells and promotes the development of effector and memory T cells. Although OX40 has been reported to be a target gene of human T-cell leukemia virus type-1 (HTLV-1) viral transactivator Tax and is overexpressed in vivo in adult T-cell leukemia (ATL) cells, an association between OX40 and HTLV-1-associated inflammatory disorders, such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), has not yet been established. Moreover, because abrogation of OX40 signals ameliorates chronic inflammation in animal models of autoimmune disease, novel monoclonal antibodies against OX40 may offer a potential treatment for HTLV-1-associated diseases such as ATL and HAM/TSP. Results: In this study, we showed that OX40 was specifically expressed in CD4+ T cells naturally infected with HTLV-1 that have the potential to produce pro-inflammatory cytokines along with Tax expression. We also showed that OX40 was overexpressed in spinal cord infiltrating mononuclear cells in a clinically progressive HAM/TSP patient with a short duration of illness. The levels of the soluble form of OX40 (sOX40) in the cerebrospinal fluid (CSF) from chronic progressive HAM/TSP patients or from patients with other inflammatory neurological diseases (OINDs) were not different. In contrast, sOX40 levels in the CSF of rapidly progressing HAM/TSP patients were higher than those in the CSF from patients with OINDs, and these patients showed higher sOX40 levels in the CSF than in the plasma. When our newly produced monoclonal antibody against OX40 was added to peripheral blood mononuclear cells in culture, HTLV-1-infected T cells were specifically removed by a mechanism that depends on antibody-dependent cellular cytotoxicity. Conclusions: Our study identified OX40 as a key molecule and biomarker for rapid progression of HAM/TSP. Furthermore, blocking OX40 may have potential in therapeutic intervention for HAM/TSP. http://www.retrovirology.com/content/10/1/51 Mineki Saito Reiko Tanaka Shiho Arishima Toshio Matsuzaki Satoshi Ishihara Takashi Tokashiki Yusuke Ohya Hiroshi Takashima Fujio Umehara Shuji Izumo Yuetsu Tanaka Retrovirology 2013, null:51 2013-05-07T00:00:00Z doi:10.1186/1742-4690-10-51 /content/figures/1742-4690-10-51-toc.gif Retrovirology 1742-4690 ${item.volume} 51 2013-05-07T00:00:00Z XML Background: HIV-2, which was transmitted to humans from a distant primate species (sooty mangabey), differs remarkably from HIV-1 in its infectivity, transmissibility and pathogenicity. We have tested the possibility that a greater susceptibility of HIV-2 capsid (CA) to the human restriction factor TRIM5alpha (hTRIM5alpha) could contribute to these differences. Results: We constructed recombinant clones expressing CA from a variety of HIV-2 viruses in the context of HIV-1 NL4-3-luciferase. CA sequences were amplified from the plasma of HIV-2 infected patients, including 8 subtype A and 7 subtype B viruses. CA from 6 non-epidemic HIV-2 subtypes, 3 HIV-2 CRF01_AB recombinants and 4 SIVsmm viruses were also tested. Susceptibility to hTRIM5alpha was measured by comparing single-cycle infectivity in human target cells expressing hTRIM5alpha to that measured in cells in which hTRIM5alpha activity was inhibited by overexpression of hTRIM5gamma.The insertion of HIV-2 CA sequences in the context of HIV-1 did not affect expression and maturation of the HIV-2 CA protein. The level of susceptibility hTRIM5alpha expressed by viruses carrying HIV-2 CA sequences was up to 9-fold higher than that of HIV-1 NL4-3 and markedly higher than a panel of primary HIV-1 CA sequences. This phenotype was found both for viruses carrying CA from primary HIV-2 sequences and viruses carrying CA from laboratory-adapted HIV-2 clones. High hTRIM5alpha susceptibility was found in all HIV-2 subtypes. In this series of viruses, susceptibility to hTRIM5alpha was not significantly affected by the presence of a proline at position 119 or by the number of prolines at positions 119, 159 or 178 in HIV-2 CA. No significant correlation was found between HIV-2 viremia and sensitivity to hTRIM5alpha. Conclusions: HIV-2 capsid sequences expressed high levels of susceptibility to hTRIM5alpha. This property, common to all HIV-2 sequences tested, may contribute in part to the lower replication and pathogenicity of this virus in humans. http://www.retrovirology.com/content/10/1/50 Junko Takeuchi Benjamin Perche Julie Migraine Séverine Mercier-Delarue Diane Ponscarme François Simon François Clavel Béatrice Labrosse Retrovirology 2013, null:50 2013-05-06T00:00:00Z doi:10.1186/1742-4690-10-50 /content/figures/1742-4690-10-50-toc.gif Retrovirology 1742-4690 ${item.volume} 50 2013-05-06T00:00:00Z PDF Background: HIV evolves rapidly at the epidemiological level but also at the within-host level. The virus' within-host evolutionary rates have been argued to be much higher than its between-host evolutionary rates. However, this conclusion relies on analyses of a short portion of the virus envelope gene. Here, we study in detail these evolutionary rates across the HIV genome. Results: We build phylogenies using a relaxed molecular clock assumption to estimate evolutionary rates in different regions of the HIV genome. We find that these rates vary strongly across the HIV genome, with higher rates in the envelope gene (env). Within-host evolutionary rates are consistently higher than between-host rates throughout the HIV genome. This difference is significantly more pronounced in env. Finally, we find weak differences between overlapping and non-overlapping regions. Conclusions: We provide a genome-wide overview of the differences in the HIV rates of molecular evolution at the within- and between-host levels. Contrary to hepatitis C virus, where differences are only located in the envelope gene, within-host evolutionary rates are higher than between-host evolutionary rates across the whole HIV genome. This supports the hypothesis that HIV strains that are less adapted to the host have an advantage during transmission. The most likely mechanism for this is storage and then preferential transmission of viruses in latent T-cells. These results shed a new light on the role of the transmission bottleneck in the evolutionary dynamics of HIV. http://www.retrovirology.com/content/10/1/49 Samuel Alizon Christophe Fraser Retrovirology 2013, null:49 2013-05-02T00:00:00Z doi:10.1186/1742-4690-10-49 /content/figures/1742-4690-10-49-toc.gif Retrovirology 1742-4690 ${item.volume} 49 2013-05-02T00:00:00Z PDF Background: Platelets, anucleate cell fragments abundant in human blood, can capture HIV-1 and platelet counts have been associated with viral load and disease progression. However, the impact of platelets on HIV-1 infection of T cells is unclear. Results: We found that platelets suppress HIV-1 spread in co-cultured T cells in a concentration-dependent manner. Platelets containing granules inhibited HIV-1 spread in T cells more efficiently than degranulated platelets, indicating that the granule content might exert antiviral activity. Indeed, supernatants from activated and thus degranulated platelets suppressed HIV-1 infection. Infection was inhibited at the stage of host cell entry and inhibition was independent of the viral strain or coreceptor tropism. In contrast, blockade of HIV-2 and SIV entry was less efficient. The chemokine CXCL4, a major component of platelet granules, blocked HIV-1 entry and neutralization of CXCL4 in platelet supernatants largely abrogated their anti-HIV-1 activity. Conclusions: Release of CXCL4 by activated platelets inhibits HIV-1 infection of adjacent T cells at the stage of virus entry. The inhibitory activity of platelet-derived CXCL4 suggests a role of platelets in the defense against infection by HIV-1 and potentially other pathogens. http://www.retrovirology.com/content/10/1/48 Theodros Solomon Tsegaye Kerstin Gnirß Niels Rahe-Meyer Miriam Kiene Annika Krämer-Kühl Georg Behrens Jan Münch Stefan Pöhlmann Retrovirology 2013, null:48 2013-05-01T00:00:00Z doi:10.1186/1742-4690-10-48 /content/figures/1742-4690-10-48-toc.gif Retrovirology 1742-4690 ${item.volume} 48 2013-05-01T00:00:00Z XML Background: Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and tropical spastic paraparesis. HTLV-1 encodes transactivator protein Tax that interacts with various cellular factors to modulate transcription and other biological functions. Additional cellular mediators of Tax-mediated transcriptional activation of HTLV-1 long terminal repeats (LTR) remain to be identified and characterized. Results: In this study, we investigated the regulatory role of group I p21-activated kinases (Paks) in Tax-induced LTR activation. Both wild-type and kinase-dead mutants of Pak3 were capable of potentiating the activity of Tax to activate LTR transcription. The effect of Paks on the LTR was attributed to the N-terminal regulatory domain and required the action of CREB, CREB-regulating transcriptional coactivators (CRTCs) and p300/CREB-binding protein. Paks physically associated with Tax and CRTCs. Paks were recruited to the LTR in the presence of Tax. siRNAs against either Pak1 or Pak3 prevented the interaction of Tax with CRTC1 and the recruitment of Tax to the LTR. These siRNAs also inhibited LTR-dependent transcription in HTLV-1-transformed MT4 cells and in cells transfected with an infectious clone of HTLV-1. Conclusion: Group I Paks augment Tax-mediated transcriptional activation of HTLV-1 LTR in a kinase-independent manner. http://www.retrovirology.com/content/10/1/47 Ching-Ping Chan Yeung-Tung Siu Kin-Hang Kok Yick-Pang Ching Hei-Man Tang Dong-Yan Jin Retrovirology 2013, null:47 2013-04-26T00:00:00Z doi:10.1186/1742-4690-10-47 /content/figures/1742-4690-10-47-toc.gif Retrovirology 1742-4690 ${item.volume} 47 2013-04-26T00:00:00Z XML Background: Expression of the cellular karyopherin TNPO3/transportin-SR2/Tnp3 is necessary for HIV-1 infection. Depletion of TNPO3 expression in mammalian cells inhibits HIV-1 infection after reverse transcription but prior to integration. Results: This work explores the role of cleavage and polyadenylation specificity factor subunit 6 (CPSF6) in the ability of TNPO3-depleted cells to inhibit HIV-1 infection. Our findings showed that depletion of TNPO3 expression inhibits HIV-1 infection, while the simultaneous depletion of TNPO3 and CPSF6 expression rescues HIV-1 infection. Several experiments to understand the rescue of infectivity by CPSF6 were performed. Our experiments revealed that the HIV-1 capsid binding ability of the endogenously expressed CPSF6 from TNPO3-depleted cells does not change when compared to CPSF6 from wild type cells. In agreement with our previous results, depletion of TNPO3 did not change the nuclear localization of CPSF6. Studies on the formation of 2-LRT circles during HIV-1 infection revealed that TNPO3-depleted cells are impaired in the integration process or exhibit a defect in the formation of 2-LTR circles. To understand whether the cytosolic fraction of CPSF6 is responsible for the inhibition of HIV-1 in TNPO3-depleted cells, we tested the ability of a cytosolic full-length CPSF6 to block HIV-1 infection. These results demonstrated that overexpression of a cytosolic full-length CPSF6 blocks HIV-1 infection at the nuclear import step. Fate of the capsid assays revealed that cytosolic expression of CPSF6 enhances stability of the HIV-1 core during infection. Conclusions: These results suggested that inhibition of HIV-1 by TNPO3-depleted cells requires CPSF6. http://www.retrovirology.com/content/10/1/46 Thomas Fricke Jose Valle-Casuso Tommy White Alberto Brandariz-Nuñez William Bosche Natalia Reszka Robert Gorelick Felipe Diaz-Griffero Retrovirology 2013, null:46 2013-04-26T00:00:00Z doi:10.1186/1742-4690-10-46 /content/figures/1742-4690-10-46-toc.gif Retrovirology 1742-4690 ${item.volume} 46 2013-04-26T00:00:00Z PDF Background: Foamy viruses (FVs) have developed a unique budding strategy within the retrovirus family. FV release requires co-expression and a highly specific interaction between capsid (Gag) and glycoprotein (Env), which cannot be complemented by heterologous Env proteins. The interaction domain in FV Env has been mapped in greater detail and resides mainly in the N-terminal tip of the cytoplasmic domain of the Env leader peptide subunit. In contrast, the corresponding domain within Gag is less well defined. Previous investigations suggest that it is located within the N-terminal part of the protein. Results: Here we characterized additional Gag interaction determinants of the prototype FV (PFV) isolate using a combination of particle release, GST pull-down and single cycle infectivity analysis assays. Our results demonstrate that a minimal PFV Gag protein comprising the N-terminal 129 aa was released into the supernatant, whereas proteins lacking this domain failed to do so. Fine mapping of domains within the N-terminus of PFV Gag revealed that the N-terminal 10 aa of PFV Gag were dispensable for viral replication. In contrast, larger deletions or structurally deleterious point mutations in C-terminally adjacent sequences predicted to harbor a helical region abolished particle egress and Gag -- Env protein interaction. Pull-down assays, using proteins of mammalian and prokaryotic origin, support the previous hypothesis of a direct interaction of both PFV proteins without requirement for cellular cofactors and suggest a potential direct contact of Env through this N-terminal Gag domain. Furthermore, analysis of point mutants within this domain in context of PFV vector particles indicates additional particle release-independent functions for this structure in viral replication by directly affecting virion infectivity. Conclusions: Thus, our results demonstrate not only a critical function of an N-terminal PFV Gag motif for the essential capsid - glycoprotein interaction required for virus budding but also point out additional functions that affect virion infectivity. http://www.retrovirology.com/content/10/1/45 Juliane Reh Annett Stange Anne Götz Marlene Rönitz Arend Große Dirk Lindemann Retrovirology 2013, null:45 2013-04-25T00:00:00Z doi:10.1186/1742-4690-10-45 /content/figures/1742-4690-10-45-toc.gif Retrovirology 1742-4690 ${item.volume} 45 2013-04-25T00:00:00Z PDF Background: The disulfide-bonded region (DSR) of HIV-1 gp41 mediates association with gp120 and plays a role in transmission of receptor-induced conformational changes in gp120 to gp41 that activate membrane fusion function. In this study, forced viral evolution of a DSR mutant that sheds gp120 was employed to identify domains within gp120-gp41 that are functionally linked to the glycoprotein association site. Results: The HIV-1AD8 mutant, W596L/K601D, was serially passaged in U87.CD4.CCR5 cells until replication was restored. Whereas the W596L mutation persisted throughout the cultures, a D601H pseudoreversion in the DSR partially restored cell-free virus infectivity and virion gp120-gp41 association, with further improvements to cell-free virus infectivity following a 2nd-site D674E mutation in the membrane-proximal external region (MPER) of gp41. In an independent culture, D601H appeared with a deletion in V4 (Thr-394-Trp-395) and a D674N substitution in the MPER, however this MPER mutation was inhibitory to W596L/K601H cell-free virus infectivity. While cell-free virus infectivity was not fully restored for the revertant genotypes, their cell-to-cell transmission approached the levels observed for WT. Interestingly, the functional boost associated with the addition of D674E to W596L/K601H was not observed for cell-cell fusion where the cell-surface expressed glycoproteins function independently of virion assembly. The W596L/K601H and W596L/K601H/D674E viruses exhibited greater sensitivity to neutralization by the broadly reactive MPER directed monoclonal antibodies, 2F5 and 4E10, indicating that the reverting mutations increase the availability of conserved neutralization epitopes in the MPER. Conclusions: The data indicate for the first time that functional crosstalk between the DSR and MPER operates in the context of assembled virions, with the Leu-596-His-601-Glu-674 combination optimizing viral spread via the cell-to-cell route. Our data also indicate that changes in the gp120-gp41 association site may increase the exposure of conserved MPER neutralization epitopes in virus. http://www.retrovirology.com/content/10/1/44 Ashraf Khasawneh Annemarie Laumaea David Harrison Anna Bellamy-McIntyre Heidi Drummer Pantelis Poumbourios Retrovirology 2013, null:44 2013-04-23T00:00:00Z doi:10.1186/1742-4690-10-44 /content/figures/1742-4690-10-44-toc.gif Retrovirology 1742-4690 ${item.volume} 44 2013-04-23T00:00:00Z XML Background: The CCR5 antagonist maraviroc (MVC) inhibits human immunodeficiency virus type 1 (HIV-1) entry by altering the CCR5 extracellular loops (ECL), such that the gp120 envelope glycoproteins (Env) no longer recognize CCR5. The mechanisms of HIV-1 resistance to MVC, the only CCR5 antagonist licensed for clinical use are poorly understood, with insights into MVC resistance almost exclusively limited to knowledge obtained from in vitro studies or from studies of resistance to other CCR5 antagonists. To more precisely understand mechanisms of resistance to MVC in vivo, we characterized Envs isolated from 2 subjects who experienced virologic failure on MVC. Results: Envs were cloned from subjects 17 and 24 before commencement of MVC (17-Sens and 24-Sens) and after virologic failure (17-Res and 24-Res). The Envs cloned during virologic failure showed broad divergence in resistance levels, with 17-Res Env exhibiting a relatively high maximal percent inhibition (MPI) of ~90% in NP2-CD4/CCR5 cells and peripheral blood mononuclear cells (PBMC), and 24-Res Env exhibiting a very low MPI of ~0 to 12% in both cell types, indicating relatively “weak” and “strong” resistance, respectively. Resistance mutations were strain-specific and mapped to the gp120 V3 loop. Affinity profiling by the 293-Affinofile assay and mathematical modeling using VERSA (Viral Entry Receptor Sensitivity Analysis) metrics revealed that 17-Res and 24-Res Envs engaged MVC-bound CCR5 inefficiently or very efficiently, respectively. Despite highly divergent phenotypes, and a lack of common gp120 resistance mutations, both resistant Envs exhibited an almost superimposable pattern of dramatically increased reliance on sulfated tyrosine residues in the CCR5 N-terminus, and on histidine residues in the CCR5 ECLs. This altered mechanism of CCR5 engagement rendered both the resistant Envs susceptible to neutralization by a sulfated peptide fragment of the CCR5 N-terminus. Conclusions: Clinical resistance to MVC may involve divergent Env phenotypes and different genetic alterations in gp120, but the molecular mechanism of resistance of the Envs studied here appears to be related. The increased reliance on sulfated CCR5 N-terminus residues suggests a new avenue to block HIV-1 entry by CCR5 N-terminus sulfopeptidomimetic drugs. http://www.retrovirology.com/content/10/1/43 Michael Roche Hamid Salimi Renee Duncan Brendan Wilkinson Kelechi Chikere Miranda Moore Nicholas Webb Helena Zappi Jasminka Sterjovski Jacqueline Flynn Anne Ellett Lachlan Gray Benhur Lee Becky Jubb Mike Westby Paul Ramsland Sharon Lewin Richard Payne Melissa Churchill Paul Gorry Retrovirology 2013, null:43 2013-04-20T00:00:00Z doi:10.1186/1742-4690-10-43 /content/figures/1742-4690-10-43-toc.gif Retrovirology 1742-4690 ${item.volume} 43 2013-04-20T00:00:00Z XML