195 In silico study on HIV-PRIs substructures to terminate proteolytic activity in HTLV

Human T-lymphotropic virus (HTLV) is RNA retrovirus, which causes CD3?+?and CD4?+?T-cell type leukemia and demyelinating diseases, like tropical spastic myelopathy. The replicative stage of the virus is one of the critical stages for the development of the disease. At present, there are no approved therapeutic agents targeting HTLV. The HTLV mechanism of malignant cell growth in adult T-cell leukemia (ATL)/lymphoma, and the HTLV-PR has been an attractive target for anticancer drug design. In comparison with other retroviruses, HTLV also encodes protease (PR) enzyme which is essential for maturation. Both the HIV and HTLV proteases show high structural similarity but known inhibitors of HIV-PR are not able to inhibit the HTLV-PR, while comparing the binding pocket of both proteases, MET37 of HTLV shows repulsive role with known HIV inhibitors. Functional analysis of M37A mutation clearly shows that MET37 is highly important for the protease function. Available inhibitors were tested against the HTLV-PR binding pocket and failed to interact with MET37. Screening of similar libraries of known compounds provides better interactions with MET37 and further validation with in vivo and in vitro studies on these screened compounds will provide more strength in discovering potent inhibitor for HTLV-PR.     

Construction of recombinant murine retroviruses that express the human T-cell leukemia virus type II and human T-cell lymphotropic virus type III trans activator genes.

Recombinant retroviruses containing the trans activator genes of human T-cell leukemia virus (HTLV) type II and human T-cell lymphotropic virus type III were constructed. The trans activator genes tat II and tat III were inserted into the murine retroviral vector pZIPNEOSV(X)1. Recombinant plasmids were transfected into the psi 2 and psi AM packaging cell lines that produce murine leukemia virions containing no retroviral RNA. Functional tat II and tat III gene products were expressed as demonstrated by trans activation of HTLV type I and II and human T-cell lymphotropic virus type III long terminal repeat-directed gene expression in the respective infected cells. Use of these recombinant vectors permits high-efficiency gene transfer into a wide variety of cells, thereby providing the opportunity to study the biochemical effects associated with tat II and tat III gene expression.     

Identification of a protective CD4+ T-cell epitope in p15gag of Friend murine leukemia virus and role of the MA protein targeting the plasma membrane in immunogenicity

Recent studies have demonstrated an essential role of Gag-specific CD4+ T-cell responses for viral control in individuals infected with human immunodeficiency virus type 1. However, little is known about epitope specificities and functional roles of the Gag-specific helper T-cell responses in terms of vaccine-induced protection against a pathogenic retroviral challenge. We have previously demonstrated that immunization with Friend murine leukemia virus (F-MuLV) Gag proteins protects mice against the fatal Friend retrovirus (FV) infection. We report here the structure of a protective T helper cell (Th) epitope, (I)VTWEAIAVDPPP, identified in the p15 (MA) region of F-MuLV Gag. In mice immunized with the Th epitope-harboring peptide or a vaccinia virus-expressed native full-length MA protein, FV-induced early splenomegaly regressed rapidly. In these mice, FV-infected cells were eliminated within 4 weeks and the production of virus-neutralizing antibodies was induced rapidly after FV challenge, resulting in strong protection against the virus infection. Interestingly, mice immunized with the whole MA mounted strong CD4+ T-cell responses to the identified Th epitope, whereas mice immunized with mutant MA proteins that were not bound to the plasma membrane failed to mount efficient CD4+ T-cell responses, despite the presence of the Th epitope. These mutant MA proteins also failed to induce strong protection against FV challenge. These data indicate the importance of the properly processible MA molecule for CD4+ T-cell priming and for the resultant induction of an effective immune response against retrovirus infections.     

Valproic acid and HIV-1 latency: beyond the sound bite

In 1977, Takatsuki and co-workers described in Japan a human malignant disease termed adult T-cell leukemia (ATL). Three years later, in 1980, Gallo and colleagues reported the identification of the first human retrovirus, human T-cell leukemia virus type I (HTLV-I), in a patient with cutaneous T-cell lymphoma. This month, Retrovirology commemorates these two land mark findings by publishing separate personal recollections by Takatsuki and Gallo respectively on the discovery of ATL and HTLV.     

Reduction of retrovirus-induced immunosuppression by in vivo modulation of T cells during acute infection

Chronic infection with Friend retrovirus is associated with suppressed antitumor immune responses. In the present study we investigated whether modulation of T-cell responses during acute infection would restore antitumor immunity in persistently infected mice. T-cell modulation was done by treatments with DTA-1 anti- glucocorticoid-induced tumor necrosis factor receptor monoclonal antibodies. The DTA-1 monoclonal antibody is nondepleting and delivers costimulatory signals that both enhance the activation of effector T cells and inhibit suppression by regulatory T cells. DTA-1 therapy produced faster Th1 immune responses, significant reductions in both acute virus loads and pathology and, most importantly, long-term improvement of CD8(+) T-cell-mediated antitumor responses.     

Biochemical and virological analysis of the 18-residue C-terminal tail of HIV-1 integrase

Background

Mouse mammary tumor virus (MMTV) encodes the Rem protein, an HIV Rev-like protein that enhances nuclear export of unspliced viral RNA in rodent cells. We have shown that Rem is expressed from a doubly spliced RNA, typical of complex retroviruses. Several recent reports indicate that MMTV can infect human cells, suggesting that MMTV might interact with human retroviruses, such as human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), and human endogenous retrovirus type K (HERV-K). In this report, we test whether the export/regulatory proteins of human complex retroviruses will increase expression from vectors containing the Rem-responsive element (RmRE).

Results

MMTV Rem, HIV Rev, and HTLV Rex proteins, but not HERV-K Rec, enhanced expression from an MMTV-based reporter plasmid in human T cells, and this activity was dependent on the RmRE. No RmRE-dependent reporter gene expression was detectable using Rev, Rex, or Rec in HC11 mouse mammary cells. Cell fractionation and RNA quantitation experiments suggested that the regulatory proteins did not affect RNA stability or nuclear export in the MMTV reporter system. Rem had no demonstrable activity on export elements from HIV, HTLV, or HERV-K. Similar to the Rem-specific activity in rodent cells, the RmRE-dependent functions of Rem, Rev, or Rex in human cells were inhibited by a dominant-negative truncated nucleoporin that acts in the Crm1 pathway of RNA and protein export.

Conclusion

These data argue that many retroviral regulatory proteins recognize similar complex RNA structures, which may depend on the presence of cell-type specific proteins. Retroviral protein activity on the RmRE appears to affect a post-export function of the reporter RNA. Our results provide additional evidence that MMTV is a complex retrovirus with the potential for viral interactions in human cells.     

Human T-cell leukemia virus type 1 envelope-mediated syncytium formation can be activated in resistant Mammalian cell lines by a carboxy-terminal truncation of the envelope cytoplasmic domain

Human T-cell leukemia virus (HTLV) envelope (Env) glycoproteins induce fusion, leading to rampant syncytium formation in a broad range of cell lines. Here, we identified murine, hamster, canine, and porcine cell lines that are resistant to HTLV-1 Env-induced syncytium formation. This resistance was not due to the absence of functional receptors for HTLV Env, as these cells were susceptible to infection with HTLV Env-pseudotyped virions. As murine leukemia virus (MLV) Env and HTLV Env present close structural homologies (F. J. Kim, I. Seiliez, C. Denesvre, D. Lavillette, F. L. Cosset, and M. Sitbon, J. Biol. Chem. 275:23417-23420, 2000), and because activation of syncytium formation by MLV Env generally requires cleavage of the R peptide in the cytoplasmic domain of the Env transmembrane (TM) component, we assessed whether truncation of the cytoplasmic domain of HTLV Env would alleviate this resistance. Indeed, in all resistant cell lines, truncation of the last 8 amino acids of the HTLV Env cytoplasmic domain (HdC8) was sufficient to overcome resistance to HTLV Env-induced syncytium formation. Furthermore, HdC8-mediated cell-to-cell infection titers varied according to the target cell lines and could be significantly higher than that observed with HTLV Env on HeLa cells. These data indicate that a determinant located within the 8 carboxy-terminal cytoplasmic amino acids of TM plays a distinct role in HTLV Env-mediated cell-to-cell infection and syncytium formation.     

Expression of glucose transporter 1 confers susceptibility to human T-cell leukemia virus envelope-mediated fusion

Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus identified and causes both adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-1-associated myelopathy, among other disorders. In vitro, HTLV-1 has an extremely broad host cell tropism in that it is capable of infecting most mammalian cell types, although at the same time viral titers remain relatively low. Despite years of study, only recently has a bona fide candidate cellular receptor, glucose transporter 1 (glut-1), been identified. Although glut-1 was shown to bind specifically to the ectodomain of HTLV-1 and HTLV-2 envelope glycoproteins, which was reversible with small interfering RNA directed against glut-1, cellular susceptibility to HTLV upon expression of glut-1 was not established. Here we show that expression of glut-1 in relatively resistant MDBK cells conferred increased susceptibility to both HTLV-1- and HTLV-2-pseudotyped particles. glut-1 also markedly increased syncytium formation in MDBK cells after exposure to HTLV-1. Another assay also demonstrated HTLV-1 envelope-cell fusion in the presence of glut-1. Taken together, these results provide additional evidence that glut-1 is a receptor for HTLV.     

Retroviral oncoprotein Tax induces processing of NF-kappaB2/p100 in T cells: evidence for the involvement of IKKalpha.

IkappaB kinase (IKK) is a key mediator of NF-kappaB activation induced by various immunological signals. In T cells and most other cell types, the primary target of IKK is a labile inhibitor of NF-kappaB, IkappaBalpha, which is responsible for the canonical NF-kappaB activation. Here, we show that in T cells infected with the human T-cell leukemia virus (HTLV), IKKalpha is targeted to a novel signaling pathway that mediates processing of the nfkappab2 precursor protein p100, resulting in active production of the NF-kappaB subunit, p52. This pathogenic action is mediated by the HTLV-encoded oncoprotein Tax, which appears to act by physically recruiting IKKalpha to p100, triggering phosphorylation-dependent ubiquitylation and processing of p100. These findings suggest a novel mechanism by which Tax modulates the NF-kappaB signaling pathway.     

Role of natural killer cells in resistance against friend retrovirus-induced leukemia

We have previously shown that immunization with a synthetic peptide that contains a single CD4(+) T-cell epitope protects mice against immunosuppressive Friend retrovirus infection. Cells producing infectious Friend virus were rapidly eliminated from the spleens of mice that had been immunized with the single-epitope peptide. However, actual effector mechanisms induced through T-helper-cell responses after Friend virus inoculation were unknown. When cytotoxic effector cells detected in the early phase of Friend retrovirus infection were separated based on their expression of cell surface markers, those lacking CD4 and CD8 but expressing natural killer cell markers were found to constitute the majority of effector cells that lysed Friend virus-induced leukemia cells. Depletion of natural killer cells by injecting anti-asialo-ganglio-N-tetraosylceramide antibody did not affect the number of CD4(+) or CD8(+) T cells in the spleen, virus antigen-specific proliferative responses of CD4(+) T cells, or cytotoxic activity against Friend virus-induced leukemia cells exerted by CD8(+) effector cells. However, the same treatment markedly reduced the killing activity of CD4(-) CD8(-) effector cells and completely abolished the effect of peptide immunization. Although the above enhancement of natural killer cell activity in the early stage of Friend virus infection was also observed in mice given no peptide, these results have demonstrated the importance and requirement of natural killer cells in vaccine-induced resistance against the retroviral infection.     

Thymic atrophy characteristic in transgenic mice that harbor pX genes of human T-cell leukemia virus type I

The human T-cell leukemia viruses (HTLV) are associated with T-cell malignancies in humans. The malignant transformation occurs after a long latency in some carriers, and its mechanism appears to be distinct from that of other classes of retroviruses which induce transformation through viral or cellular oncogenes. A widely postulated explanation is that the products of novel pX genes transactivate endogenous cellular genes which lead to tumor development in T cells. To directly examine the pathological effects of pX genes in vivo, we produced transgenic mice harboring the HTLV type I pX genes under several regulatory units: HTLV type I long terminal repeat, immunoglobulin enhancer-simian virus 40 promoter, and mouse mammary tumor virus long terminal repeat. Atrophy of the thymus was characteristic in these mice no matter which regulatory unit directed the expression of the genes.     

Interactions of human retroviruses with the host cell cytoskeleton

As obligate cell parasites, viruses have evolved into professional manipulators of host cell functions. Accordingly, viruses often remodel the cytoskeleton of target cells in order to convert one of the cell's barriers to viral replication into a vehicle for the virus that facilitates the generation of infectious progeny. Surprisingly little is known about the mechanisms employed by two major human pathogens, HIV and human T-cell leukaemia virus (HTLV), to exploit host cell cytoskeletal dynamics. New studies have begun to unravel how these retroviruses remodel cytoskeletal structures to facilitate entry into, transport within and egress from target cells. Exciting progress has been made in understanding how HIV and HTLV polarize actin and also control microtubule organization to spread from donor to target cells in close cell-contacts termed virological synapses.     

Distinct mechanisms of CD4+ and CD8+ T-cell activation and bystander apoptosis induced by human immunodeficiency virus type 1 virions

Apoptosis of uninfected bystander T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection. HIV-1 envelope/receptor interactions and immune activation have been implicated as contributors to bystander apoptosis. To better understand the relationship between T-cell activation and bystander apoptosis during HIV-1 pathogenesis, we investigated the effects of the highly cytopathic CXCR4-tropic HIV-1 variant ELI6 on primary CD4(+) and CD8(+) T cells. Infection of primary T-cell cultures with ELI6 induced CD4(+) T-cell depletion by direct cell lysis and bystander apoptosis. Exposure of primary CD4(+) and CD8(+) T cells to nonreplicating ELI6 virions induced bystander apoptosis through a Fas-independent mechanism. Bystander apoptosis of CD4(+) T cells required direct contact with virions and Env/CXCR4 binding. In contrast, the apoptosis of CD8(+) T cells was triggered by a soluble factor(s) secreted by CD4(+) T cells. HIV-1 virions activated CD4(+) and CD8(+) T cells to express CD25 and HLA-DR and preferentially induced apoptosis in CD25(+)HLA-DR(+) T cells in a CXCR4-dependent manner. Maximal levels of binding, activation, and apoptosis were induced by virions that incorporated MHC class II and B7-2 into the viral membrane. These results suggest that nonreplicating HIV-1 virions contribute to chronic immune activation and T-cell depletion during HIV-1 pathogenesis by activating CD4(+) and CD8(+) T cells, which then proceed to die via apoptosis. This mechanism may represent a viral immune evasion strategy to increase viral replication by activating target cells while killing immune effector cells that are not productively infected.     

Subsets of T cells in healthy rhesus macaques (Macaca mulatta) infected with simian T-lymphotropic virus type 1

Simian T-lymphotropic virus type 1 (STLV-1) is a C-type retrovirus of nonhuman primates that is genetically and antigenically related to human T-lymphotropic virus type 1 (HTLV-1). Infection with STLV-1 has been reported in many species of Old World monkeys and apes, including rhesus macaques (Macaca mulatta). Similar to HTLV infection in humans, STLV infection has been associated with T-cell lymphoproliferative disease or lymphoma in a small proportion of infected animals, predominantly African species. There are conflicting reports of T-cell subset alterations in healthy HTLV-1 carriers. To the authors' knowledge, analysis of T-cell subsets in healthy STLV-1 carrier rhesus macaques has not been reported. Subsets of T cells in peripheral blood from healthy, STLV-1-seropositive rhesus macaques (n = 17) and seronegative controls matched for age and sex (n = 17) were determined by use of fluorescence-activated cell sorter analysis. Parameters measured included CD3, CD4, CD8, CD25, CD28, CD38, and HLA-DR cell sets. Significant differences in T-cell subsets or hematologic parameters were not observed between healthy STLV-seropositive and seronegative groups.     

Pseudotyping with human T-cell leukemia virus type I broadens the human immunodeficiency virus host range.

Several epidemiologic and clinical studies suggest that patients coinfected with human immunodeficiency virus (HIV), the primary etiologic agent in AIDS, and other viruses, such as cytomegalovirus or human T-cell leukemia virus (HTLV), have a more severe clinical course than those infected with HIV alone. Cells infected with two viruses can, in some cases, give rise to phenotypically mixed virions with altered or broadened cell tropism and could therefore account for some of these findings. Such pseudotypes could alter the course of disease by infecting more tissues than are normally infected by HIV. We show here that HIV type 1 (HIV-1) efficiently incorporates the HTLV type I (HTLV-I) envelope glycoprotein and that both HIV-1 and HTLV-II accept other widely divergent envelope glycoproteins to form infectious pseudotype viruses whose cellular tropisms and relative abilities to be transmitted by cell-free virions or by cell contact are determined by the heterologous envelope. We also show that the mechanism by which virions incorporate heterologous envelope glycoproteins is independent of the presence of the homologous glycoprotein or heterologous gag proteins. These results may have important implications for the mechanism of HIV pathogenesis.