Gp120 V3-dependent impairment of R5 HIV-1 infectivity due to virion-incorporated CCR5

Entry of R5 human immunodeficiency virus type 1 (HIV-1) into target cells requires sequential interactions of the envelope glycoprotein gp120 with the receptor CD4 and the coreceptor CCR5. We investigated replication of 45 R5 viral clones derived from the HIV-1JR-FLan library carrying 0-10 random amino acid substitutions in the gp120 V3 loop. It was found that 6.7% (3/45) of the viruses revealed >or=10-fold replication suppression in PM1/CCR5 cells expressing high levels of CCR5 compared with PM1 cells expressing low levels of CCR5. In HIV-1V3L#08, suppression of replication was not associated with entry events and viral production but with a marked decrease in infectivity of nascent progeny virus. HIV-1V3L#08, generated from infected PM1/CCR5 cells, was 98% immunoprecipitated by anti-CCR5 monoclonal antibody T21/8, whereas the other infectious viruses were only partially precipitated, suggesting that incorporation of larger amounts of CCR5 into the virions caused impairment of viral infectivity in HIV-1V3L#08. The results demonstrate the implications of an alternative influence of CCR5 on HIV-1 replication.     

Modulation of human immunodeficiency virus type 1 infectivity through incorporation of tetraspanin proteins

Accumulating evidence indicates that human immunodeficiency virus type 1 (HIV-1) acquires various cellular membrane proteins in the lipid bilayer of the viral envelope membrane. Although some virion-incorporated cellular membrane proteins are known to potently affect HIV-1 infectivity, the virological functions of most virion-incorporated membrane proteins remain unclear. Among these host proteins, we found that CD63 was eliminated from the plasma membranes of HIV-1-producing T cells after activation, followed by a decrease in the amount of virion-incorporated CD63, and in contrast, an increase in the infectivity of the released virions. On the other hand, we found that CD63 at the cell surface was preferentially embedded on the membrane of released virions in an HIV-1 envelope protein (Env)-independent manner and that virion-incorporated CD63 had the potential to inhibit HIV-1 Env-mediated infection in a strain-specific manner at the postattachment entry step(s). In addition, these behaviors were commonly observed in other tetraspanin proteins, such as CD9, CD81, CD82, and CD231. However, L6 protein, whose topology is similar to that of tetraspanins but which does not belong to the tetraspanin superfamily, did not have the potential to prevent HIV-1 infection, despite its successful incorporation into the released particles. Taken together, these results suggest that tetraspanin proteins have the unique potential to modulate HIV-1 infectivity through incorporation into released HIV-1 particles, and our findings may provide a clue to undiscovered aspects of HIV-1 entry.     

A CD63 mutant inhibits T-cell tropic human immunodeficiency virus type 1 entry by disrupting CXCR4 trafficking to the plasma membrane

We have discovered that an N-terminal deletion mutant of a membrane protein, CD63, (CD63ΔN) blocks entry of CXCR4-using, T-cell tropic human immunodeficiency virus type 1 (X4 HIV-1) by suppressing CXCR4 surface expression. This suppression was observed for CXCR4 but not for CD4, CCR5, CD25, CD71 or other tetraspanin proteins. The suppression of CXCR4 expression on the plasma membrane appeared to be caused by mislocalization of CXCR4 and exclusive transportation of CXCR4 toward intracellular organelles, mainly late endosomes/lysosomes. Our data suggest that CXCR4 trafficking can be modified in terms of its recruitment to the plasma membrane without enhancing the degradation or arresting vesicular transport of CXCR4.     

Induction of protective immune responses against R5 human immunodeficiency virus type 1 (HIV-1) infection in hu-PBL-SCID mice by intrasplenic immunization with HIV-1-pulsed dendritic cells: possible involvement of a novel factor of human CD4(+) T-cell origin

The potential of a dendritic cell (DC)-based vaccine against human immunodeficiency virus type 1 (HIV-1) infection in humans was explored with SCID mice reconstituted with human peripheral blood mononuclear cells (PBMC). HIV-1-negative normal human PBMC were transplanted directly into the spleens of SCID mice (hu-PBL-SCID-spl mice) together with autologous mature DCs pulsed with either inactivated HIV-1 (strain R5 or X4) or ovalbumin (OVA), followed by a booster injection 5 days later with autologous DCs pulsed with the same respective antigens. Five days later, these mice were challenged intraperitoneally with R5 HIV-1(JR-CSF). Analysis of infection at 7 days postinfection showed that the DC-HIV-1-immunized hu-PBL-SCID-spl mice, irrespective of the HIV-1 isolate used for immunization, were protected against HIV-1 infection. In contrast, none of the DC-OVA-immunized mice were protected. Sera from the DC-HIV-1- but not the DC-OVA-immunized mice inhibited the in vitro infection of activated PBMC and macrophages with R5, but not X4, HIV-1. Upon restimulation with HIV-1 in vitro, the human CD4(+) T cells derived from the DC-HIV-1-immunized mice produced a similar R5 HIV-1 suppressor factor. Neutralizing antibodies against human RANTES, MIP-1alpha, MIP-1beta, alpha interferon (IFN-alpha), IFN-beta, IFN-gamma, interleukin-4 (IL-4), IL-10, IL-13, IL-16, MCP-1, MCP-3, tumor necrosis factor alpha (TNF-alpha), or TNF-beta failed to reverse the HIV-1-suppressive activity. These results show that inactivated HIV-1-pulsed autologous DCs can stimulate splenic resident human CD4(+) T cells in hu-PBL-SCID-spl mice to produce a yet-to-be-defined, novel soluble factor(s) with protective properties against R5 HIV-1 infection.     

Quantification of the virus-host interaction in human T lymphotropic virus I infection

Background

Cellular infection with human immunodeficiency virus (HIV) both in vitro and in vivo requires a member of the chemokine receptor family to act as a co-receptor for viral entry. However, it is presently unclear to what extent the interaction of HIV proteins with chemokine receptors generates intracellular signals that are important for productive infection.

Results

In this study we have used a recently described family of chemokine inhibitors, termed BSCIs, which specifically block chemokine-induced chemotaxis without affecting chemokine ligands binding to their receptors. The BSCI termed Peptide 3 strongly inhibited CCR5 mediated HIV infection of THP-1 cells (83 ± 7% inhibition assayed by immunofluoresence staining), but had no effect on gp120 binding to CCR5. Peptide 3 did not affect CXCR4-dependent infection of Jurkat T cells.

Conclusion

These observations suggest that, in some cases, intracellular signals generated by the chemokine coreceptor may be required for a productive HIV infection.     

Neuraminidase enhances the initial steps of human T-cell leukemia virus type 1 replication

Human T-cell leukemia virus type 1 (HTLV-1) infection is involved in the development of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. A high HTLV-1 proviral load in circulating lymphocytes of HTLV-1 carriers is a risk factor for HTLV-1-related diseases. The virus–cell interaction is linked to viral tropism and pathogenesis. Characterization of the factors that affect HTLV-1 infection is important for preventing HTLV-1 infection. HTLV-1 virions are believed to be weakly infectious under cell culture conditions; however, we found that the treatment of HTLV-1 virions with microbial neuraminidase, an enzyme catalyzing the removal of sialic acid residues from various glycoconjugates, enhanced the number of proviral DNAs in infected cells in a dose-dependent manner. Neuraminidase treatment of virions, but not target cells, enhanced viral binding and entry into cells and viral infectivity; treatment of target cells prior to infection had no effect. Moreover, the number of HTLV-1-mediated syncytia was higher in the presence of neuraminidase. Our results suggest a possible contribution of microbial agents carrying neuraminidase activity to HTLV-1 pathogenesis.     

Immunostimulation by induced expression of NKG2D and its MIC ligands in HTLV-1-associated neurologic disease

The NKG2D receptor costimulates effector/memory CD8 T cells and is normally absent on CD4 T cells but can be induced by T cell antigen receptor complex stimulation and interleukin-15 (IL-15). Among its ligands are the human major histocompatibility complex class I-related MICA and MICB, which have a restricted tissue distribution but are frequently associated with malignancies and some microbial infections. Moreover, aberrant expression of MIC may promote autoimmune disease progression. Human T cell lymphotropic virus type I (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic inflammatory disease of the central nervous system that resembles multiple sclerosis. Disease progression involves production of IL-15 and its receptor through transactivation by the viral Tax regulator protein, an activated immune response state, and local cytokine production and T cell fratricide by Tax-specific cytotoxic T lymphocytes (CTL). This study shows that as with CD8 T cells, substantial proportions of HAM/TSP patient CD4 T cells are positive for NKG2D and that large numbers of T cells from both subsets express MIC, which can be transactivated by Tax independent of nuclear factor κB. Engagement of MIC by NKG2D promotes spontaneous HAM/TSP T cell proliferation and, apparently, CTL activities against HTLV-1-infected T cells. These results reveal a viral strategy that may exploit immune stimulatory mechanisms to negotiate a balance between promotion and limitation of infected host T cell expansions.     

Cross-linking cell surface chemokine receptors leads to isolation, activation, and differentiation of monocytes into potent dendritic cells

Monocytes express on the cell surface several kinds of chemokine receptors that facilitate chemotaxis followed by differentiation in target tissues. In the present study, we found that a large number of monocytes from peripheral blood mononuclear cells (PBMCs) tightly adhered to plastic cell culture plates precoated with a monoclonal antibody (mAb, clone T312) specific for human CCR5 but not an isotype control after overnight incubation. Soluble T312 did not induce such adhesion, indicating that cross-linking of CCR5 is required for the enhanced adhesion of monocytes. The adhesion was blocked by a PI3-K inhibitor and an anti-CD18 blocking mAb. Following the cross-linking of CCR5, monocytes synthesized high levels of M-CSF, RANTES, MIP-1 alpha, and MIP-1 beta associated with a readily detectable down modulation of CD14, CD4, CCR5, and CXCR4 expression. The T312-enriched monocytes differentiated into dendritic cells (DCs) in the presence of interleukin-4 alone. After maturation with beta-interferon, the T312-induced DCs stimulated proliferation of allogeneic na?ve CD4(+) T cells accompanied by the synthesis of high levels of gamma-interferon in vitro. Furthermore, the T312-induced DCs were capable of stimulating antigen-specific human T- and B-cell immune responses in our hu-PBL-SCID mouse system. Finally, screening of other anti-chemokine receptor mAbs showed that select clones of mAbs against CXCR4 and CCR3 were also capable of facilitating enrichment of monocytes similar to T312. These results show that cross-linking of chemokine receptors on monocytes by appropriate mAbs leads to activation and differentiation of monocytes and that the method described herein provides an alternate simple strategy for adherence-based isolation of monocytes and generation of functional DCs.