Identification of the cellular prohibitin 1/prohibitin 2 heterodimer as an interaction partner of the C-terminal cytoplasmic domain of the HIV-1 glycoprotein.

Our studies aim to elucidate the functions carried out by the very long, and in its length highly conserved, C-terminal cytoplasmic domain (Env-CT) of the HIV-1 glycoprotein. Mass spectrometric analysis of cellular proteins bound to a tagged version of the HIV Env-CT led to the identification of the prohibitin 1 and 2 proteins (Phb1 and Phb2). These ubiquitously expressed proteins, which exist as stable heterodimers, have been shown to have multiple functions within cells and to localize to multiple cellular and extracellular compartments. The specificity of binding of the Phb1/Phb2 complex to the Env-CT was confirmed in various manners, including coimmunoprecipitation with authentic provirally encoded, full-length Env. Strong binding was dependent on Env residues 790 to 800 and could be severely inhibited by the double mutation L799R/L800Q but not by mutation of these amino acids individually. Analysis of the respective mutant virions revealed that their different abilities to bind Phb1/Phb2 correlated with their replicative properties. Thus, mutated virions with single mutations [HIV-Env-(L799R) and HIV-Env-(L800Q)] replicated similarly to wild-type HIV, but HIV-Env-(L799R/L800Q) virions, which cannot bind Phb1/Phb2, exhibited a cell-dependent replicative phenotype similar to that of HIV-Env-Tr712, lacking the entire Env-CT domain. Thus, replicative spread was achieved, although somewhat delayed, in "permissive" MT-4 cells but failed to occur in "nonpermissive" H9 T cells. These results point to binding of the Phb1/Phb2 complex to the Env-CT as being of importance for replicative spread in nonpermissive cells, possibly by modulating critical Phb-dependent cellular process(es).

Cell-free infectivity of HIV type 1 produced in nonpermissive cells is only moderately impacted by C-terminal Env truncation despite abrogation of viral spread.

Mutant HIV virions, encoding C-terminally truncated Env proteins, exhibit a cell-specific replication defect, i.e., they can replicate in a few T cell lines (termed permissive cells) but not in the majority of T cell lines (termed nonpermissive cells). We have studied the properties of two mutant virions (pNL-Tr712 and pNL-Tr752), encoding Envs with C-terminal truncations of 144 and 104 amino acids, respectively. We show that although unable to give rise to a spreading infection in nonpermissive H9 cells, both cell-free pNL-Tr712 and pNL-Tr752 virions, produced in these cells, still exhibit relatively high levels of infectivity (30-80% of wildtype) when tested in nonpermissive target cells. Compatible with this high remaining infectivity, we observed that the levels of Env incorporation into mutant virions, produced in nonpermissive cells, were not drastically reduced as has been reported by others. The high remaining infectivity of cell-free mutant virions in nonpermissive cells is difficult to reconcile with the complete lack of spreading infection in these cells. We demonstrate that nonpermissive cells are less susceptible to single-round infection with cell-free virus than permissive cells. It is thus conceivable that in these cells other transmission routes, e.g., cell-cell transmission, may be more important for total virus spread and that this route may be more severely impacted by the C-terminal Env truncations.

Generation of H9 T-cells stably expressing a membrane-bound form of the cytoplasmic tail of the Env-glycoprotein: lack of transcomplementation of defective HIV-1 virions encoding C-terminally truncated Env.

H9-T-cells do not support the replication of mutant HIV-1 encoding Env protein lacking its long cytoplasmic C-terminal domain (Env-CT). Here we describe the generation of a H9-T-cell population constitutively expressing the HIV-1 Env-CT protein domain anchored in the cellular membrane by it homologous membrane-spanning domain (TMD). We confirmed that the Env-TMD-CT protein was associated with cellular membranes, that its expression did not have any obvious cytotoxic effects on the cells and that it did not affect wild-type HIV-1 replication. However, as measured in both a single-round assay as well as in spreading infections, replication competence of mutant pNL-Tr712, lacking the Env-CT, was not restored in this H9 T-cell population. This means that the Env-CT per se cannot transcomplement the replication block of HIV-1 virions encoding C-terminally truncated Env proteins and suggests that the Env-CT likely exerts its function only in the context of the complete Env protein.

Effects of signal peptide exchange on HIV-1 glycoprotein expression and viral infectivity in mammalian cells.

In certain cell systems, exchange of the human immunodeficiency virus (HIV) Env signal peptide (SP) sequence with that of heterologous SPs has been shown to increase gp120 transport and secretion. Here we demonstrate that exchange of the HIV-Env-SP with those from erythropoietin or tissue plasminogen activator in the proviral context does not increase wild-type membrane-bound Env expression or incorporation into released virions. In fact, virion infectivity was decreased. These infectivity decreases were largely due to effects on Env transport and/or function and only to a minor extent to cis effects as a result of the sequence exchanges themselves. Thus, in fact, it is not advantageous to employ heterologous SPs to achieve high-level expression of functional cell surface membrane- or virion-associated HIV-Env.

Selection and characterization of a replication-competent human immunodeficiency virus type 1 variant encoding C-terminally truncated env.

A long cytoplasmic C-terminus (Env-CT) on the human immunodeficiency virus type 1 (HIV-1) Env protein is a highly conserved feature in vivo. Mutant HIV lacking the Env-CT cannot replicate in PBMCs and in the majority of T cell lines (nonpermissive cells, e.g., H9 cells) in vitro. We report here that a single amino acid change (N750K) in the context of the mutant virus pNL-Tr752 lacking 104 C-terminal Env amino acids gives rise to a virus variant pNL-Tr752(N750K), which can now replicate in nonpermissive H9 cells and, albeit to a lower extent, in PBMCs. We have analyzed the properties of replication-competent pNL-Tr752(N750K) in comparison to its defective counterpart pNL-Tr752 and to wild-type virus in H9 cells. In all cases, the respective glycoproteins were functional in inducing membrane fusion and were incorporated into particles. In comparison to pNL-Tr752 and pNL-Wt, pNL-Tr752(N750K) glycoprotein exhibited increased fusion induction and 2- to 3-fold increased incorporation into particles, properties that may contribute to the observed replication competence.

Incorporation of chimeric HIV-SIV-Env and modified HIV-Env proteins into HIV pseudovirions.

Low level incorporation of the viral glycoprotein (Env) into human immunodeficiency virus (HIV) particles is a major drawback for vaccine strategies against HIV/AIDS in which HIV particles are used as immunogen. Within this study, we have examined two strategies aimed at achieving higher levels of Env incorporation into non-infectious pseudovirions (PVs). First, we have generated chimeric HIV/SIV Env proteins containing the truncated C-terminal tail region of simian immunodeficiency virus (SIV)mac239-Env767(stop), which mediates strongly increased incorporation of SIV-Env into SIV particles. In a second strategy, we have employed a truncated HIV-Env protein (Env-Tr752(N750K)) which we have previously demonstrated to be incorporated into HIV virions, generated in infected T-cells, to a higher level than that of Wt-HIV-Env. Although the chimeric HIV/SIV Env proteins were expressed at the cell surface and induced increased levels of cell-cell fusion in comparison to Wt-HIV-Env, they did not exhibit increased incorporation into either HIV-PVs or SIV-PVs. Only Env-Tr752(N750K) exhibited significantly higher (threefold) levels of incorporation into HIV-PVs, an improvement, which, although not dramatic, is worthwhile for the large-scale preparation of non-infectious PVs for vaccine studies aimed at inducing Env humoral responses.

HTLV-1 Tax protects against CD95-mediated apoptosis by induction of the cellular FLICE-inhibitory protein (c-FLIP).

The HTLV-1 transactivator protein Tax is essential for malignant transformation of CD4 T cells, ultimately leading to adult T-cell leukemia/lymphoma (ATL). Malignant transformation may involve development of apoptosis resistance. In this study we investigated the molecular mechanisms by which HTLV-1 Tax confers resistance toward CD95-mediated apoptosis. We show that Tax-expressing T-cell lines derived from HTLV-1-infected patients express elevated levels of c-FLIP(L) and c-FLIP(S). The levels of c-FLIP correlated with resistance toward CD95-mediated apoptosis. Using an inducible system we demonstrated that both resistance toward CD95-mediated apoptosis and induction of c-FLIP are dependent on Tax. In addition, analysis of early cleavage of the BH3-only Bcl-2 family member Bid, a direct caspase-8 substrate, revealed that apoptosis is inhibited at a CD95 death receptor proximal level in Tax-expressing cells. Finally, using siRNA we directly showed that c-FLIP confers Tax-mediated resistance toward CD95-mediated apoptosis. In conclusion, our data suggest an important mechanism by which expression of HTLV-1 Tax may lead to immune escape of infected T cells and, thus, to persistent infection and transformation.

Inter-retroviral fusion mediated by human immunodeficiency virus or murine leukemia virus glycoproteins: independence of cellular membranes and membrane vesicles.

We have recently demonstrated for the first time that inter-retroviral membrane fusion, i.e., membrane fusion between individual retroviral particle populations with incorporated HIV-1 Env and cellular receptors, respectively, can occur (Sparacio et al. 2000, Virology 271: 248-252). We have extended these analyses here and confirmed that fusion between particles can occur in the extracellular medium independent of any cellular membranes and that luciferase transduction, mediated by the fused structures, is independent of significant potential contribution by contaminating membrane vesicles. We have additionally analyzed whether membrane fusion between HIV-like particles can be mediated by amphotropic murine leukemia virus (MuLV) glycoprotein and its respective cellular receptor, PiT-2. We demonstrate that PiT-2 can be incorporated into HIV-like particles and can fuse with MuLV-Env-carrying particles. This occurs only in the situation in which the incorporated MuLV-Env protein has been activated to fusion activity by HIV protease-mediated removal of the C-terminal R-peptide and is completely inhibited when the respective particles are generated in the presence of the HIV protease inhibitor, Saquinavir.