HIV-1 Vpr and the Induction of G2 Cell-Cycle Arrest.
The 96–amino acid HIV-1 protein Vpr induces G2 cell-cycle arrest in proliferating human T cells. This arrest enhances viral replication since the HIV-1 long terminal repeat is more active in G2. Despite considerable study, the molecular basis for Vpr-induced G2 arrest has remained elusive. Using time-lapse video fluorescence microscopy to study synchronized cultures of HeLa cells expressing specific G2 cell-cycle regulators (Wee1, Cdc25C, and cyclin B1) fused to the green or red fluorescent protein, we discovered that Vpr induces dynamic changes in the architecture and integrity of the nuclear envelope. These changes are manifested by the successive projection and retraction of nuclear envelope herniations (Figure 1). These herniations intermittently rupture, leading to the admixture of proteins normally sequestered in the nucleus or cytoplasm. These DNA-filled herniations are caused by Vpr-induced disruptions (often local) in the structure of the nuclear lamina underlying the nuclear envelope. Mutants of Vpr that fail to induce G2 arrest do not induce such changes, suggesting that these abnormalities of the nuclear lamina and/or the secondary ruptures they induce may centrally contribute to the observed cell-cycle arrest. Nuclear herniations are also detected after HIV-1 infection of cells. Indeed, the small quantities of Vpr present in the incoming HIV virions appear sufficient to support this response. Coupled with the fact that Vpr markedly enhances the replication of HIV-1 in nondividing macrophages, this latter result raises the intriguing possibility that nuclear entry of the large HIV preintegration complex (PIC) may be facilitated by its passage through these Vpr-induced ruptures in the nuclear envelope. However, in collaborative studies with Drs. Daniel Eckstein and Mark Goldsmith, we have found that, in contrast to macrophages, Vpr is not required for HIV-1 infection of nondividing resting T cells. The productive infection of these resting T cells apparently involves import of the viral PIC through nuclear pores. This process likely involves the recognized nuclear targeting signals present in integrase and matrix proteins or the DNA flap formed as an intermediate of reverse transcription.
HIV-1 Nef and the Enhancement of Viral Infectivity.
The 25–27-kDa regulatory protein Nef plays a key, although poorly understood, role in accelerating the progression of HIV-1-induced clinical disease in infected hosts. Nef exerts several different biological effects in vitro, including enhancement of virion infectivity, downregulation of CD4 and MHC class I receptor expression, and modulation of various intracellular signaling pathways. The enhancing effect of Nef on virion infectivity requires its expression in the producer cell, although its effect is only manifested after infection of the subsequent target cell. HIV-1 enters target cells by two routes. Fusion at the cell surface often leads to productive infection, and endocytosis generally results in a dead end form of infection. Our recent studies revealed that Nef increases accumulation of p24 Gag in the cytoplasm of target cells, suggesting that Nef enhances the entry of virions into cells via the productive pathway of infection. These findings indicated that Nef acts at a more proximal point in the retroviral life cycle than was previously appreciated, leading to enhanced viral infectivity.
HIV-1 Nef as a Mediator of Viral Immune Evasion.
Pathogenic infection of lymphatic tissues with HIV-1 or simian immunodeficiency virus type 1 (SIV-1) results in significant apoptosis; however, the dying cells are often uninfected. This observation raises the possibility that programmed cell death may be specifically attenuated in infected cells. Since some of the dying bystander cells are virus-specific cytotoxic T lymphocytes (CTLs), this apoptosis likely contributes to a form of immune evasion. Xu and colleagues have shown that the Nef proteins of HIV and SIV activate transcription and surface expression of Fas ligand (FasL) (see J. Exp. Med. 186:7, 1997, and J. Exp. Med. 189:1489, 1999). This ligand engages Fas present on neighboring CTLs, triggering death signaling. This finding contributes to the diminished number of CTLs found in monkeys infected with SIV encoding wildtype Nef versus a mutant form of Nef. It remains unknown how the HIV-infected host cell, which expresses both FasL and Fas and is exposed to both circulating and membrane-bound forms of tumor necrosis factor (TNF), avoids a similar apoptotic death.
Our recent studies have revealed that HIV-1 Nef assembles with and inhibits the enzymatic activity of apoptosis-signal regulating kinase 1 (ASK1) (Figure 2). This kinase is believed to play a central role in death signaling initiated through both the human Fas and TNF-1 receptors. We further demonstrated that HIV-1 Nef inhibits Fas- and TNF-mediated induction of JNK activity and significantly suppresses the induction of apoptosis by these agonists. These antiapoptotic activities of Nef are compromised by mutation of its amino terminal myristilation signal and the arginine at position 106 in the NL4-3 Nef allele. We also found that the levels of Nef produced during the course of HIV infection of primary human CD4 T cells are sufficient to impair Fas-induced apoptosis. Conversely, infection of these cells with HIV carrying the R106A mutation in Nef failed to provide protection from Fas-induced apoptosis. Additionally, in HIV-infected SupT1 cells, we have detected the physical assembly of Nef and ASK1 and have shown that Nef blocks TNFa activation of the ASK1 enzyme. These studies have provided new insights into how the HIV infected cell may be protected from FasL/Fas and TNF-mediated apoptosis while this same death pathway is utilized to induce killing of bystander cells, including cytotoxic T cells with anti-HIV activity.
