HIV-1, the virus responsible for the AIDS pandemic is a retrovirus belonging to the lentivirus genus. The viral genome consists of two copies of linear, positive-sense ssRNA approximately 9,700 nucleotides long, encoding 14 proteins. In the cytoplasm of infected cells, the viral ssRNA is copied to a dsDNA proviral molecule by viral reverse-transcriptase which is transported to the nucleus and integrated into the host cell genome.
The ability to initiate infection by transfecting cultured cells with dsDNA HIV-1 proviruses greatly facilitated development of experimental systems for the investigation of HIV-1 molecular biology: Mutant viruses can readily be constructed in the laboratory and their replication properties compared with the isogenic wild type. Investigation of recombinant HIV-1 constructs from which the vpu gene was deleted led to Vpu being labeled an "accessory" protein of HIV-1 because it is not essential for virus replication in vitro. Nevertheless, a number of reports have shown that Vpu significantly enhances the release of progeny virions from infected cells. The magnitude of this effect is dependent on both the virus strain and the type of cell infected. Replication of vpu knockout strains is only marginally decreased (0-4-fold) in cultured CD4+ T-cells or cell lines derived from T-lymphocytes. However, at the other extreme, using infected macrophages, one study has reported a decrease greater than 1,000-fold in the release of new virus particles when Vpu was ablated, compared to the isogenic wild-type HIV-1 strain [see (Schubert et al., 1999) and references therein]. Interestingly, it has been reported (Du et al., 1993) that vpu can induce a significant constraint upon the host range and growth potential of HIV-1 and it was noted that adaptation of cells, such as T-cell lines, to in vitro replication can involve selection of viruses that have ablated vpu gene expression by mutation.
As revealed by more detailed probing of the effects of vpu deletion on HIV-1 biology, an intriguing characteristic of the Vpu protein is that it exerts a number of effects on apparently disparate cellular processes (Kerkau et al., 1997; Emerman and Malim, 1998; Schubert et al., 1998). Thus, in the past 10 years it has been discovered that Vpu:
1. binds to the CD4 protein in the ER and induces its degradation via the ubiquitin-proteosome pathway;
2. controls the trafficking of the HIV-1 glycoprotein gp160, and certain other glyco-proteins, to the plasma membrane;
3. reduces HIV-1 cytopathicity by reducing virus-induced syncytium formation;
4. downregulates expression of MHC class 1 on the cell surface; and
5. augments the budding of newly assembled virus particles from the host-cell plasma membrane.
A major scientific challenge is to understand fully the molecular mechanisms underlying the diverse range of physiological outcomes involving Vpu. In this regard, mutagenesis studies have shown that two Vpu-mediated activities, CD4 degradation and enhancement of virion budding, reside in two separate domains of the protein. Mutations that prevent phos-phorylation of the conserved serine residues in the cytoplasmic domain revealed that phos-phorylation is essential for CD4 degradation, as well as inhibition of gp120 trafficking, and reduced syncytium formation. Yet, an absence of phosphorylation had only a minimal impact upon augmentation of virus release. Further, a mutant in which the sequence of the first 27 N-terminal amino acids of Vpu was randomized resulted in a protein (Vpu-RND) which had lost its ability to enhance virion budding, although the CD4 degradation activity was not affected (Schubert et al., 1996a). The TM domain of Vpu expressed without the cytoplasmic domain, was still able to augment virus budding, but not degradation of CD4. Thus, it was concluded that the C-terminal hydrophilic cytoplasmic domain mediates CD4 degradation and that the N-terminal membrane-spanning domain (amino acids 1-27) modulates virus particle release (Schubert et al., 1996a), though the mechanisms by which Vpu exerts these activities remain to be elucidated.
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