The role of the transmembrane and cytoplasmic domains of the VPU protein of human immunodeficiency virus type 1 (HIV-1) in the acute CD4+ T cell loss and disease in the simian human immunodeficiency virus (SHIV)/macaque model

The Vpu protein from the human immunodeficiency virus type 1 (HIV-1) enhances virus release from infected cells and down regulates CD4 from the cell surface. In this dissertation, I have shown that Vpu may be expressed at higher levels than previously reported and that there appears to be a strong selective pressure to maintain the vpu and env genes in separate reading frames to produce a functional Vpu protein. Previous studies have associated enhanced virus release with the ion channel activity of the transmembrane domain of Vpu and that large deletions in this region of Vpu produce a virus that is less pathogenic to pig-tailed macaques. It was therefore hypothesized that Vpu and specifically the transmembrane domain play an important role in virus release and pathogenesis in the simian human immunodeficiency virus (SHIV) macaque model. A novel virus was constructed (SHIVTM) in which the transmembrane domain of the Vpu protein was scrambled to disrupt any ion channel properties of this domain. Following inoculation into pig-tailed macaques, the virus replicated with reduced kinetics, the macaques maintained CD4+T cell levels in the normal range and there was no tissue pathology. Thus, I have shown for the first time that the transmembrane domain of Vpu contributes to the pathogenicity in pig-tailed macaques. To determine if the transmembrane domain of Vpu could be functionally substituted with that of the prototypical viroporin, the M2 protein of influenza A, a novel virus was constructed (SHIVM2) expressing a chimeric Vpu protein with the transmembrane domain of M2 from Influenza A. The SHIV M2 proved to be pathogenic in macaques and sensitive to the M2 inhibitor, rimantadine. Lastly a mutant (SHIVA19H) was constructed in which a histidine was introduced at position 19 in the transmembrane domain of the wild type Vpu. The SHIVVpuA19H replicated with similar kinetics compared to SHIVKU-1bMC33 but was sensitive to rimantadine. Thus, I have shown that a pathogenic SHIV can be converted into a rimantadine sensitive virus by alteration of a single amino acid. These results show that the transmembrane domain of Vpu can be a target for novel anti-HIV drugs.