Previous studies have shown that the transmembrane (TM) domain of the subtype B Vpu enhances virion release from cells and some studies have shown that this domain may form an oligomeric structure with properties of an ion channel. To date, no studies have been performed to assess the role of this domain in virus pathogenesis in a macaque model of disease. Using a pathogenic molecular clone of simian human immunodeficiency virus (SHIVKU-1bMC33), we have generated a novel virus in which the transmembrane domain of the Vpu protein was scrambled but maintained hydrophobic in nature (SHIVTM), which presumably would disrupt any ion channel TM properties of this protein. Vectors expressing the Vpu as a fusion protein with the enhanced green fluorescent protein (VpuTMEGFP) indicate that it was transported to the same intracellular compartment as the unmodified Vpu protein but did not down-regulate cell surface expression of CD4. To assess the pathogenicity of SHIVTM, three pig-tailed macaques were inoculated with the SHIVTM and monitored for 6-8 months for CD4+ T cell levels, viral loads and the stability of the sequence of the vpu gene. Our results indicated that unlike the parental SHIVKU-1bMC33, inoculation of macaques with SHIVTM did not cause a severe CD4+ T cell loss over the course of their infections. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that the scrambled TM was stable during the course of infection. At necropsy, examination of tissues revealed low viral loads and none of the pathology commonly observed in lymphoid and non-lymphoid tissues following inoculation with the pathogenic parental SHIVKU-1bMC33 virus. Thus, these results show for the first time that the TM domain of Vpu contributes to the pathogenicity of SHIVKU-1bMC33 in pig-tailed macaques.

The structure of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) is composed of a short Nterminal domain (NTD), a transmembrane domain (TM), and a cytoplasmic domain (CD). Previous studies have shown that the Vpu protein from subtype B HIV-1 is transported predominantly to the rough endoplasmic reticulum (RER)/Golgi complex compartments of the cell and is not incorporated into virions. Using a previously described VpuEGFP reporter system in which the Vpu protein was fused to the gene for enhanced green fluorescent protein (EGFP), we showed that the subtype B Vpu fusion protein was localized to the RER/Golgi region of the cell, similar to the native protein. In the present study, we show that fusion of the subtype C Vpu to EGFP results in a fusion protein that is transported to the cell surface. Using this reporter system, chimeric Vpu proteins in which the CD of the subtype B and C proteins were exchanged showed that the CD was sufficient for targeting the subtype B protein to the Golgi complex of the cell. Following identification of the cytoplasmic domain as being responsible for intracellular targeting, we then generated a series of mutants in which 13, 23, 31, 38, 51, and 56 amino acids were deleted from the cytoplasmic domain of subtype B Vpu. These deletion mutants were analyzed by SDS-PAGE for size, for membrane localization, and intracellular localization by confocal fluorescence microscopy. Our results indicate that the mutant with the carboxyl-terminal 13 amino acids deleted was still localized to the Golgi complex but mutants with 23, 31, 38, 51, and 56 amino acids from the carboxyl-terminus of the subtype B Vpu were transported to the cell surface. These results suggest that a signal for the retention of the subtype B Vpu within the Golgi complex resides in the second alpha-helical domain.

The Vpu protein of human immunodeficiency virus type 1 has been shown to shunt the CD4 receptor molecule to the proteasome for degradation and to enhance virus release from infected cells. The exact mechanism by which the Vpu protein enhances virus release is currently unknown but some investigators have shown that this function is associated with the transmembrane domain and potential ion channel properties. In this study, we determined if the transmembrane domain of Vpu could be functionally substituted with that of the prototypical viroporin, the M2 protein of influenza A virus. We constructed chimeric vpu gene in which the transmembrane domain of Vpu was replaced with that of the M2 protein of influenza. This chimeric vpu gene was substituted for the vpu gene in the genome of a pathogenic simian human immunodeficiency virus, SHIVKU-1bMC33. The resulting virus, SHIVM2, synthesized a Vpu protein that had a slightly different Mr compared to the parental SHIVKU-1bMC33, reflecting the different sizes of the two Vpu proteins. The SHIVM2 was shown to replicate with slightly reduced kinetics when compared to the parental SHIVKU-1bMC33 but electron microscopy revealed that the site of maturation was similar to the parental virus SHIVKU1bMC33. We show that the replication and spread of SHIVM2 could be blocked with the antiviral drug rimantadine, which is known to target the M2 ion channel. Our results indicate a dose dependent inhibition of SHIVM2 with 100 microM rimantadine resulting in a >95% decrease in p27 released into the culture medium. Rimantadine did not affect the replication of the parental SHIVKU-1bMC33. Examination of SHIVM2-infected cells treated with 50 microM rimantadine revealed numerous viral particles associated with the cell plasma membrane and within intracytoplasmic vesicles, which is similar to HIV-1 mutants lacking a functional vpu. To determine if SHIVM2 was as pathogenic as the parental SHIVKU-1bMC33 virus, two pig-tailed macaques were inoculated and followed for up to 8 months. Both pig-tailed macaques developed severe CD4+ T cell loss within 1 month of inoculation, high viral loads, and histological lesions consistent with lymphoid depletion similar to the parental SHIVKU-1bMC33. Taken together, these results indicate for the first time that the TM domain of the Vpu protein can be functionally substituted with the TM of M2 of influenza A virus, and shows that compounds that target the TM domain of Vpu protein of HIV-1 could serve as novel anti-HIV-1 drugs.

The Vpu protein is the smallest of the proteins encoded by human immunodeficiency virus type 1 (HIV-1). This transmembrane protein interacts with the CD4 molecule in the rough endoplasmic reticulum (RER), resulting in its degradation via the proteasome pathway. Vpu also has been shown to enhance virion release from infected cells. While much has been learned about the function of Vpu in cell culture systems, its exact role in HIV-1 pathogenesis is still unknown. This has been primarily due to the lack of a suitable primate model system since vpu is found only in HIV-1 and simian immunodeficiency viruses isolated from chimpanzees (SIVcpz), and three species of old world monkeys within the genus Cercopithecus. Several laboratories have developed pathogenic molecular clones of simian-human immunodeficiency virus (SHIV) in which the tat, rev, vpu and env genes of HIV-1 are expressed in the genetic background of SIV. The availability of such clones has allowed investigators to assess the role of Vpu in pathogenesis using a relevant animal model. This review will focus on the current understanding of the structure-function relationships of Vpu protein and recent advances using the SHIV model to assess the role of Vpu in HIV-1 pathogenesis.

Antiviral effectors such as Natural Killer (NK) cells have impaired functions in chronic hepatitis B (CHB) patients. The molecular mechanism responsible for this dysfunction remains poorly characterized. We show that decreased cytokine production capacity of peripheral NK cells from CHB patients was associated with reduced expression of NKp30 and CD16, and defective mTOR pathway activity. Transcriptome analysis of patients NK cells revealed an enrichment for transcripts expressed in exhausted T cells suggesting that NK cell dysfunction and T cell exhaustion employ common mechanisms. In particular, the transcription factor TOX and several of its targets were over-expressed in NK cells of CHB patients. This signature was predicted to be dependent on the calcium-associated transcription factor NFAT. Stimulation of the calcium-dependent pathway recapitulated features of NK cells from CHB patients. Thus, deregulated calcium signalling could be a central event in both T cell exhaustion and NK cell dysfunction occurring during chronic infections.