Substitution of the transmembrane domain of Vpu in simian–human immunodeficiency virus (SHIVKU1bMC33) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques

Abstract

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.

Identification of amino acids within the second alpha helical domain of the human immunodeficiency virus type 1 Vpu that are critical for preventing CD4 cell surface expression

“…None of the mutations were found to disrupt the α-helical region (data not shown). Previously, we showed that fusion of the Vpu protein to enhanced green fluorescent protein (EGFP) still resulted in the ability to down-modulate cell surface CD4 (Hout et al, 2005; 2006; Singh et al, 2003). We analyzed cell surface CD4 expression of the ten Vpu mutant proteins with the unmodified subtype B (VpuEGFP) as a positive control and a Vpu protein with both serine residues of the two casein kinase II sites changed to glycine residues (Vpu S52,56G EGFP) as a negative control.…”

Determinants of Tetherin Antagonism in the Transmembrane Domain of the Human Immunodeficiency Virus Type 1 Vpu Protein

“…Since scrambling the TM domain blocks both ion channel activity and efficient virus release, it was thought that these activities were related (10,57). Indeed, amiloride-based drugs that block Vpu channel function have been shown to display antiviral activity against HIV-1 in (tetherin-positive) macrophages (9), and other studies report that chimeric Vpu proteins bearing the TM domain of the influenza A virus proton channel M2 were functional for virus release and sensitive to rimantadine (19,20). However, since we found that mutation of S23, known to be required for channel activity (60) and the potential target for amilorides (26), had no effect on tetherin inactivation in our assays, there appears to be a discrepancy in directly correlating cation transport and virus release (tetherin antagonism).…”

The Vpu Protein: New Concepts in Virus Release and CD4 Down-Modulation

“…Substitution of the M2 TMD histidine residue with an alanine resulted in a constitutively open channel, indicating its importance to channel activation [32, 33, 68, 78]. Using this as a basis, we first made a simian human immunodeficiency virus (SHIV) in which the TMD of Vpu was exchanged with the TMD of M2 [34]. This virus, SHIV M2 , was capable of causing a severe loss of CD4 + T cells and AIDS when inoculated into macaques.…”

“…SIV cpz CAM13, SIV cpz ANT, and SIV cpz TAN1 only contain a single casein kinase II site. All four vpu isolates were fused in frame to the gene for enhanced green fluorescence protein (EGFP) and expressed under the control of a CMV promoter, similar to the HIV-1 subtype B vpu genes our laboratory has previously analyzed [34, 35, 74]. All four SIV cpz Vpu fusion proteins were membrane-associated, partially co-localized with DsRed-ER and ECFP-Golgi marker proteins, and completely co-localized with an ECFP-Membrane marker.…”