Preservation of Tetherin and CD4 Counter-Activities in Circulating Vpu Alleles despite Extensive Sequence Variation within HIV-1 Infected Individuals

Abstract

The HIV-1 Vpu protein is expressed from a bi-cistronic message late in the viral life cycle. It functions during viral assembly to maximise infectious virus release by targeting CD4 for proteosomal degradation and counteracting the antiviral protein tetherin (BST2/CD317). Single genome analysis of vpu repertoires throughout infection in 14 individuals infected with HIV-1 clade B revealed extensive amino acid diversity of the Vpu protein. For the most part, this variation in Vpu increases over the course of infection and is associated with predicted epitopes of the individual's MHC class I haplotype, suggesting CD8+ T cell pressure is the major driver of Vpu sequence diversity within the host. Despite this variability, the Vpu functions of targeting CD4 and counteracting both physical virus restriction and NF-κB activation by tetherin are rigorously maintained throughout HIV-1 infection. Only a minority of circulating alleles bear lesions in either of these activities at any given time, suggesting functional Vpu mutants are heavily selected against even at later stages of infection. Comparison of Vpu proteins defective for one or several functions reveals novel determinants of CD4 downregulation, counteraction of tetherin restriction, and inhibition of NF-κB signalling. These data affirm the importance of Vpu functions for in vivo persistence of HIV-1 within infected individuals, not simply for transmission, and highlight its potential as a target for antiviral therapy.

Author Summary

The accessory protein Vpu, encoded by HIV-1, performs at least two major roles in the virus life cycle, namely the degradation of newly synthesized CD4 molecules and the counteraction of a host antiviral protein, tetherin. These activities promote the release of infectious viruses from host cells, and recent evidence suggests that Vpu function has been crucial for the cross-species transmission of HIV-1 from chimpanzees, and its subsequent pandemic spread in humans. Here we studied the functional variation in Vpu in infected individuals. We found that the Vpu amino acid sequence can be highly variable within an individual, and that this variation is likely to result from host immune responses targeting antigens derived from Vpu. However, despite this variation, Vpu's major functions are preserved, with only a minority of circulating alleles showing defects throughout the course of infection. These data suggest that defective Vpu proteins are selected against within the infected individual, implying that Vpu functions are critical for HIV-1 replication throughout natural infection, not simply at transmission. Therefore Vpu may represent a novel target for antiviral therapy to augment current treatment strategies for HIV/AIDS.