Activities of Transmitted/Founder and Chronic Clade B HIV-1 Vpu and a C-Terminal Polymorphism Specifically Affecting Virion Release

Abstract

Acute HIV-1 infection is characterized by a type I interferon response, resulting in the induction of host restriction factors. HIV-1 has evolved to counteract these factors, and one such adaptation, the ability of Vpu to counteract BST2/tetherin, is associated with the evolution of simian immunodeficiency virus (SIV cpz ) into pandemic group M human immunodeficiency virus type 1 (HIV-1). During transmission between individuals, very few viruses or even a single virus, the "transmitted/founder" (T/F) virus, gives rise to the new infection, but in the new host the selective pressure of the immune response yields the diverse "quasispecies" of chronic infection. Here we examine the functional characteristics of Vpu proteins encoded by T/F viruses compared to acute and chronic viruses from longitudinally sampled subjects. The studied T/F Vpu proteins showed a trend toward optimized CD4 downregulation compared to chronic Vpu proteins but did not differ substantially in their ability to downregulate BST2 or enhance virion release, although individual clones from each group were impaired in these activities. Analysis of the functionally impaired clones identified a C-terminal residue, W76, as important specifically for Vpu enhancement of virion release. Primary Vpu clones encoding a W76G polymorphism, or site-directed mutants encoding a W76G substitution, were impaired in their ability to enhance virion release, but they were not defective for BST2 surface downregulation. Conversely, the virion release function of impaired primary clones was restored by creating a G76W substitution. The identification of W76 as important for virion release enhancement that is independent of BST2 surface downregulation supports the potential to mechanistically separate these functions of Vpu. IMPORTANCETo establish infection in a host, HIV-1 must evade the host's immune response, including the production of antiviral factors. HIV-1 encodes proteins that antagonize these defenses, including Vpu. Vpu counteracts the host protein BST2, which blocks the release of progeny viruses from the host cell. To determine the importance of Vpu activity to HIV-1 transmission, this study assessed the functionality of Vpu from viruses isolated soon after transmission ("transmitted/founder" viruses) compared to isolates from chronic infection. Although the anti-BST2 activity of Vpu proteins from the tested transmitted/founder viruses did not differ from the activity of the chronic Vpu proteins, the transmitted/founder Vpu proteins trended toward having superior activity against another host protein, CD4. Further, this study identified an amino acid near the C terminus of Vpu that is specifically important for Vpu's ability to enhance the release of progeny virus from the host cell, supporting the notion of a new mechanism for this function of Vpu.

Resistance to type 1 interferons is a major determinant of HIV-1 transmission fitness

“…HIV-1 counteracts tetherin using its Vpu protein, which binds tetherin and prevents its expression on the cell surface (62)(63)(64). However, TF Vpu proteins do not seem to counteract tetherin more effectively than the Vpu protein of chronic viruses (65). Moreover, TF-infected CD4 + T cells were shown to produce more cell-free virions even in the absence of Vpu (54).…”

Membrane Anchoring by a C-terminal Tryptophan Enables HIV-1 Vpu to Displace Bone Marrow Stromal Antigen 2 (BST2) from Sites of Viral Assembly

“…The source of this residual activity has been attributed to the ability of Vpu to displace BST2 from virion-assembly sites in the plane of the membrane, an activity that has been roughly mapped to the C-terminal portion of the cytoplasmic domain of Vpu (16). During an analysis of naturally occurring Vpu proteins, we found the converse phenotype: the preserved ability to degrade and down-regulate BST2 yet impaired enhancement of virion release (17). This phenotype mapped to a W76G polymorphism, a mutation that presumably arises in infected hosts to provide escape from cytotoxic T lymphocytes.…”

“…This phenotype mapped to a W76G polymorphism, a mutation that presumably arises in infected hosts to provide escape from cytotoxic T lymphocytes. Site-directed mutagenesis confirmed that optimal enhancement of virion release depends upon Trp-76, which is near the C terminus of Vpu and is well conserved in certain HIV-1 clades (17). Substitution of a glycine for Trp-76 impairs virion release without affecting the ability of Vpu to co-localize with BST2, to downregulate BST2 from the cell surface, or to reduce the steadystate amount of cellular BST2.…”

“…Plasmids, Cells, and Reagents-Proviral plasmids pNL4-3, pNL4-3⌬Vpu (pNL4 -3/Udel (23)), pNL4-3-Vpu-S52N,S56N (24), pNL4-3-Vpu-W76G (17), pNL4-3-S52N,S56N ϩ W76G (17), and pNL4-3-Vpu-A10F,A14F,A18F (25) have been described previously. Rev-dependent FLAG-tagged Vpu expression constructs for NL4-3-Vpu, Vpu-S52N,S56N, Vpu-W76G, Vpu-S52N,S56N, and Vpu-S52N,S56N ϩ W76G were described previously (17).…”

“…Rev-dependent FLAG-tagged Vpu expression constructs for NL4-3-Vpu, Vpu-S52N,S56N, Vpu-W76G, Vpu-S52N,S56N, and Vpu-S52N,S56N ϩ W76G were described previously (17). Expression plasmids encoding Vpu-W76F and Vpu-S52N,S56N ϩ W76F with a C-terminal FLAG tag were constructed by site-directed mutagenesis of expression constructs for NL4-3-Vpu and Vpu-S52N,S56N, respectively, using a QuikChange II site-directed mutagenesis kit (Agilent Technologies).…”