Role of Viral Protein U (Vpu) in HIV-1 Infection and Pathogenesis

Human immunodeficiency virus (HIV)-1 and HIV-2 originated from cross-species transmission of simian immunodeficiency viruses (SIVs). Most of these transfers resulted in limited spread of these viruses to humans. However, one transmission event involving SIVcpz from chimpanzees gave rise to group M HIV-1, with M being the principal strain of HIV-1 responsible for the AIDS pandemic. Vpu is an HIV-1 accessory protein generated from Env/Vpu encoded bicistronic mRNA and localized in cytosolic and membrane regions of cells capable of being infected by HIV-1 and that regulate HIV-1 infection and transmission by downregulating BST-2, CD4 proteins levels, and immune evasion. This review will focus of critical aspects of Vpu including its zoonosis, the adaptive hurdles to cross-species transmission, and future perspectives and broad implications of Vpu in HIV-1 infection and dissemination.


Introduction
Human immunodeficiency virus (HIV) is a lentivirus that belongs to the Retroviridae family. The HIV retrovirus contains two RNA molecules with three prototypic genes that encode group-specific antigen (gag), envelope (env), and polymerase (pol) proteins [1][2][3]. HIV isolates have been classified into two types: HIV-type 1 (HIV-1) and HIV-type 2 (HIV-2). HIV-1 is the causative agent of HIV/AIDS, while HIV-2 is constrained to some welldefined Central and Western Africa regions; relative to HIV-1, HIV-2 has weak transmission capabilities [4,5]. Based on phylogenetic analysis, HIV-1 shows substantial similarities to the virus SIVgor that infects gorillas (Gorilla gorilla) and the virus SIVcpz that infects chimpanzees (Pan troglodytes troglodytes). HIV-2 resembles the virus SIVsmm that infects sooty mangabey monkeys (Cercocebus atys). HIV-1 evolved from chimpanzees and/or gorillas by independent cross-species transmissions of SIVs [4,5].
The HIV-1 virus has been subdivided into M, N, O, and P subtypes according to their origin and distribution patterns within the human population. HIV-1 M is globally distributed and is a major factor causing the pandemic disease AIDS. HIV-1 N is rare (non-major/non-outlier) and originated from chimpanzees [5]. HIV-1 O and P groups have close relationships to the virus SIVgor isolated from gorillas; geographically, the O group virus is constrained to Cameroon and surrounding countries [5]. The P group virus was discovered to come from two individuals in Cameroon. Vpu protein is encoded by all groups of HIV-1, but biological differences have been noted between the various sources of Vpu proteins. Pandemic M group viruses contain Vpu proteins that are more highly active than do other groups [5]. Clearly, one needs to understand the functions of Vpu proteins to understand their roles in controlling HIV-1 infection and HIV/AIDS disease pathogenesis.

Vpu Gene and Its Diversification
Vpu was initially characterized as an U open reading frame (ORF) product localized in the HIV-1 genome between the env and tat exons [6]. The Vpu protein is translated from bicistronic mRNA of env-vpu presumably through leaky scanning of ribosomes from the initiation codon of the vpu gene [7,8]. The vpu gene is encoded in the HIV-1 genome, but it is not present in the genomes of HIV-2 and of SIVs such as SIV from rhesus macaques (SIVmac) and SIV from sooty mangabey (SIVsmm) [4,5,9]. However, structural homologs of Vpu have been identified in SIV from chimpanzee (SIVcpz), as well as in SIV from the greater spot-nosed monkey (Cercopithecus nictitans; SIVgsn), the mona monkey (Cercopithecus mona; SIVmon), the mustached monkey (Cervicopithecus Cephus; SIVmus), Dent's mona monkey (Cercopithecus mona denti; SIVdent), and recently in gorilla (Gorilla gorilla; SIVgor) [9][10][11][12][13].

Vpu Protein and Its Cellular Distribution
Vpu is a multimeric integral membrane phosphoprotein with 81 amino acids [14,15]. It has three distinct alpha-helices: the N-terminus proximal transmembrane domain (Helix1-TMD: 6-29 residues) and two C-terminus domains, Helix 2 (32-52 residues) and Helix 3 (57-72 residues) [16-19] ( Figure 1). Helix-2 is amphipathic and is hydrophobic with polar residues on the sides. The hydrophobic portion is buried in plasma membranes, while the hydrophilic region is cytoplasmic [20]. Helix-3 contains acidic amino acids interconnected by two phosphorylated Serine residues: S52 and S56 [21]. Protein kinase casein kinase 2 (CK-2) catalyzes the phosphorylation of the serines ( Figure 1) and these posttranslational modifications regulate associations between Helix 3 and beta-TrCP/ubiquitin ligase complexes [22,23]. Vpu once oligomerized can form pentameric pore-like structures through which selective monovalent cations can pass [24-26]. Vpu protein is composed of three different distinct alpha helices: the N-terminus proximal transmembrane domain (Helix1-TMD: 6-29 residues) and a cytoplasmic domain that consists of two alpha helices (Helix 2: 32-52 residues, Helix 3: 57-72 residues). The first cytoplasmic helix shows amphipathic behavior with hydrophobic and polar residues on the sides. The hydrophobic portion is buried in the cell membrane, while the hydrophilic region is exposed to the cytoplasmic side. The second cytoplasmic helix is formed by acidic amino acids. Two phosphorylated serine residues, S52 and S56, interconnect these cytoplasmic helices.
Vpu proteins localize to the plasma membranes, endoplasmic reticulum (ER), and trans-Golgi network (TGN) [27][28][29][30]. Sequence analysis of the cytosolic domain of Vpu shows the presence of putative trafficking signals that carry variations in amino acid residues among different subtypes of Vpu. These signals include YXXΦ, a conserved tyrosine-based sorting motif (where Φ represents a hydrophobic residue), and a ([D/E] XXXL [/I/V]) sorting motif consisting of acidic residue/dileucine-based sequences that are present in the hinge portion between the cytosolic domain and the TMD. The latter is involved in endocytosis and the targeting of transmembrane host proteins to lysosomes [31]. Another ([D/E] XXXL [/I/V]) motif is present in the second alpha-helix of the cytoplasmic domain [29]. Several primary isolates of HIV-1 and laboratory-adapted viruses carry polymorphisms of the vpu gene that are based on variations of putative trafficking signal sequences [32] and these polymorphisms regulate subcellular distribution patterns and biological activities of the Vpu protein.
BST-2 is a 30-36 kDa type II integral membrane protein that is expressed constitutively as well as following induction by type-I interferon or other pro-inflammatory signals [65]. It consists of a short N-terminal cytoplasmic tail inter-linked to a transmembrane domain and an extracellular domain anchored in the membrane via its glycosylphosphatidylinositol (GPI) moiety in the C-terminal region [73]. BST-2 is distributed mainly in cholesterol-rich microdomains of the cell membrane and intracellular compartments such as the trans-Golgi-network (TGN) and endosomes [73,74].
BST-2 can physically tether de novo generated virion particles at the cell membrane of infected cells, thereby decreasing virus release [75,76]. This tethering occurs following formation of homodimers via parallel disulfide-bonding and cross-linking with virions particles and plasma membranes through its membrane anchoring N-terminal domains [75,77]. BST-2 makes "axial" arrangements in which the BST-2 GPI anchors remain connected to the membrane of infected cells. Vpu downregulates BST-2 and interactions between Vpu and BST-2 utilize their respective transmembrane domains. The Ala14, Ala18, and Trp22 residues of the Vpu TMD are crucial for BST-2 downregulation from the cell membrane through direct interaction with specific residues on BST-2 (Val, Iso, Leu, and Leu) [78,79]. These residues are involved in TMD-TMD interactions, create an anti-parallel helix-helix interface [80], and maintain the interaction between these proteins [81,82]. The antagonistic effect of Vpu on BST-2 activity takes place by three sequential steps: downregulation from the cell surface, restriction of BST-2 recycling, and decline in intracellular BST-2 levels. Downregulation of BST-2 at the cell surface is mediated by clathrin-coated vesicles through direct interaction of the AP2 (clathrin adaptor complex) with a Y6XY8 motif (non-canonical dual Tyrosine residues) present in the cytoplasmic tail of BST-2 [74,83]. Vpu restricts the recycling of internalized BST-2 to the cell membrane and blocks the translocation of de novo generated BST-2 to the cell membrane [84][85][86][87].
BST-2 is degraded by ubiquitination following recruitment of the SCF-β-TrCP-E3 ligase complex to the DS52GxxS56 motif that is present in the cytosolic region of Vpu [88,89] ( Figure 3). Vpu enhances ubiquitination of BST-2 through lysine/serine and threonine amino acid residues present in the cytoplasmic tail of Vpu [89]. Vpu induces ubiquitination and degradation of both BST-2 and CD4 by identical molecular mechanisms, although the outcomes are different. Instead of targeting BST-2 to proteasomes, Vpu induces the β-TrCP-dependent sorting of BST-2 to lysosomes [77,84,88]. Moreover, it has been shown that the ESCRT complex and Rab7 are critical components of the endo-lysosomal trafficking involved in the degradation of BST-2 [90,91] (Figure 3).
Of the four HIV-1 groups, the M group is the most highly pathogenic and transmissible because Vpu is highly active, and this increases the ability of the virus to disseminate from one cell to another by counteracting the host protein BST-2 and evading the immune system (Table 1).

Immune Evasion to Virus Fitness and Survival
Innate immune responses play a significant role in host defenses against viral infections. Innate immune cells like natural killer (NK) and dendritic cells respond to invading viruses and contribute to controlling viral infection and replication during the initial stages of infection [92][93][94]. However, Vpu stabilizes HIV-1 infection and replication by evading immune responses by CD1d and NTB-A downmodulation [42,54]. Vpu also downregulates MHCII molecules from the cell surface to inhibit antigen presentation [39]. Together, these responses help HIV-1-infected cells escape cytotoxic and natural killer cells' ability to kill the infected cell.

Regulation of Ion Channel Activity
Vpu can oligomerize its transmembrane domain and form pentamer ion channel pores selective for monovalent cations [16,24,26]. Vpu ion channel activity is regulated by serine (S23) amino acids that are conserved in HIV-1 M group viruses [106,107]. TASK-1, a mammalian two-pore potassium channel protein with structural homology protein to Vpu, stabilizes cell membrane potential [108,109]. Vpu interacts with TASK-1 proteins, inhibits its ion channel activity, and depolarizes plasma membranes to enhance cellular secretions [108][109][110].

Vpu Effects on HIV-1 LTR Activity
HIV-1 gene activation is dependent on host transcription factors including NF-κB, NFAT, and Ap-1 [111]. Vpu and its structural homolog TASK-1 inhibit transcription of unintegrated HIV-1 DNA in an NF-κB-dependent manner [112]. Vpu mutants (replaced transmembrane domain of Vpu with its structural homologs) also suppress virus production by reducing LTR activity by an unknown mechanism [112,113]. Thus, Vpu appears to be capable of regulating LTR activity to control virus production in infected cells possibly through the involvement of zinc finger proteins and histone deacetylase (HDAC) [114][115][116].
Autophagy can enhance virus release and secretions from infected macrophage or monocyte cells [133][134][135]. HIV-1 enhances autophagy, while HIV-1 Nef blocks autophagy by direct interactions with Beclin and TFEB sequestration [136,137]. However, very little is currently known about the effects of Vpu on endolysosome degradation or autophagy pathways.

Conclusions
Vpu is an HIV-1 protein that counteracts host factors crucial for disseminating virus and disease progression. The primary targets of Vpu are cell surface host proteins that promote ubiquitination and proteasomal degradation processes [138][139][140]. Vpu might be targeted therapeutically to block the formation of heterooligomeric interactions between Vpu and host proteins at the cell surface as well as to suppress the progression of HIV-1 infection [141]. Moreover, Vpu disturbs the ubiquitination of host proteins by interacting with cellular factor β-TrCP through the cytosolic DSGxxS motif [138].

Hence, the transmembrane domain and DSxxSG motif in the cytosolic domain of Vpu may be targeted therapeutically against HIV-1 infection and disease progression.
Author Contributions: All authors contributed equally. All authors have read and agreed to the published version of the manuscript.

Funding:
We gratefully acknowledge different research funding support provided by the National Institute of Health (NIH), USA; P30GM100329, U54GM115458, R01MH100972, R01MH105329, R01MH119000, 2R01NS065957, and 2R01DA032444. We would like to thank Parinaz Ghanbari for designing the illustrations contained in this article.  [CrossRef]