1.A.40 The Human Immunodeficiency Virus Type I, HIV-1 (Retrovirdiac) Vpu Channel (Vpu-C) Family
The mechanisms and functions of viral channel proteins have been reviewed by Fischer and Hsu (2011) and Fischer et al. (2012). The Vpu-C channel protein is expressed in the subcellular membranes of infected cells but not in the membrane envelope of the virion. It plays multiple roles in the life cycle of HIV-1: it triggers virus release, probably dependent on channel formation, and it binds residues 402-420 of CD4 of the host cell. CD4 is the virion receptor for HIV-1, and Vpu promotes CD4 degradation. It is an 81 aa type I membrane protein, phosphorylated on serine residues 52 and 56. It forms a homopentameric channel, but can form multiple oligomers. Its channel activity has been reconstituted in planar lipid bilayers. It is a general ion conducting channel that prefers monovalent cations over anions. Viral channel-forming peptides/proteins have been reviewed, and Vpu has been modeled (Fischer and Hsu 2011). Phosphorylation of serine in Vpu promotes interactions with clathrin adapter proteins, AP1 (P61966) and AP2 (Q962W1.2) (Stoneham et al. 2017).
Reconstitution of both full length Vpu(1-81) and a short transmembrane (TM) domain comprising peptide Vpu(1-32) into bilayers under a constant electric field results in an asymmetric orientation of those channels. In both cases, channel activity with similar kinetics is observed (Mehnert et al. 2007). Channels can open and remain open within a broad series of conductance states even if a small or no electric potential is applied. The mean open time for Vpu peptide channels is voltage-independent. The rate of channel opening shows a biphasic voltage activation, suggesting that the gating is influenced by the interaction of the dipole moments of the TM helices with an electric field (Mehnert et al. 2007).
Synthetic proteins with 4 or 5 TMSs, all derived from the TMS of Vpu, have been constructed (Becker et al., 2004). They form discrete ion channels with conductances of 42 and 76 pS, respectively. This suggests that self assembly of monomers forms the physiological channel, most likely a pentamer. A variety of oligomers coexist in phospholipid bilayers, so that a unique supramolecular structure can not be defined. Nonetheless, oligomers of various sizes have similar intermolecular contacts and orientations (Lu et al., 2010). Interactions of Vpu with host cellular constituents have been reviewed (González 2015).
Although it is clear that Vpu can form channels, it is not certain that Vpu-mediated channel formation is the mechanism by which it exerts its physiological function. Vpu and the N-terminal 40 residue region of the mammalian background K+ channel, TASK-1, have structural and functional similarity, and these two proteins physically interact (Hsu et al., 2004). Vpu abolished the TASK-1 current, and overexpression of TASK-1 led to impairment of Vpu''''''''s ability to enhance viral particle release. Thus, it is possible that Vpu, a multifunctional protein, functions to control the current of TASK-1.
While the C-terminal domain of HIV-1 Vpu is critical for CD4 degradation, the transmembrane domain (TM) mediates ion channel activity, enhances virus release and is essential for counteracting CD317/Bst-2/Tetherin. Bolduan et al. (2011) analyzed whether the ion channel activity of Vpu is required to antagonize CD317-mediated restriction of virion release. Not the ion channel activity, but its ability to remove CD317 from the cell surface is required to augment HIV-1 release.
The role of histidine in channel-forming transmembrane (TM) helices was investigated by comparing the TM helices from Virus protein ''''u'''' (Vpu) and the M2 proton channel. Both proteins are members of the viroporin family of small membrane proteins that exhibit ion channel activity, and have a single TM helix that is capable of forming oligomers. The TM helices from both proteins have a conserved tryptophan towards the C-terminus. Alanine 18 of Vpu has been mutated to histidine in order to artificially introduce the same HXXXW motif that is central to the proton channel activity of M2. The mutated Vpu TMS resulted in an increase in helix tilt angle of 11° in lipid bilayers compared to the wild-type Vpu TMS. Wang et al. (2013) found the reverse when histidine 37 of the HXXXW motif in M2 was mutated to alanine; it decreased the helix tilt by 10° from that of wild-type M2. The tilt change was independent of both the helix length and the presence of tryptophan. Compared to wild-type M2, the H37A mutant displayed a lowered sensitivity to the proton concentration. The solvent accessibility of histidine-containing M2 was greater than without histidine. This suggests that the TM helix may increase solvent exposure by changing its tilt angle in order to accommodate a polar/charged residue within the hydrophobic membrane region. The comparative results of M2, Vpu and their mutants demonstrated the significance of histidine in a transmembrane helix and the plasticity of the function and structure of ion channels stemming from changes at a single amino acid site.
A fine-grained docking protocol was used to generate a bundle-like structure of the bitopic membrane protein Vpu, a type I membrane protein with 81 amino acids. Vpu forms ion- and substrate-conducting bundles in the plasma membrane in the infected cell. The Vpu1-32 peptide that includes the TMS assembles into homo-pentameric bundles around prepositioned Na, K, Ca or Cl ions. For bundles with the lowest energy, the TMSs generate a hydrophobic pore. Bundles in which Ser-24 faces the pore have higher energy. The tilt of the helices in the lowest energy bundles is larger than bundles with serines facing the pore. Left-handed bundles are lowest in energy where the ions are located at the serines (Li et al. 2013).
The transmembrane domain of Vpu has dual functions: it counteracts the human restriction factor tetherin and forms a cation channel. These two functions are causally unrelated. Greiner et al. 2016 examined structure and function correlates of different Vpu homologs from HIV-1 and SIV and showed that ion channel activity is an evolutionary conserved property of Vpu proteins. An electrophysiological testing of Vpus from different HIV-1 groups (N and P) and SIVs from chimpanzees (SIVcpz), and greater spot-nosed monkeys (SIVgsn) showed that they all possess channel activity.
The generalized transport reaction catalyzed by Vpu is:
ions (in) ions (out)