FIV Gag: virus assembly and host-cell interactions

Abstract

Infection of domestic cats with virulent strains of the feline immunodeficiency virus (FIV) leads to an acquired immunodeficiency syndrome (AIDS), similar to the pathogenesis induced in humans by infection with human immunodeficiency virus type 1 (HIV-1). Thus, FIV is a highly relevant model for anti-HIV therapy and vaccine development. FIV is not infectious in humans, so it is also a potentially effective non-toxic gene therapy vector. To make better use of this model, it is important to define the cellular machinery utilized by each virus to produce virus particles so that relevant similarities can be identified. It is well understood that all replication-competent retroviruses encode gag, pol, and env genes, which provide core elements for virus replication. As a result, most antiretroviral therapy targets pol-derived enzymes (protease, reverse transcriptase, and integrase) orenv-derived glycoproteins that mediate virus attachment and entry. However, resistance to drugs against these targets is a persistent problem, and novel targets must be identified to produce more effective drugs that can either substitute or be combined with current therapy. Elements of the gag gene (matrix, capsid, nucleocapsid, and "late" domains) have yet to be exploited as antiviral targets, even though the Gag precursor polyprotein is self-sufficient for the assembly and release of virus particles from cells. This process is far better understood in primate lentiviruses, especially HIV-1. However, there has been significant progress in recent years in defining how FIV Gag is targeted to the cellular plasma membrane, assembles into virions, incorporates FIV Env glycoproteins, and utilizes host cell machinery to complete virus release. Recent discoveries of intracellular restriction factors that target HIV-1 and FIV capsids after virus entry have also opened exciting new areas of research. This review summarizes currently known interactions involving HIV-1 and FIV Gag that affect virus release, infectivity, and replication.

Figure 1. Overview of HIV-1 and FIV Gag domains and the virus assembly and release pathway

A) Gag is synthesized as a precursor protein (HIV-1 Pr55^Gag = FIV Pr50_Gag) that exists in both monomeric and oligomeric forms in the cytoplasm. Interactions between Gag and host factors target Gag to the plasma membrane (PM) in most cell types. Membrane binding facilitates higher-order Gag multimerization that leads to virus assembly and budding. Release of virus particles is driven by interactions with host cell factors. Maturation of the virus occurs during or shortly after virus release and is required for virus infectivity. Env is a highly glycosylated protein, synthesized in the endoplasmic reticulum (ER), that follows the secretory pathway through the trans-Golgi network to the PM. Env is incorporated into budding virus particles via interactions between the matrix (MA) domain of Gag and the Env cytoplasmic tail (Env-CT). Adapted from (Joshi and Freed, 2007) with permission of Future Medicine Ltd. B) Domains of the Gag precursor protein from HIV-1 and FIV are shown, including known or predicted functions and interactions. The MA domain is covalently modified with myristate (myr) at the N-terminus. Two patches of basic residues (+) in MA are modestly conserved. Capsid (CA) folds into an N-terminal domain (NTD), which contains a conserved cyclophilin A (CypA) binding loop, and a C-terminal domain (CTD). Nucleocapsid (NC) contains two conserved zinc-coordinating motifs (Zn), which are important for interaction of Gag with viral RNA. NC is flanked by two spacer peptides (sp) in both HIV-1 and FIV, which must be cleaved from CA and NC during maturation for full viral infectivity. C-terminal domains of Gag in HIV-1 (p6) and FIV (p2) contain late domains important for virus release. C) An alignment of C-terminal domains of several lentiviral Gag proteins is shown, highlighting a highly conserved PSAPP motif that binds the ubiquitin E2-variant (UEV) domain of tumor-susceptibility gene 101 (Tsg101). Equine infectious anemia virus (EIAV) and HIV-1 share an LYPX_nL motif that binds the V domain of apoptosis-linked-gene-2-interacting protein X (Alix). BIV, bovine immunodeficiency virus; SIVagm, simian immunodeficiency virus from African green monkey.

Figure 2. Relationship between retrovirus release and ESCRT machinery

Retroviruses utilize late domains in Gag to recruit endosomal-sorting complexes required for transport (ESCRT-0, I, II, III), which facilitate virus release from the plasma membrane (PM). An example of the normal function of ESCRT is demonstrated by the downregulation of epidermal growth factor receptor (EGFR). Upon binding its ligand (EGF) at the PM, the EGFR is monoubiquitinated and internalized by endocytosis. Attachment of ubiquitin (Ub) to EGFR targets Ub-EGFR to the multivesicular body (MVB) through an endosomal sorting process, and results in the degradation of EGFR in lysosomes. First, Ub-EGFR bound to an endocytic vesicle is internalized into early endosomes by ESCRT-0 through a direct interaction with hepatocyte growth factor regulated tyrosine kinase substrate (Hrs). ESCRT-I (I) is recruited to Ub-EGFR through a PSAP motif in Hrs, which binds the UEV domain of Tsg101. The Tsg101 UEV domain binds Ub-EGFR to complete the transfer from Hrs. Tsg101 itself is bound to ESCRT-I through C-terminal Stalk and Head domains. ESCRT-I bound to Ub-EGFR then traffics from the early endosome to the MVB. Through a series of interactions with ESCRT-II, III, Alix, and Vps4, an intraluminal vesicle (ILV) containing EGFR is formed, which buds into the MVB. HIV-1 and FIV Gag are each ubiquitinated proteins with a PT/SAP motif, which mimic Hrs and recruit ESCRT machinery to stimulate virus release from the PM. TSG-5' and TSG-3' constructs are dominant-negative inhibitors of Tsg101 that inhibit HIV-1 and FIV release, because each lacks components found in the endogenous full-length protein. Alix is an ESCRT-associated protein that binds many cellular factors in the domains indicated, including Tsg101 and ESCRT-III. HIV-1 and EIAV Gag each contain a YPX_nL motif that binds the Alix V domain. Association of Gag with Alix recruits ESCRT machinery, which enhances virus release. Expression of Alix-V alone, without ESCRT-(I,III)-binding domains, inhibits HIV-1 and EIAV Gag release but has no effect on FIV. Adapted from (Demirov and Freed, 2004) with permission from Elsevier.