Avian Sarcoma Virus and Human Immunodeficiency Virus, Type 1 Use Different Subsets of ESCRT Proteins to Facilitate the Budding Process

Abstract

Members of the Nedd4 family of E3 ubiquitin ligases bind the L domain in avian sarcoma virus (ASV) Gag and facilitate viral particle release. Translational fusion of ASV Gag with an L domain deletion (⌬p2b) to proteins that comprise ESCRT-I, -II, and -III (the endocytic sorting complexes required for transport) rescued both Gag ubiquitination and particle release from cells. The ESCRT-I factors Vps37C or Tsg101 were more effective in rescue of Gag/⌬p2b budding than the ESCRT-II factor Eap20 or the ESCRT-III component CHMP6. Thus ESCRT components can substitute for Nedd4 family members in ASV Gag release. Unlike wild type, ASV Gag/⌬p2b -ESCRT chimeras failed to co-immunoprecipitate with co-expressed hemagglutinin-tagged Nedd4, indicating that Nedd4 was not stably associated with these Gag fusions. Release of the Gag-ESCRT-I or -II fusions was inhibited by a dominant negative mutant of Vps4 ATPase similar to wild type ASV Gag. In contrast to ASV Gag, HIV-1 Gag containing an L domain inactivating mutation (P7L) was efficiently rescued by fusion to a component of ESCRT-III (Chmp6) but not ESCRT-II (Eap20). Depletion of the endogenous pool of Eap20 (ESCRT-II) had little effect on HIV-1 Gag release but blocked ASV Gag release. In contrast, depletion of the endogenous pool of Vps37C (ESCRT-I) had little effect on ASV but blocked HIV-1 Gag release. Furthermore, an N-terminal fragment of Chmp6 inhibited both HIV-1 and ASV Gag release in a dominant negative manner. Taken together, these results indicate that ASV and HIV-1 Gag utilize different combinations of ESCRT proteins to facilitate the budding process, although they share some common elements.Retroviruses, and many other enveloped viruses, evolved mechanisms to exploit components of the endocytic sorting pathway to bud from cells efficiently. The retroviral Gag precursor polyprotein contains the major structural components of the virus, including late assembly domains that function as docking sites for host cell factors that promote the release of virus-like particles (VLPs) 4 from the plasma membrane (1-7). Although distinct classes of L domain sequences exist among retroviruses (with core motifs of PTAP, PPXY, and YPXL), each functions as a binding site to recruit different components of the vacuolar protein sorting (Vps) machinery. Class E Vps proteins function in the process of sorting cargo proteins into the luminal vesicles of multivesicular bodies (MVBs). MVBs are carrier vesicles that deliver cargo designated for lysosomal degradation from early to late endosomes (8). Functionally conserved from yeast to mammalian cells, most class E Vps proteins are organized into high molecular weight cytoplasmic complexes called ESCRT (endocytic sorting complexes required for transport) -I, -II, and -III. Although the mechanistic details of MVB biogenesis remain poorly characterized, current studies suggest that the ESCRT complexes are sequentially recruited to endosomal compartments to remodel and invaginate the limiting endosomal membrane and sort ubiquitinated protein ...

Vps4 and the ESCRT-III complex are required for the release of infectious hepatitis C virus particles

“…A DN ATPase-defective mutant of Vps4, in which the active site glutamic acid has been mutated to glutamine, leads to defective MVB sorting and formation of aberrant endosomes in BHK cells (Bishop & Woodman, 2000) and also in Huh7 cells. Production of infectious particles of HIV-1 (Garrus et al, 2001), HSV-1 (Crump et al, 2007), HBV (Lambert et al, 2007) and ASV (Pincetic et al, 2008) is inhibited upon expression of the Vps4DN protein. The wild-type or DN forms of Vps4, tagged with GFP (Vps4WT-GFP or Vps4DN-GFP) were transfected into Huh7 cells, the distribution of Vps4DN-GFP was distinct from Vps4WT ( Fig.…”

“…In the absence of Vps4 activity, the ESCRT complex cannot function and aberrant endosomes are formed. Numerous enveloped RNA viruses such as human immunodeficiency virus type 1 (HIV-1) (Garrus et al, 2001), Ebola virus (Martin-Serrano et al, 2001) and avian sarcoma virus (ASV) (Pincetic et al, 2008) and, more recently, enveloped DNA viruses including hepatitis B virus (HBV) (Lambert et al, 2007) and herpes simplex virus 1 (HSV-1) (Crump et al, 2007) have been shown to utilize ESCRT complexes during virion morphogenesis. In most cases viruses interact with the ESCRT machinery via late (L) domains, short motifs normally found in capsid or matrix proteins, which act as docking sites for ESCRT components.…”

Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions

“…Thereafter, budding occurs concurrently with the release of ESCRT components from the MVB membrane induced by an AAA-ATPase, Vps4, which is present in humans as two isoforms, Vps4A and Vps4B (Babst et al, 1998;Katzmann et al, 2002). Numerous enveloped RNA viruses such as human immunodeficiency virus type 1 (HIV-1) (Garrus et al, 2001), Ebola virus (Martin-Serrano et al, 2001) and avian sarcoma virus (ASV) (Pincetic et al, 2008) and, more recently, hepatitis C virus (HCV) (Ariumi et al, 2011;Corless et al, 2010;Lai et al, 2010) have been shown to utilize ESCRT complexes during virion morphogenesis. Furthermore, enveloped DNA viruses including hepatitis B virus (HBV) (Lambert et al, 2007;Watanabe et al, 2007) and herpes simplex virus 1 (HSV-1) (Crump et al, 2007) have been reported to exploit the MVB machinery.…”

The Roles of ESCRT Proteins in Healthy Cells and in Disease

“…Recent studies have shown that ESCRT-III and Vps4 can be recruited independently of either Tsg101 or Alix by the herpes simplex virus type-1 (Pawliczek & Crump et al, 2009) and the hepatitis C virus (Corless et al, 2010). ESCRT-II was found not to be essential for HIV-budding (Langelier et al, 2006), however ESCRT-II was discovered recently to be essential for release of the avian sarcoma virus (Pincetic et al, 2008). Other viruses such as the rabies virus can indirectly recruit the ESCRTs' by using the PPxY motif to specifically recruit WW-domain-containing E3 ubiquitin ligases of the Nedd4 family (Kikonyogo et al, 2001).…”