NEDD4L Overexpression Rescues the Release and Infectivity of Human Immunodeficiency Virus Type 1 Constructs Lacking PTAP and YPXL Late Domains

Abstract

The cellular ESCRT pathway functions in membrane remodeling events that accompany endosomal protein sorting, cytokinesis, and enveloped RNA virus budding. In the last case, short sequence motifs (termed late domains) within human immunodeficiency virus type 1 (HIV-1) p6Gag bind and recruit two ESCRT pathway proteins, TSG101 and ALIX, to facilitate virus budding. We now report that overexpression of the HECT ubiquitin E3 ligase, NEDD4L/NEDD4-2, stimulated the release of HIV-1 constructs that lacked TSG101-and ALIX-binding late domains, increasing infectious titers >20-fold. Furthermore, depletion of endogenous NEDD4L inhibited the release of these crippled viruses and led to cytokinesis defects. Stimulation of virus budding was dependent upon the ubiquitin ligase activity of NEDD4L and required only the minimal HIV-1 Gag assembly regions, demonstrating that Gag has ubiquitin-dependent, cis-acting late domain activities located outside of the p6 region. NEDD4L stimulation also required TSG101 and resulted in ubiquitylation of several ESCRT-I subunits, including TSG101. Finally, we found that TSG101/ESCRT-I was required for efficient release of Mason-Pfizer monkey virus, which buds primarily by using a PPXY late domain to recruit NEDD4-like proteins. These observations suggest that NEDD4L and possibly other NEDD4-like proteins can ubiquitylate and activate ESCRT-I to function in virus budding.The structural proteins of many enveloped RNA viruses, including human immunodeficiency virus type 1 (HIV-1) Gag, contain short cis-acting sequence motifs, termed "late domains," that recruit host factors to facilitate virus budding (5,29,35,59). To date, three well-characterized late-domain sequences and their cellular binding partners have been described: PT(S)AP late domains bind TSG101 (6, 12, 31, 57), YPXL late domains bind ALIX/AIP1 (51) (as well as the AP-2 adaptor complex [2]), and PPXY late domains bind various members of the large family of mammalian NEDD4 ubiquitin E3 ligases (hereafter called NEDD4-like proteins; reviewed in references 22, 29, and 46). Each of the late domain binding partners can be linked to the cellular ESCRT (endosomal sorting complex required for transport) pathway, indicating that this pathway functions in virus release.Within the cell, the ESCRT pathway also plays important roles in helping to sort membrane proteins into vesicles that bud into late endosomes/multivesicular bodies (MVB) (21, 38, 61). These vesicles carry membrane proteins from the limiting endosomal membrane into the endosomal lumen, where their contents can ultimately be degraded when MVBs fuse with lysosomes. The ESCRT pathway can also be recruited to function in the abscission stage of cytokinesis (1), a process in which the thin membranous midbody that connects two dividing cells is severed to complete the process of cell division. In addition to their shared dependence on the ESCRT pathway, the processes of MVB vesicle formation, abscission, and virus budding are similar in that the membrane fission events required for ve...

The ESCRT machinery at a glance

“…ESCRT-II is apparently not involved in HIV-1 budding, nor is the Vps20 subunit of ESCRT-III, which couples ESCRT-II and ESCRT-III in MVB biogenesis. Ubiquitylation by the ligase NEDD4L is also involved in ESCRT-mediated HIV-1 budding (Chung et al, 2008;Usami et al, 2008). In summary, ESCRT-I and ALIX appear to act as adaptors that connect the viral Gag protein to ESCRT-III, which in turn is directly responsible for membrane cleavage and virion release.…”

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

“…For example, viruses containing the PPxY motif are able to recruit Nedd4L and subsequently co-opt the ESCRT machinery in an as yet uncharacterized manner. Furthermore, despite not containing the PPxY motif, HIV-1 Gag protein deficient in Tsg101-and Alix-binding L-domains, can be rescued by overexpression of Nedd4L (Chung et al, 2008;Usami et al, 2008), highlighting the inter-changeability and flexibility inherent in L-domains. Thus, it is feasible that HCV gains entry to the ESCRT machinery by an as yet uncharacterized interaction, in a manner that is Tsg101-and Alix-independent.…”

Multiple Interactions between the ESCRT Machinery and Arrestin-Related Proteins: Implications for PPXY-Dependent Budding

“…For example, it is now established that Nedd4L can stimulate the release and infectivity of HIV-1 viruses that lack the PTAP and LYPXL late domains, and this activity requires the enzymatic activity encoded by Nedd4L (8,57,60). Additionally, Itch is recruited by MLV Gag in a PPXY-independent manner to promote viral egress, and, as shown for PPXY-dependent budding, this activity also requires the core ESCRT machinery (25).…”