Angiomotin functions in HIV-1 assembly and budding

Many retroviral Gag proteins contain PPXY late assembly domain motifs that recruit proteins of the NEDD4 E3 ubiquitin ligase family to facilitate virus release. Overexpression of NEDD4L can also stimulate HIV-1 release but in this case the Gag protein lacks a PPXY motif, suggesting that NEDD4L may function through an adaptor protein. Here, we demonstrate that the cellular protein Angiomotin (AMOT) can bind both NEDD4L and HIV-1 Gag. HIV-1 release and infectivity are stimulated by AMOT overexpression and inhibited by AMOT depletion, whereas AMOT mutants that cannot bind NEDD4L cannot function in virus release. Electron microscopic analyses revealed that in the absence of AMOT assembling Gag molecules fail to form a fully spherical enveloped particle. Our experiments indicate that AMOT and other motin family members function together with NEDD4L to help complete immature virion assembly prior to ESCRT-mediated virus budding.

Late Assembly Domain Function Can Exhibit Context Dependence and Involves Ubiquitin Residues Implicated in Endocytosis

ABSTRACT Retroviral Gag polyproteins contain regions that promote the separation of virus particles from the plasma membrane and from each other. These Gag regions are often referred to as late assembly (L) domains. The L domain of human immunodeficiency virus type 1 (HIV-1) is in the C-terminal p6 gag domain and harbors an essential P(T/S)APP motif, whereas the L domains of oncoretroviruses are in the N-terminal half of the Gag precursor and have a PPXY core motif. We recently observed that L domains induce the ubiquitination of a minimal HIV-1 Gag construct and that point mutations which abolish L domain activity prevent Gag ubiquitination. In that study, a peptide from the Ebola virus L domain with overlapping P(T/S)APP and PPXY motifs showed exceptional activity in promoting Gag ubiquitination and the release of virus-like particles. We now show that a substitution which disrupts the PPXY motif but leaves the P(T/S)APP motif intact abolishes L domain activity in the minimal Gag context, but not in the context of a near full-length HIV-1 Gag precursor. Our results reveal that the P(T/S)APP motif does not function autonomously and indicate that the HIV-1 nucleocapsid-p1 region, which is proximal to p6 gag , can cooperate with the conserved L domain core motif. We have also examined the effects of ubiquitin mutants on virus-like particle production, and the results indicate that residues required for the endocytosis function of ubiquitin are also involved in virus budding.

Efficient Particle Production by Minimal Gag Constructs Which Retain the Carboxy-Terminal Domain of Human Immunodeficiency Virus Type 1 Capsid-p2 and a Late Assembly Domain

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Gag precursor Pr55 gag by itself is capable of assembling into retrovirus-like particles (VLP). In the present study, we attempted to identify the minimal Gag sequences required for the formation of VLP. Our results show that about 80% of Pr55 gag can be either deleted or replaced by heterologous sequences without significantly compromising VLP production. The smallest chimeric molecule still able to efficiently form VLP was only about 16 kDa. This minimal Gag construct contained the leucine zipper domain of the yeast transcription factor GCN4 to substitute for the assembly function of nucleocapsid (NC), followed by a P-P-P-P-Y motif to provide late budding (L) domain function, and retained only the myristylation signal and the C-terminal capsid-p2 domain of Pr55 gag . We also show that the L domain function of HIV-1 p6 gag is not dependent on the presence of an active viral protease and that the NC domain of Pr55 gag is dispensable for the incorporation of Vpr into VLP.

Equine Infectious Anemia Virus Gag Polyprotein Late Domain Specifically Recruits Cellular AP-2 Adapter Protein Complexes during Virion Assembly

ABSTRACT We have identified an interaction between the equine infectious anemia virus (EIAV) late assembly domain and the cellular AP-2 clathrin-associated adapter protein complex. A YXXL motif within the EIAV Gag late assembly domain was previously characterized as a sequence critical for release of assembling virions. We now show that this YXXL sequence interacts in vitro with the AP-50 subunit of the AP-2 complex, while the functionally interchangeable late assembly domains carried by the Rous sarcoma virus p2b protein and human immunodeficiency virus type 1 p6 protein, which utilize PPPY and PTAPP L domains, respectively, do not bind AP-50 in vitro. In addition, EIAV late domain mutants containing mutations that have previously been shown to abrogate budding also exhibit marked decreases in AP-50 binding efficiencies. A role for AP-2 complex in viral assembly is supported by immunofluorescence analysis of EIAV-infected equine dermal cells demonstrating specific colocalization of the α adaptin subunit of AP-2 with the EIAV p9 protein at sites of virus budding on the plasma membrane. These data provide strong evidence that EIAV utilizes the cellular AP-2 complex to accomplish virion assembly and release.