Budding of Retroviruses Utilizing Divergent L Domains Requires Nucleocapsid

Abstract

eWe recently reported that human immunodeficiency virus type 1 (HIV-1) carrying PTAP and LYPX n L L domains ceased budding when the nucleocapsid (NC) domain was mutated, suggesting a role for NC in HIV-1 release. Here we investigated whether NC involvement in virus release is a property specific to HIV-1 or a general requirement of retroviruses. Specifically, we examined a possible role for NC in the budding of retroviruses relying on divergent L domains and structurally homologous NC domains that harbor diverse protein sequences. We found that NC is critical for the release of viruses utilizing the PTAP motif whether it functions within its native Gag in simian immunodeficiency virus cpzGAB2 (SIVcpzGAB2) or SIVsmmE543 or when it is transplanted into the heterologous Gag protein of equine infectious anemia virus (EIAV). In both cases, virus release was severely diminished even though NC mutant Gag proteins retained the ability to assemble spherical particles. Moreover, budding-defective NC mutants, which displayed particles tethered to the plasma membrane, were triggered to release virus when access to the cell endocytic sorting complex required for transport pathway was restored (i.e., in trans expression of Nedd4.2s). We also examined the role of NC in the budding of EIAV, a retrovirus relying exclusively on the (L)YPX n L-type L domain. We found that EIAV late budding defects were rescued by overexpression of the isolated Alix Bro1 domain (Bro1). Bro1-mediated rescue of EIAV release required the wild-type NC. EIAV NC mutants lost interactions with Bro1 and failed to produce viruses despite retaining the ability to self-assemble. Together, our studies establish a role for NC in the budding of retroviruses harboring divergent L domains and evolutionarily diverse NC sequences, suggesting the utilization of a common conserved mechanism and/or cellular factor rather than a specific motif.

Identification of the HIV-1 NC Binding Interface in Alix Bro1 Reveals a Role for RNA

“…However, clues into the necessity for interactions between NC and Bro1 come from the latter's ability to recruit CHMP4, which links Gag directly to membrane fission-inducing ESCRT-III members. Consistent with this notion, Bro1 can function as the smallest unit of Alix as its ectopic expression promoted virus release, provided it retained binding to both NC and CHMP4 (4,10,11). Moreover, an Alix mutant lacking binding to either NC or p6 failed to replace cellular Alix and promote EIAV budding (Fig.…”

“…Since the discovery of NC-Bro1 interactions, questions regarding their role in virus release arose. Several lines of evidence underscored the importance of NC-Bro1 interactions, including the findings that a functional NC is required for Alix-mediated virus release (4,10,11,31), and mutant NC viruses that fail to bind the Bro1 domain were also defective in virus budding (4,10,11). The role of Bro1-NC interactions in virus release was further strengthened by the identification of residues that mediate binding.…”

“…3C). Moreover, mutation of basic residues in NC also eliminated binding to Bro1 (4,10,36), implying the involvement of a negatively charged factor. RNA, however, was excluded from Bro1-NC interactions in a previous report (31).…”

“…The N-terminally Flag-tagged Nedd4.1 was described by Sette et al (35) and the C-terminally HA-tagged APOBEC3G by Huthhoff and Malim (16). The glutathione S-transferase (GST) fusion plasmids encoding the EIAV UK NC-p9 region, HIV-1 NC-p6, and its mutants, NC RKI -p6 and NC RKII -p6, were previously described (4,10,36).Virus release analysis. 293T cells were maintained and transfected as previously described (36).…”

Interaction of Human Parainfluenza Virus Type 3 Nucleoprotein with Matrix Protein Mediates Internal Viral Protein Assembly

“…For RSV, a transcription antiterminator, M2-1, mediates the association of RNPs with the M protein and is required for the incorporation of RNPs into virions (17), and further structural analysis showed that M2-1 is located between the RNP and M in isolated viral particles (18). However, for viruses belonging to Paramyxovirinae subfamilies and some other enveloped viruses, such as retroviruses and filoviruses, N has been described to be a mediator of virion assembly and budding (6,14,(19)(20)(21). At least two reports suggested that N of influenza virus plays a critical role in virion assembly, possibly through its interaction with M1.…”