Budding of retroviruses utilizing divergent L domains requires nucleocapsid.

We 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.

Late domain-independent rescue of a release-deficient Moloney murine leukemia virus by the ubiquitin ligase itch.

Moloney murine leukemia virus (MoMLV) Gag utilizes its late (L) domain motif PPPY to bind members of the Nedd4-like ubiquitin ligase family. These interactions recruit components of the cell's budding machinery that are critical for virus release. MoMLV Gag contains two additional L domains, PSAP and LYPAL, that are believed to drive residual MoMLV release via interactions with cellular proteins Tsg101 and Alix, respectively. We found that overexpression of Tsg101 or Alix failed to rescue the release of PPPY-deficient MoMLV via these other L domains. However, low-level expression of the ubiquitin ligase Itch potently rescued the release and infectivity of MoMLV lacking PPPY function. In contrast, other ubiquitin ligases such as WWP1, Nedd4.1, Nedd4.2, and Nedd4.2s did not rescue this release-deficient virus. Efficient rescue required the ubiquitin ligase activity of Itch and an intact C2 domain but not presence of the endophilin-binding site. Additionally, we found Itch to immunoprecipitate with MoMLV Gag lacking the PPPY motif and to be incorporated into rescued MoMLV particles. The PSAP and LYPAL motifs were dispensable for Itch-mediated virus rescue, and their absence did not affect the incorporation of Itch into the rescued particles. Itch-mediated rescue of release-defective MoMLV was sensitive to inhibition by dominant-negative versions of ESCRT-III components and the VPS4 AAA ATPase, indicating that Itch-mediated correction of MoMLV release defects requires the integrity of the host vacuolar sorting protein pathway. RNA interference knockdown of Itch suppressed the residual release of the MoMLV lacking the PPPY motif. Interestingly, Itch stimulation of the PPPY-deficient MoMLV release was accompanied by the enhancement of Gag ubiquitination and the appearance of new ubiquitinated Gag proteins in virions. Together, these results suggest that Itch can facilitate MoMLV release in an L domain-independent manner via a mechanism that requires the host budding machinery and involves Gag ubiquitination.

Two distinct binding modes define the interaction of Brox with the C-terminal tails of CHMP5 and CHMP4B.

Interactions of the CHMP protein carboxyl terminal tails with effector proteins play important roles in retroviral budding, cytokinesis, and multivesicular body biogenesis. Here we demonstrate that hydrophobic residues at the CHMP4B C-terminal amphipathic α helix bind a concave surface of Brox, a mammalian paralog of Alix. Unexpectedly, CHMP5 was also found to bind Brox and specifically recruit endogenous Brox to detergent-resistant membrane fractions through its C-terminal 20 residues. Instead of an α helix, the CHMP5 C-terminal tail adopts a tandem ß-hairpin structure that binds Brox at the same site as CHMP4B. Additional Brox:CHMP5 interface is furnished by a unique CHMP5 hydrophobic pocket engaging the Brox residue Y348 that is not conserved among the Bro1 domains. Our studies thus unveil a ß-hairpin conformation of the CHMP5 protein C-terminal tail, and provide insights into the overlapping but distinct binding profiles of ESCRT-III and the Bro1 domain proteins.