Functional replacement of a retroviral late domain by ubiquitin fusion.
Retroviral Gag polyprotein precursors are both necessary and sufficient for the assembly and release of virus-like particles (VLPs) from infected cells. It is well established that small Gag-encoded motifs, known as late domains, promote particle release by interacting with components of the cellular endosomal sorting and ubiquitination machinery. The Gag proteins of a number of different retroviruses are ubiquitinated; however, the role of Gag ubiquitination in particle egress remains undefined. In this study, we investigated this question by using a panel of equine infectious anemia virus (EIAV) Gag derivatives bearing the wild-type EIAV late domain, heterologous retroviral late domains or no late domain. Ubiquitin was fused in cis to the C-termini of these Gag polyproteins, and the effects on VLP budding were measured. Remarkably, fusion of ubiquitin to EIAV Gag lacking a late domain (EIAV/DeltaYPDL-Ub) largely rescued VLP release. We also determined the effects of ubiquitin fusion on the sensitivity of particle release to budding inhibitors and to depletion of key endosomal sorting factors. Ubiquitin fusion rendered EIAV/DeltaYPDL-Ub sensitive to depletion of cellular endosomal sorting factors Tsg101 and Alix and to overexpression of dominant-negative fragments of Tsg101 and Alix. These findings demonstrate that ubiquitin can functionally compensate for the absence of a retroviral late domain and provide insights into the host-cell machinery engaged by ubiquitin during particle egress.
Joshi
Anjali
A
Virus-Cell Interaction Section, HIV Drug Resistance Program, National Cancer Institute, Frederick, MD 21702-1201, USA.
Munshi
Utpal
U
Ablan
Sherimay D
SD
Nagashima
Kunio
K
Freed
Eric O
EO
D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015683:Gene Products, gag / Q000378:metabolism*; D004860:Infectious Anemia Virus, Equine / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D061988:Proteasome Inhibitors; D025801:Ubiquitin / Q000378:metabolism*
D015853:Cysteine Proteinase Inhibitors; D015683:Gene Products, gag; D061988:Proteasome Inhibitors; D025801:Ubiquitin