Studies of deltaretrovirus assemby and release

by Wang, Huating

Abstract (Summary)
The assembly of retrovirus particles requires the expression of the Gag polyprotein precursor (PrGag), which is used as a principle building scaffold for retrovirus assembly and budding from infected cells. The retrovirus Gag polyprotein has all the necessary information to mediate intracellular transport to the cell membrane, to package full-length retroviral genomes, to direct assembly of virus particles, and to catalyze the final budding process. This dissertation was focused on the virus particle assembly of Deltaretroviruses, namely human T-cell leukemia virus type-1 (HTLV-1) and bovine leukemia virus (BLV). These viruses replicate to low titers in their natural hosts and are poorly infectious in cells culture. Information regarding the molecular details of their life cycles, including virus assembly, is limited. Virus-like particle (VLP) assay model systems were developed for BLV and HTLV-1 to test five hypotheses. The first hypothesis was that the myristylation signal in the matrix (MA) domain of BLV Gag was required for membrane targeting and binding of BLV Gag. Mutations that disrupted the amino-terminal glycine residue (which would block the addition of myristic acid) led to a drastic reduction in VLP production but did not eliminate Gag membrane localization, suggesting that other residues in Gag were involved in membrane targeting and binding. The second hypothesis was that the PPPY domain within BLV MA was required for virus release. Mutation of the PPPY motif significantly reduced VLP production and this reduction was more severe in the presence of an active viral protease. Examination of particles by electron microscopy revealed an abundance of particles that began to pinch off from the plasma membrane but were not completely released from the cell surface, indicating that the PPPY motif functions as a late domain (L domain). The third hypothesis tested was that the MA and nucleocapsid (NC) domains were protein determinants of BLV RNA packaging. Mutagenesis of conserved basic residues as well as residues of the zinc-finger domains in the BLV NC domain of PrGag revealed residues that led to a reduction in viral RNA packaging. Interestingly, when conserved basic residues in the BLV MA domain of PrGag were mutated to alanine or glycine, but not when mutated to another basic residue, reductions in viral RNA packaging were also observed. The ability of PrGag to be targeted to the cell membrane was not affected by these mutations in MA, indicating that these basic residues in the MA domain of PrGag influence RNA packaging, without influencing Gag membrane localization. It was further observed that i) a MA/NC double mutant had a more severe RNA packaging defect than either mutant alone, and ii) RNA packaging was not found to be associated with transient localization of Gag in the nucleus. These observations indicate that both the MA and NC domains of BLV Gag are involved in RNA packaging. The fourth hypothesis tested was that both the PPPY and PTAP motifs in the C-terminus of HTLV-1 MA function as the L domain and influence virus release. Mutation of either motif (i.e., PPPY changed to APPY or PTAP changed to PTRP) reduced budding efficiencies. Further analysis revealed that PPPY plays an essential role in HTLV-1 particle budding from the plasma membrane and could not be replaced by other late domain motifs, i.e. PTAP or YPDL, whereas the PTAP motif plays a subtler role in the virus release. The fifth hypothesis tested was that HTLV-1 particles could assembly at both the plasma membrane and multivesicular bodies (MVBs) and PTAP directs the Gag to the plasma membrane. I demonstrated that when HTLV-1 PTAP motif was altered, an accumulation of Gag proteins and virus particles were observed in intracellular compartments. These compartments were CD63-positive multi-vesicular bodies (MVBs). Further analysis excluded the possibility that these particles accumulated inside MVBs were a result of re-internalization of extracellular particles. It was further fou nd that (i) the particle-containing MVBs traffic along microtubules using dynein-dynactin complexes recruited by RILP and these particles could therefore exit the cell by exocytosis. (ii) PI3P is likely to be a Gag receptor on MVBs as Inhibition of PI3K disrupted the MVB pathway.
Bibliographical Information:


School:The Ohio State University

School Location:USA - Ohio

Source Type:Master's Thesis



Date of Publication:01/01/2004

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