Graduate Thesis Or Dissertation
 

Identification of intracellular trafficking signals contained within the vaccinia virus F13L protein

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  • The F13L protein is the major envelope antigen of vaccinia virus, the prototypic member of the Orthopoxvirus genus. F13L is 372 residues in length and is essential for the formation of wrapped forms of virus. F13L contains a number of potential functional domains including a palmitylation site, a phospholipase domain and several tyrosine-based sorting signals (YxxΦ) including three YxxI motifs and a canonical YxxL late (L) domain-like motif. L domains have been identified in several other viruses, including HIV and EIAV, and function in the assembly and release of viral particles from infected cells. Since F13L is required for the formation of wrapped viral particles, it was hypothesized that one or more of these tyrosine-based motifs may possess L domain activity.Sequence analysis revealed that the YxxL motif is very highly conserved throughout the Chordopoxvirus subfamily suggesting that this sequence may convey some biological advantage to the virus. In contrast, the YxxI domains were much less conserved.A trans complementation assay was developed to analyze which residues within the candidate tyrosine-based motifs were required for functional F13L. Based upon targeted mutagenesis of residues within the YxxΦ domains, F13L function was shown to require the tyrosine residue and Φ residue within the YxxL motif and within one of the YxxI motifs. This is consistent with what has been published regarding L domains in other viral systems. The degree of complementation associated with each mutation was measured by both the size and number of plaques produced as well as by an enrichment of a viral population deficient in F13L expression.Co-immunoprecipitation experiments demonstrated a physical interaction between F13L and a cellular trafficking component, Alix. Recombinant viruses containing mutations within the YxxL motif displayed an accumulation phenotype that inversely related binding affinity to the rescue ability demonstrated by each mutation in the trans complementation assay. Furthermore, gradient fractionation of cells infected with the recombinant mutants revealed the presence of wrapped forms of virus. This indicated that the block in the release of enveloped virions associated with the tyrosine and leucine mutants occurred subsequent to the formation of intracellular enveloped virus.
  • Keywords: Late (L) assembly domains, Vaccinia, tyrosine-based sorting signals, YxxL, YxxI motifs, Viral proteins, Vaccinia virus F13L, Viral envelopes
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