The N-terminal quarter of reovirus cell attachment protein sigma 1 possesses intrinsic virion-anchoring function.
Previously the receptor recognition domain of the reovirus serotype 3 (T3) cell attachment protein (sigma 1) was mapped to the C-terminal half of the protein using deletion mutagenesis of the reovirus S1 gene. A similar approach has been adopted in the present study to map the domain on T3 sigma 1 that is responsible for incorporation into the virion (i.e., the anchoring domain). Restriction enzymes which divide the T3 S1 cDNA into four segments (5'-I-II-III-IV-3') of similar size were used to generate four mutants, each with a particular segment deleted. The mutants were cloned into SV40 expression vectors and used to transfect COS-1 cells which were subsequently with reovirus serotype 1. Progeny viral particles with truncated T3 sigma 1 proteins incorporated were then identified by radioimmunoprecipitation with a serotype-specific anti-T3 sigma 1 serum. It was found that the mutant lacking I (mutant dl) was totally incapable of being incorporated into the virion, whereas the mutant lacking domain II (mutant dII) was incorporated efficiently. Due to altered antigenicities of the mutants lacking domain III (mutant dIII) or domain IV (mutant dIV), incorporation of these two proteins into virions was less detectable using the above assay. Evidence that domain I (the N-terminal 121 amino acids) alone dictates the incorporation of sigma 1 into the virion came from the subsequent demonstration that a chimeric protein containing domain I fused to chloramphenicol acetyltransferase (CAT) was incorporated into the virion (detectable with an anti-CAT serum) as efficiently as the full-length sigma 1 protein.
Mah
D C
DC
Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Alberta, Canada.
Leone
G
G
Jankowski
J M
JM
Lee
P W
PW
D000818:Animals; D001483:Base Sequence; D036022:Capsid Proteins; D002460:Cell Line; D002872:Chromosome Deletion; D005814:Genes, Viral; D007739:L Cells / Q000502:physiology; D012089:Mammalian orthoreovirus 3 / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010957:Plasmids; D015183:Restriction Mapping; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014771:Virion / Q000235:genetics / Q000502:physiology*
D036022:Capsid Proteins; D015345:Oligonucleotide Probes; D014764:Viral Proteins; C031728:sigma 1 protein, reovirus