Lethality of PE2 incorporation into Sindbis virus can be suppressed by second-site mutations in E3 and E2.
Sindbis virions contain two glycoproteins, E1 and E2. E2 is produced initially as a precursor, PE2, from which the amino-terminal 64 amino acids are cleaved by a cellular protease at a late stage in virion maturation. A mutation at E2 position 1 (Arg to Asn) was placed into Sindbis virus AR339 by site-directed mutagenesis of a full-length AR339 cDNA clone, pTRSB, to produce pTRSB-N. The mutation created a signal for N-linked glycosylation immediately adjacent to the PE2 cleavage signal. Virions derived from pTRSB-N were glycosylated at E2 position 1, and they quantitatively incorporated PE2 in place of E2. When pTRSB-N transcripts were electroporated into BHK-21 cells, TRSB-N particles were released with nearly normal efficiency; however, the specific infectivity of TRSB-N particles was very low. Analysis of seven infectious revertants of TRSB-N revealed that reversion was linked to (i) mutations that eliminated the signal for N-linked glycosylation and thus restored the PE2 cleavage phenotype or (ii) conservation of the PE2 cleavage defect combined with incorporation of suppressor mutations in E3 or E2. The genotype of each revertant was reconstructed in the genetic background of TRSB-N, and each reverting mutation also was replaced individually into the genetic background of wild-type virus (TRSB). Each PE2-containing revertant was attenuated in newborn CD-1 mice and replicated poorly in cultured mosquito cells (C6/36). Reverting mutations in the genetic background of TRSB did not reduce virulence in mice or growth in mosquito cells, suggesting that the phenotypes of attenuation in mice and reduced growth in mosquito cells were linked to failure of PE2 cleavage and not to the reverting mutations themselves.
Heidner
H W
HW
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27599-7290.
McKnight
K L
KL
Davis
N L
NL
Johnston
R E
RE
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D009033:Culicidae / Q000166:cytology; D006031:Glycosylation; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017422:Sequence Analysis, DNA; D012845:Sindbis Virus / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity*; D013489:Suppression, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism; D014774:Virulence / Q000235:genetics
D011498:Protein Precursors; D014759:Viral Envelope Proteins; C064950:glycoprotein E2, Sindbis virus