Selection of RNA replicons capable of persistent noncytopathic replication in mammalian cells.
The natural life cycle of alphaviruses, a group of plus-strand RNA viruses, involves transmission to vertebrate hosts via mosquitoes. Chronic infections are established in mosquitoes (and usually in mosquito cell cultures), but infection of susceptible vertebrate cells typically results in rapid shutoff of host mRNA translation and cell death. Using engineered Sindbis virus RNA replicons expressing puromycin acetyltransferase as a dominant selectable marker, we identified mutations allowing persistent, noncytopathic replication in BHK-21 cells. Two of these adaptive mutations involved single-amino-acid substitutions in the C-terminal portion of nsP2, the viral helicase-protease. At one of these loci, nsP2 position 726, numerous substitution mutations were created and characterized in the context of RNA replicons and infectious virus. Our results suggest a direct correlation between the level of viral RNA replication and cytopathogenicity. This work also provides a series of alphavirus replicons for noncytopathic gene expression studies (E. V. Agapov, I. Frolov, B. D. Lindenbach, B. M. Prágai, S. Schlesinger, and C. M. Rice, Proc. Natl. Acad. Sci. USA 95:12989-12994, 1998) and a general strategy for selecting RNA viral mutants adapted to different cellular environments.
Frolov
I
I
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.
Agapov
E
E
Hoffman
T A
TA
Prágai
B M
BM
Lippa
M
M
Schlesinger
S
S
Rice
C M
CM
D000220:Adaptation, Biological; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001191:Arylamine N-Acetyltransferase; D015415:Biomarkers; D002460:Cell Line; D002642:Chick Embryo; D002874:Chromosome Mapping; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000235:genetics; D003588:Cytopathogenic Effect, Viral; D008322:Mammals; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010641:Phenotype; D011499:Protein Processing, Post-Translational; D011691:Puromycin / Q000494:pharmacology; D012367:RNA, Viral / Q000096:biosynthesis* / Q000502:physiology; D012093:Replicon; D017386:Sequence Homology, Amino Acid; D012845:Sindbis Virus / Q000235:genetics* / Q000502:physiology; D017735:Virus Latency; D014779:Virus Replication
D015415:Biomarkers; D012367:RNA, Viral; D011691:Puromycin; D001191:Arylamine N-Acetyltransferase; D003546:Cysteine Endopeptidases; C078306:nsP2 proteinase