Helices alpha2 and alpha3 of West Nile virus capsid protein are dispensable for assembly of infectious virions.
The internal hydrophobic sequence within the flaviviral capsid protein (protein C) plays an important role in the assembly of infectious virions. Here, this sequence was analyzed in a West Nile virus lineage I isolate (crow V76/1). An infectious cDNA clone was constructed and used to introduce deletions into the internal hydrophobic domain which comprises helix alpha2 and part of the loop intervening helices alpha2 and alpha3. In total, nine capsid deletion mutants (4 to 14 amino acids long) were constructed and tested for virus viability. Some of the short deletions did not significantly affect growth in cell culture, whereas larger deletions removing almost the entire hydrophobic region significantly impaired viral growth. Efficient growth of the majority of mutants could, however, be restored by the acquisition of second-site mutations. In most cases, these resuscitating mutations were point mutations within protein C changing individual amino acids into more hydrophobic residues, reminiscent of what had been observed previously for another flavivirus, tick-borne encephalitis virus. However, we also identified viable spontaneous pseudorevertants with more than one-third of the capsid protein removed, i.e., 36 or 37 of a total of 105 residues, including all of helix alpha3 and a hydrophilic segment connecting alpha3 and alpha4. These large deletions are predicted to induce formation of large, predominantly hydrophobic fusion helices which may substitute for the loss of the internal hydrophobic domain, underlining the unrivaled structural and functional flexibility of protein C.
Schlick
Petra
P
Intercell AG, Vienna, Austria. pschlick@intercell.com
Taucher
Christian
C
Schittl
Beate
B
Tran
Janina L
JL
Kofler
Regina M
RM
Schueler
Wolfgang
W
von Gabain
Alexander
A
Meinke
Andreas
A
Mandl
Christian W
CW
D000595:Amino Acid Sequence; D000818:Animals; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D017353:Gene Deletion; D016679:Genome, Viral / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014771:Virion / Q000737:chemistry*; D019065:Virus Assembly; D014902:West Nile virus / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D036022:Capsid Proteins; D018076:DNA, Complementary; D011486:Protein C; D012367:RNA, Viral