The Src family kinase c-Yes is required for maturation of West Nile virus particles.
The role of cellular genes in West Nile virus (WNV) replication is not well understood. Examination of cellular transcripts upregulated during WNV infection revealed an increase in the expression of the src family kinase (SFK) c-Yes. WNV-infected cell lines treated with the SFK inhibitor PP2 demonstrated a 2- to 4-log decrease in viral titers, suggesting that SFK activity is required for completion of the viral replication cycle. RNA interference mediated knock-down of c-Yes, but not c-Src, and similarly reduced virus yield, specifically implicating c-Yes in WNV production. Interestingly, PP2 treatment did not reduce intracellular levels of either viral RNA or protein, suggesting that the drug does not act on the early stages of replication. However, endoglycosidase H (endoH) digestion of the viral envelope (E) glycoprotein revealed that the acquisition of endoH-resistant glycans by E, but not endogenous major histocompatibility complex class I, was reduced in PP2-treated cells, demonstrating that E specifically does not traffic beyond the endoplasmic reticulum in the absence of SFK activity. Electron microscopy further revealed that PP2-treated WNV-infected cells accumulated an increased number of virions in the ER compared to untreated cells. Therefore, we conclude that inhibition of SFK activity did not interfere with virus assembly but prevented transit of virions through the secretory pathway. These results identify c-Yes as a cellular protein that is involved in WNV assembly and egress.
Hirsch
Alec J
AJ
Vaccine and Gene Therapy Institute, Oregon Health & Sciences University, 505 N.W. 185th Avenue, Beaverton, Oregon 97006, USA. hirschal@ohsu.edu
Medigeshi
Guruprasad R
GR
Meyers
Heather L
HL
DeFilippis
Victor
V
Früh
Klaus
K
Briese
Thomas
T
Lipkin
W Ian
WI
Nelson
Jay A
JA
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008854:Microscopy, Electron; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D051575:Proto-Oncogene Proteins c-yes; D011743:Pyrimidines / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis; D029589:Seminal Plasma Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D014709:Vero Cells; D014764:Viral Proteins / Q000096:biosynthesis; D019065:Virus Assembly / Q000187:drug effects / Q000502:physiology; D014779:Virus Replication / Q000187:drug effects / Q000502:physiology; D014902:West Nile virus / Q000187:drug effects / Q000502:physiology*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*
C412373:AG 1879; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D011743:Pyrimidines; D034741:RNA, Small Interfering; D012367:RNA, Viral; D029589:Seminal Plasma Proteins; D014764:Viral Proteins; D011505:Protein-Tyrosine Kinases; D051575:Proto-Oncogene Proteins c-yes; C489834:YES1 protein, human; D019061:src-Family Kinases