Double-stranded RNA-binding protein E3 controls translation of viral intermediate RNA, marking an essential step in the life cycle of modified vaccinia virus Ankara.
Infection of human cells with modified vaccinia virus Ankara (MVA) activates the typical cascade-like pattern of viral early-, intermediate- and late-gene expression. In contrast, infection of human HeLa cells with MVA deleted of the E3L gene (MVA-DeltaE3L) results in high-level synthesis of intermediate RNA, but lacks viral late transcription. The viral E3 protein is thought to bind double-stranded RNA (dsRNA) and to act as an inhibitor of dsRNA-activated 2'-5'-oligoadenylate synthetase (2'-5'OA synthetase)/RNase L and protein kinase (PKR). Here, it is demonstrated that viral intermediate RNA can form RNase A/T1-resistant dsRNA, suggestive of activating both the 2'-5'OA synthetase/RNase L pathway and PKR in various human cell lines. Western blot analysis revealed that failure of late transcription in the absence of E3L function resulted from the deficiency to produce essential viral intermediate proteins, as demonstrated for vaccinia late transcription factor 2 (VLTF 2). Substantial host cell-specific differences were found in the level of activation of either RNase L or PKR. However, both rRNA degradation and phosphorylation of eukaryotic translation initiation factor-2alpha (eIF2alpha) inhibited the synthesis of VLTF 2 in human cells. Moreover, intermediate VLTF 2 and late-protein production were restored in MVA-DeltaE3L-infected mouse embryonic fibroblasts from Pkr(0/0) mice. Thus, both host-response pathways may be involved, but activity of PKR is sufficient to block the MVA molecular life cycle. These data imply that an essential function of vaccinia virus E3L is to secure translation of intermediate RNA and, thereby, expression of other viral genes.
Ludwig
Holger
H
Department of Virology, Paul-Ehrlich-Institut, Paul-Ehrlich-Straße 51-59, 63225 Langen, Germany.
Suezer
Yasemin
Y
Department of Virology, Paul-Ehrlich-Institut, Paul-Ehrlich-Straße 51-59, 63225 Langen, Germany.
Waibler
Zoe
Z
Department of Immunology, Paul-Ehrlich-Institut, Paul-Ehrlich-Straße 51-59, 63225 Langen, Germany.
Kalinke
Ulrich
U
Department of Immunology, Paul-Ehrlich-Institut, Paul-Ehrlich-Straße 51-59, 63225 Langen, Germany.
Schnierle
Barbara S
BS
Department of Virology, Paul-Ehrlich-Institut, Paul-Ehrlich-Straße 51-59, 63225 Langen, Germany.
Sutter
Gerd
G
Department of Virology, Paul-Ehrlich-Institut, Paul-Ehrlich-Straße 51-59, 63225 Langen, Germany.
D015088:2',5'-Oligoadenylate Synthetase / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D015967:Gene Expression Regulation, Viral; D006801:Humans; D051379:Mice; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics*; D013045:Species Specificity; D014157:Transcription Factors / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics / Q000502:physiology*; D014764:Viral Proteins / Q000235:genetics*; D014779:Virus Replication
C081374:E3L protein, Vaccinia virus; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D011494:Protein Kinases; D015088:2',5'-Oligoadenylate Synthetase