Norovirus genome circularization and efficient replication are facilitated by binding of PCBP2 and hnRNP A1.
Sequences and structures within the terminal genomic regions of plus-strand RNA viruses are targets for the binding of host proteins that modulate functions such as translation, RNA replication, and encapsidation. Using murine norovirus 1 (MNV-1), we describe the presence of long-range RNA-RNA interactions that were stabilized by cellular proteins. The proteins potentially responsible for the stabilization were selected based on their ability to bind the MNV-1 genome and/or having been reported to be involved in the stabilization of RNA-RNA interactions. Cell extracts were preincubated with antibodies against the selected proteins and used for coprecipitation reactions. Extracts treated with antibodies to poly(C) binding protein 2 (PCBP2) and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 significantly reduced the 5'-3' interaction. Both PCBP2 and hnRNP A1 recombinant proteins stabilized the 5'-3' interactions and formed ribonucleoprotein complexes with the 5' and 3' ends of the MNV-1 genomic RNA. Mutations within the 3' complementary sequences (CS) that disrupt the 5'-3'-end interactions resulted in a significant reduction of the viral titer, suggesting that the integrity of the 3'-end sequence and/or the lack of complementarity with the 5' end is important for efficient virus replication. Small interfering RNA-mediated knockdown of PCBP2 or hnRNP A1 resulted in a reduction in virus yield, confirming a role for the observed interactions in efficient viral replication. PCBP2 and hnRNP A1 induced the circularization of MNV-1 RNA, as revealed by electron microscopy. This study provides evidence that PCBP2 and hnRNP A1 bind to the 5' and 3' ends of the MNV-1 viral RNA and contribute to RNA circularization, playing a role in the virus life cycle.
Departamento de Infectómica y Patogénesis Molecular.
Eduardo
E
López-Manríquez
Surender
S
Vashist
Luis
L
Ureña
Ian
I
Goodfellow
Pedro
P
Chavez
José Eduardo
JE
Mora-Heredia
Clotilde
C
Cancio-Lonches
Efraín
E
Garrido
Ana Lorena
AL
Gutiérrez-Escolano
D000818:Animals; D047369:Chromatin Immunoprecipitation; D055785:Gene Knockdown Techniques; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000235:genetics / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D008854:Microscopy, Electron; D029322:Norovirus / Q000502:physiology*; D020871:RNA Stability; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; C501904:Pcbp2 protein, mouse; D012367:RNA, Viral; D016601:RNA-Binding Proteins