Norovirus Genome Circularization and Efficient Replication Are Facilitated by Binding of PCBP2 and hnRNP A1

Abstract

c 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.

Human Norovirus NS3 Has RNA Helicase and Chaperoning Activities

“…For instance, during vRNA replication of RNA viruses, vRI-dsRNAs must be efficiently unwound, thereby releasing nascently synthesized progeny vRNAs from vRNA templates. Moreover, viral genomic and antigenomic RNAs contain multiple cis-acting elements, highly structured RNA regions that usually play pivotal roles in the replication, translation, and encapsidation of vRNAs (11,51). Sometimes, a specific RNA structure required for one vRNA function, like translation or encapsidation, may need disruption and refolding to facilitate another vRNA function, like vRNA replication, and vice versa.…”

“…Sometimes, a specific RNA structure required for one vRNA function, like translation or encapsidation, may need disruption and refolding to facilitate another vRNA function, like vRNA replication, and vice versa. For example, genomic RNAs or mRNAs of some RNA viruses, including norovirus (13,52,53), flavivirus (12), hantavirus (54), and reovirus (55), can undergo circularization, resulting in the base pairing of vRNA 5= and 3= ends to form panhandle-like structures that may facilitate vRNA translation, encapsidation, or RdRP recruitment to the 3= ends of vRNAs (51,56). However, for initiation of RNA replication, these cyclized vRNA structures need to be disrupted to make the 3= ends of the vRNAs accessible by RdRPs; an example of this type of activity is the chaperoning effect of cypovirus VP5 on cyclized cypoviral mRNAs (25).…”

PCBP2 promotes the development of glioma by regulating FHL3/TGF‐β/Smad signaling pathway

“…Dysregulation of PCBP2 may damage multiple biological processes through RNAbinding pathways. For instance, PCBP2 could regulate the genome circularization and replication of various RNA viruses, like norovirus (Lopez-Manriquez et al, 2013), severe acute respiratory syndrome coronaviruses (SARS-CoV; Shi et al, 2014), poliovirus (Walter, Parsley, Ehrenfeld, & Semler, 2002), and hepatitis C virus (Li, Masaki, Shimakami, & Lemon, 2014). The upregulation of PCBP2 may contribute to the formation of ribosomal initiation complex, thus enhancing the replication of virus (Asnani, Pestova, & Hellen, 2016).…”

Quantitative interactome reveals that porcine reproductive and respiratory syndrome virus nonstructural protein 2 forms a complex with viral nucleocapsid protein and cellular vimentin

“…According to the results of Trinkle-Mulcahy et al, the hnRNP proteins, heat shock proteins, ribosomal proteins and multiple cytoskeletal proteins are potential non-specific contaminants using pull-down or immunoprecipitation strategies [31]. However, many studies have revealed that these "nonspecific contaminants" are associated with virus infection through the interactions with viral proteins [52][53][54][55][56][57][58]. Furthermore, previous studies have demonstrated that PCBP2, one of the hnRNP proteins, interacts directly with PRRSV nsp1β [48,49]; inhibition of Hsp90 or Hsp70 attenuates PRRSV replication [50,51].…”