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.

Negative effect of heat shock on feline calicivirus release from infected cells is associated with the control of apoptosis.

FCV infection causes rapid cytopathic effects, and its replication results in the induction of apoptosis changes in cultured cells. It is well established that the survival of apoptotic cells can be enhanced by the expression of heat-shock proteins (Hsp) to prevent damage or facilitate recovery. Hsps can act as molecular chaperones, but they can also have anti-apoptotic roles by binding to apoptotic proteins and inhibiting the activation of caspases, the primary mediators of apoptosis. Because apoptosis occurs during FCV infection and heat shock (HS) treatment has a cytoprotective role due to the expression of Hsps, we studied the effect of the HS response to hyperthermia during FCV infection in cultured cells. We found that FCV infection does not inhibit the expression of Hsp70 induced by HS and that non-structural and structural protein synthesis was not modified during HS treatment. However, HS caused a delay in the appearance of a cytopathic effect in infected cells, as well as a reduction in the extracellular but not in the cell-associated viral yield. This antiviral effect of HS correlates with the inhibition of caspase-3 activation. Thus, the HS-induced reduction in virus production appeared to be associated with the control of apoptosis, supporting previous data that indicate that apoptosis is necessary for FCV release.