Mechanism of cap-snatching in hantaviruses

Abstract

Hantaviruses are enveloped RNA viruses belong to the Family Bunyaviridae, which cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) in humans. The hantavirus genome consists of three negative-strand RNA segments, S, M and L, which encodes nucleocapsid protein (N), glycoprotein precursor (GPC), RNA-dependent RNA polymerase (RdRp), and in some genus, a non-structural protein (NSs). Hantaviruses initiate transcription by cap-snatching mechanism. Host mRNAs are processed by viral RNP and used as primers for transcription initiation. N proteins protect those cap donors and enrich them in P-bodies. We showed that in P-body deficient cells, capped oligos were also protected but not effectively used, suggesting that caps concentrated within P-bodies are preferred for cap-snatching. We also characterized the determinants for a capped transcript to be an efficient cap donor using test transcripts. We showed hantavirus more efficiently snatches caps from mRNAs not engaged in translation and the hantavirus RNP preferentially cleaves the cap donor mRNA after the G residue located 14 nucleotides from 5' cap. Moreover, better sequence complementarity between the 3' terminus of vRNA and cap donor favors cap-snatching. These results indicate hantavirus snatches caps from nontranslating mRNA, and the viral RNP may recognize and process host-capped oligos based on position and complementarity of the annealing between to vRNA and capped oligos. We also explored requirement of other viral proteins for RdRp function. We found N protein interacts with RdRp in virus-infected cells. We mapped the RdRp binding domain to the N-terminal region of the N that excluded the RNA binding domain. Similarly, the N-binding pocket is located at the C-terminus of RdRp. We demonstrate that an N-RdRp interaction is required for RdRp function during the course of virus infection in the host cell. Taken together, we characterized the preferred capped primer used for efficient cap-snatching. We also provided insights to the specificity of the hantavirus RdRp endonuclease, by showing N-RdRp interaction in the absence of RNA binding domain. Our findings have provided insight into the understanding of the mechanism hantavirus cap-snatching and will shed light on the identification of anti-viral targets for the treatment of hantavirus-caused diseases.