Hantavirus Nucleocapsid Protein Has Distinct m7G Cap- and RNA-binding Sites

Abstract

Hantaviruses, members of the Bunyaviridae family, are emerging category A pathogens that carry three negative stranded RNA molecules as their genome. Hantavirus nucleocapsid protein (N) is encoded by the smallest S segment genomic RNA (viral RNA). N specifically binds mRNA caps and requires four nucleotides adjacent to the cap for high affinity binding. We show that the N peptide has distinct cap-and RNA-binding sites that independently interact with mRNA cap and viral genomic RNA, respectively. In addition, N can simultaneously bind with both mRNA cap and vRNA. N undergoes distinct conformational changes after binding with either mRNA cap or vRNA or both mRNA cap and vRNA simultaneously. Hantavirus RNA-dependent RNA polymerase (RdRp) uses a capped RNA primer for transcription initiation. The capped RNA primer is generated from host cell mRNA by the cap-snatching mechanism and is supposed to anneal with the 3 terminus of vRNA template during transcription initiation by single G-C base pairing. We show that the capped RNA primer binds at the cap-binding site and induces a conformational change in N. The conformationally altered N with a capped primer loaded at the cap-binding site specifically binds the conserved 3 nine nucleotides of vRNA and assists the bound primer to anneal at the 3 terminus. We suggest that the cap-binding site of N, in conjunction with RdRp, plays a key role during the transcription and replication initiation of vRNA genome.Hantaviruses cause two types of serious human illnesses when transmitted to humans from rodent hosts: hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome (1, 2). The spherical hantavirus particles harbor three negative sense genomic RNA segments (S, L, and M segments) within a lipid bilayer (3). The mRNAs derived from S, L, and M segments encode viral nucleocapsid protein (N), viral RNA-dependent RNA polymerase (RdRp), 2 and glycoproteins (G1 and G2), respectively. The characteristic feature of the hantaviral genome is the partially complementary sequence at the 5Ј and 3Ј termini of each of the three genome segments that undergo base pairing and form panhandle structures (4 -6). N is a multifunctional protein playing a vital role in multiple processes of virus replication cycle and has been found to undergo trimerization both in vivo and in vitro (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). During encapsidation, the three viral RNA (vRNA) molecules are specifically recognized by N inside the host cell and targeted for packaging. Multiple in vitro studies have shown that N preferentially binds vRNA compared with complementary RNA (cRNA) or nonviral RNA (13, 20 -25). It has been proposed that the specific binding of N with either the panhandle or the sequence at the 5Ј terminus alone selectively facilitates the encapsidation of vRNA to generate three nucleocapsids that are packaged into infectious virions (25, 26). The RNA-binding domain of Hantaan virus N protein has been mapped to the central conserved region corresponding to amino acids f...

The Lysine Residues within the Human Ribosomal Protein S17 Sequence Naturally Inserted into the Viral Nonstructural Protein of a Unique Strain of Hepatitis E Virus Are Important for Enhanced Virus Replication

“…One interesting example of a viral protein interacting with host ribosomal proteins is the case of the hantavirus nucleocapsid (N) protein, which interacts with the RPS19 protein (47)(48)(49). It has been shown that RPS19 interaction with N protein facilitates ribosome loading onto the viral RNA transcripts, leading to preferential translation of viral transcripts.…”

Characterization of the Interaction between Hantavirus Nucleocapsid Protein (N) and Ribosomal Protein S19 (RPS19)

“…We also used continuous variation plots to verify the binding stoichiometry results as reported previously (14,59,60). This is a reliable method to determine the binding stoichiometry of protein-protein complexes.…”

“…Calculation of Binding Stoichiometry-As described previously (14,59), the binding stoichiometry (expressed in terms of the number of RPS19 molecules bound per molecule of N) was estimated from the intersection of two straight lines of a least square fit plot of ⌬F/⌬F max against the ratio of input concentrations of N and 1,5-IAEDANS-labeled RPS19. We also used continuous variation plots to verify the binding stoichiometry results as reported previously (14,59,60).…”

A genome-wide RNAi screen reveals that mRNA decapping restricts bunyaviral replication by limiting the pools of Dcp2-accessible targets for cap-snatching

“…Rather, all segmented negative-sense RNA viruses (bunyaviruses, arenaviruses, and orthomyxoviruses) ''cap-snatch'' the 59 ends of host mRNAs, simultaneously defending the 59 end of their mRNAs from exonucleases and facilitating translation. Bunyavirus-encoded N protein specifically binds the 59 caps of host mRNAs (Mir et al 2010). The viral RNAdependent RNA polymerase (L), which has endonucleolytic activity (Patterson et al 1984;Reguera et al 2010), then cleaves host mRNAs 10-18 nucleotides (nt) downstream from the 59 cap and uses this primer as a template for viral mRNA transcription (Patterson et al 1984;Bouloy et al 1990;Simons and Pettersson 1991).…”

“…Of these, influenza A virus, an orthomyxovirus, is the best studied and snatches the 59 end of pre-mRNAs in the nucleus (Herz et al 1981;Plotch et al 1981). Since bunyaviruses and arenaviruses replicate in the cytoplasm, they must use a distinct pool of mRNAs; however, while our mechanistic understanding of bunyaviral cap-snatching is increasing (van Poelwijk et al 1996;van Knippenberg et al 2005;Mir et al 2010;Morin et al 2010;Reguera et al 2010;Cheng and Mir 2012), little is known about whether the host can combat this replication step or what pool of endogenous mRNAs are being targeted for this process.…”