The minus strand and ambisense segmented RNA viruses include multiple important human pathogens and are divided into three families, the Orthomyxoviridae, the Bunyaviridae, and the Arenaviridae. These viruses all initiate viral transcription through the process of ''cap-snatching,'' which involves the acquisition of capped 5 oligonucleotides from cellular mRNA. Hantaviruses are emerging pathogenic viruses of the Bunyaviridae family that replicate in the cytoplasm of infected cells. Cellular mRNAs can be actively translated in polysomes or physically sequestered in cytoplasmic processing bodies (P bodies) where they are degraded or stored for subsequent translation. Here we show that the hantavirus nucleocapsid protein binds with high affinity to the 5 cap of cellular mRNAs, protecting the 5 cap from degradation. We also show that the hantavirus nucleocapsid protein accumulates in P bodies, where it sequesters protected 5 caps. P bodies then serve as a pool of primers during the initiation of viral mRNA synthesis by the viral polymerase. We propose that minus strand segmented viruses replicating in the cytoplasm have co-opted the normal degradation machinery of P bodies for storage of cellular caps. Our data also indicate that modification of the cap-snatching model is warranted to include a role for the nucleocapsid protein in cap acquisition and storage.bunyavirus ͉ minus strand RNA virus ͉ RNA degradation ͉ viral transcription ͉ RNA translation T he paradigm for transcription initiation involving cap-snatching is based on the orthomyxovirus influenza and posits that the heterotrimeric viral RNA-dependent RNA polymerase (RdRp) acquires 5Ј caps through the endonuclease activity of the PB1 subunit of the influenza RdRp (1, 2). This general mechanism of cap-snatching has been assumed for all minus strand segmented RNA viruses including the bunyaviruses and arenaviruses. However, one rather than three genes encode the RdRp of bunyaviruses and arenaviruses, and RdRp-associated endonuclease activity has yet to be established. Moreover, whereas influenza viruses carry out cap-snatching and transcription in the nucleus of infected cells, bunyavirus and arenavirus transcription and genome replication is cytoplasmic (3-8).Cellular mRNA degradation begins with removal of the poly(A) tail. Two alternative pathways that are both dependent on prior deadenylation then further degrade mRNA (9-11). mRNA can undergo 3Ј to 5Ј exonucleolytic decay, catalyzed by cytoplasmic exosomes under the control of peptides of the SKI complex. Alternatively, the 5Ј mRNA cap can be removed by the decapping enzyme DCP2/DCP1, rendering the mRNA susceptible to 5Ј to 3Ј digestion by the exonuclease XRN1. Decapping and XRN1-dependent 5Ј to 3Ј degradation is the predominant pathway for turnover of cellular mRNAs. Moreover, the components of the 5Ј to 3Ј decay machinery, including DCP2/DCP1 and XRN1, as well as a host of other peptides that function in RNA degradation and RNA regulation, are located in discreet cytoplasmic foci called processing bodie...

The eIF4F cap‐binding complex mediates the initiation of cellular mRNA translation. eIF4F is composed of eIF4E, which binds to the mRNA cap, eIF4G, which indirectly links the mRNA cap with the 43S pre‐initiation complex, and eIF4A, which is a helicase necessary for initiation. Viral nucleocapsid proteins (N) function in both genome replication and RNA encapsidation. Surprisingly, we find that hantavirus N has multiple intrinsic activities that mimic and substitute for each of the three peptides of the cap‐binding complex thereby enhancing the translation of viral mRNA. N binds with high affinity to the mRNA cap replacing eIF4E. N binds directly to the 43S pre‐initiation complex facilitating loading of ribosomes onto capped mRNA functionally replacing eIF4G. Finally, N obviates the requirement for the helicase, eIF4A. The expression of a multifaceted viral protein that functionally supplants the cellular cap‐binding complex is a unique strategy for viral mRNA translation initiation. The ability of N to directly mediate translation initiation would ensure the efficient translation of viral mRNA.

Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is encoded by the smallest of the three genome segments (S). N protein is the principal structural component of the viral capsid and is central to the hantavirus replication cycle. We examined intermolecular N-protein interaction and RNA binding by using bacterially expressed Sin Nombre virus N protein. N assembles into di-and trimeric forms. The mono-and dimeric forms exist transiently and assemble into a trimeric form. In contrast, the trimer is highly stable and does not efficiently disassemble into the mono-and dimeric forms. The purified N-protein trimer is able to discriminate between viral and nonviral RNA molecules and, interestingly, recognizes and binds with high affinity the panhandle structure composed of the 3 and 5 ends of the genomic RNA. In contrast, the mono-and dimeric forms of N bind RNA to form a complex that is semispecific and salt sensitive. We suggest that trimerization of N protein is a molecular switch to generate a protein complex that can discriminate between viral and nonviral RNA molecules during the early steps of the encapsidation process.

Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is the principal structural component of the viral capsid. N forms a stable trimer that specifically recognizes the panhandle structure formed by the viral RNA termini. We used trimeric glutathione S-transferase (GST)-N protein and small RNA panhandles to examine the requirements for specific recognition by Sin Nombre hantavirus N. Trimeric GST-N recognizes the panhandles of the three viral RNAs (S, M, and L) with high affinity, whereas the corresponding plus-strand panhandles of the complementary RNA are recognized with lower affinity. Based on analysis of nucleotide substitutions that alter either the higher-order structure of the panhandle or the primary sequence of the panhandle, both secondary structure and primary sequence are necessary for stable interaction with N. A panhandle 23 nucleotides long is necessary and sufficient for high-affinity binding by N, and stoichiometry calculations indicate that a single N trimer interacts with a single panhandle. Surprisingly, displacement of the panhandle structure away from the terminus does not eliminate recognition by N. The binding of N to the panhandle is an entropy-driven process resulting in initial stable N-RNA interaction followed by a conformational change in N. Taken together, these data provide insight into the molecular events that take place during interaction of N with the panhandle and suggest that specific high-affinity interaction between an RNA binding domain of trimeric N and the panhandle is required for encapsidation of the three viral RNAs.The members of the Hantavirus genus of the family Bunyaviridae are spherical, enveloped viruses containing tripartite negative-sense RNA as their genome (37). The three genomic RNA segments, designated L, M, and S, encode an RNAdependent RNA polymerase, envelope glycoproteins (G1 and G2), and nucleocapsid (N) protein, respectively. Hantavirus infections can cause two serious and often fatal human diseases, hemorrhagic fever with renal syndrome and hantaviral pulmonary syndrome, characterized by lung damage and cardiac dysfunction (36). Humans are infected with hantaviruses from rodent reservoirs that are persistently infected without signs of disease (38).The initial steps in hantavirus infection require binding of the G1 and/or G2 protein to ␤3 integrin (9) or other cell surface receptors, followed by virus entry and uncoating. After entry, L-protein mediates transcription of minus-strand RNA, resulting in plus-stranded mRNA in the cytoplasm, apparently with an orthomyxovirus-like cap-snatching mechanism (7,8,16). Following viral mRNA translation, transcription shifts from mRNA to plus-strand complementary RNA and de novo minus-strand viral RNA synthesis concomitant with the formation of ribonucleoprotein structures (7,16,37). The ribonucleoproteins appear to be composed of viral RNA, N protein, and presumably viral polymerase and accumulate on the cytoplasmic side of intracellular me...

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...