Sin Nombre Virus mRNA Synthesis

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...

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

The Hantavirus Nucleocapsid Protein Recognizes Specific Features of the Viral RNA Panhandle and Is Altered in Conformation upon RNA Binding

Mir

Panganiban

2005

“…The use of capped primers following a "prime and realign" mechanism has been suggested for the Bunyaviridae transcription initiation (24). Transcription termination signals have been identified in Hantaan and Sin Nombre virus (SNV) mRNAs (27). SNV S and L segment mRNAs are not polyadenylated; however, M segment-derived mRNA is polyadenylated and synthesis is terminated at a (U)8 polyadenylation transcription termination signal (27).…”

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confidence: 99%

Signatures of Host mRNA 5′ Terminus for Efficient Hantavirus Cap Snatching

Cheng

Mir

2012

Hantaviruses, similarly to other negative-strand segmented RNA viruses, initiate the synthesis of translation-competent capped mRNAs by a unique cap-snatching mechanism. Hantavirus nucleocapsid protein (N) binds to host mRNA caps and requires four nucleotides adjacent to the 5′ cap for high-affinity binding. N protects the 5′ caps of cellular transcripts from degradation by the cellular decapping machinery. The rescued 5′ capped mRNA fragments are stored in cellular P bodies by N, which are later efficiently used as primers by the hantaviral RNA-dependent RNA polymerase (RdRp) for transcription initiation. We showed that N also protects the host mRNA caps in P-body-deficient cells. However, the rescued caps were not effectively used by the hantavirus RdRp during transcription initiation, suggesting that caps stored in cellular P bodies by N are preferred for cap snatching. We examined the characteristics of the 5′ terminus of a capped test mRNA to delineate the minimum requirements for a capped transcript to serve as an efficient cap donor during hantavirus cap snatching. We showed that hantavirus RdRp preferentially snatches caps from the nonsense mRNAs compared to mRNAs engaged in translation. Hantavirus RdRp preferentially cleaves the cap donor mRNA at a G residue located 14 nucleotides downstream of the 5′ cap. The sequence complementarity between the 3′ terminus of viral genomic RNA and the nucleotides located in the vicinity of the cleavage site of the cap donor mRNA favors cap snatching. Our results show that hantavirus RdRp snatches caps from viral mRNAs. However, the negligible cap-donating efficiency of wild-type mRNAs in comparison to nonsense mRNAs suggests that viral mRNAs will not be efficiently used for cap snatching during viral infection due to their continuous engagement in protein synthesis. Our results suggest that efficiency of an mRNA to donate caps for viral mRNA synthesis is primarily regulated at the translational level.

“…The general replication cycle starts with G1 and G2 binding to B3 integrins (7,8) or other cell surface receptors, followed by virus entry and uncoating. After entry, L protein-mediated primary transcription of mRNAs occurs in the cytoplasm, apparently using an orthomyxovirus-like capsnatching mechanism (4,14). Following mRNA translation, transcription shifts from mRNA to cRNA and viral RNA synthesis, and ribonucleoprotein (RNP) structures are formed (4,14,24).…”

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confidence: 99%

“…After entry, L protein-mediated primary transcription of mRNAs occurs in the cytoplasm, apparently using an orthomyxovirus-like capsnatching mechanism (4,14). Following mRNA translation, transcription shifts from mRNA to cRNA and viral RNA synthesis, and ribonucleoprotein (RNP) structures are formed (4,14,24). The RNPs appear to be composed of viral RNAs, N proteins, and presumably L proteins and accumulate on the cytoplasmic sides of cellular membranes, possibly through interactions with the G1 and G2 proteins (9,21).…”

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confidence: 99%

Hantavirus Nucleocapsid Protein Oligomerization

Alfadhli

Love

Arvidson

et al. 2001

Hantaviruses are enveloped, negative-strand RNA viruses which can be lethal to humans, causing either a hemorrhagic fever with renal syndrome or a hantaviral pulmonary syndrome. The viral genomes consist of three RNA segments: the L segment encodes the viral polymerase, the M segment encodes the viral surface glycoproteins G1 and G2, and the S segment encodes the nucleocapsid (N) protein. The N protein is a 420-to 430-residue, 50-kDa protein which appears to direct hantavirus assembly, although mechanisms of N protein oligomerization, RNA encapsidation, budding, and release are poorly understood. We have undertaken a biochemical and genetic analysis of N protein oligomerization. Bacterially expressed N proteins were found by gradient fractionation to associate not only as large multimers or aggregates but also as dimers or trimers. Chemical cross-linking of hantavirus particles yielded N protein cross-link products with molecular masses of 140 to 150 kDa, consistent with the size of an N trimer. We also employed a genetic, yeast two-hybrid method for monitoring N protein interactions. Analyses showed that the C-terminal half of the N protein plus the N-terminal 40 residues permitted association with a full-length N protein fusion. These N-terminal 40 residues of seven different hantavirus strains were predicted to form trimeric coiled coils. Our results suggest that coiled-coil motifs contribute to N protein trimerization and that nucleocapsid protein trimers are hantavirus particle assembly intermediates. Hantaviruses, such as the Sin Nombre hantavirus (SNV) andProspect Hill virus (PHV), are members of the bunyavirus class of viruses (3,20,24). They are enveloped, negative-strand RNA viruses and carry three genomic RNA segments: the L segment, which encodes an RNA-dependent RNA polymerase; the M segment, which encodes envelope glycoproteins G1 and G2; and the S segment, which encodes the viral nucleocapsid (N) protein (24). Hantaviruses are of medical importance, because many of them cause either a hemorrhagic fever with renal syndrome or a hantaviral pulmonary syndrome, which is characterized by lung damage and cardiac dysfunction (25).Models for hantavirus replication at the cellular level have been based on direct experiments and by inference from work on other bunyaviruses. The general replication cycle starts with G1 and G2 binding to B3 integrins (7,8) or other cell surface receptors, followed by virus entry and uncoating. After entry, L protein-mediated primary transcription of mRNAs occurs in the cytoplasm, apparently using an orthomyxovirus-like capsnatching mechanism (4, 14). Following mRNA translation, transcription shifts from mRNA to cRNA and viral RNA synthesis, and ribonucleoprotein (RNP) structures are formed (4, 14, 24). The RNPs appear to be composed of viral RNAs, N proteins, and presumably L proteins and accumulate on the cytoplasmic sides of cellular membranes, possibly through interactions with the G1 and G2 proteins (9, 21). Evidence suggests that RNPs use microfilaments for transport to ...