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1.
The hantavirus nucleocapsid protein recognizes specific features of the viral RNA panhandle and is altered in conformation upon RNA binding 总被引:2,自引:0,他引:2 下载免费PDF全文
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. 相似文献
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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. 相似文献
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Jonsson CB Gallegos J Ferro P Severson W Xu X Schmaljohn CS Fero P 《Protein expression and purification》2001,23(1):134-141
Sin Nombre virus is a member of the Hantavirus genus, family Bunyaviridae, and is an etiologic agent of hantavirus pulmonary syndrome. The hantavirus nucleocapsid (N) protein plays an important role in the encapsidation and assembly of the viral negative-sense genomic RNA. The Sin Nombre N protein was expressed as a C-terminal hexahistidine fusion in Escherichia coli and initially purified by nickel-affinity chromatography. We developed methods to extract the soluble fraction and to solubilize the remainder of the N protein using denaturants. Maximal expression of protein from native purification was observed after a 1.5-h induction with IPTG (2.4 mg/L). The zwitterionic detergent Chaps did not enhance the yield of native purifications, but increased the yield of protein obtained from insoluble purifications. Both soluble and insoluble materials, purified by nickel-affinity chromatography, were also subjected to Hi Trap SP Sepharose fast-flow (FF) chromatography. Both soluble and insoluble proteins had a similar A(280) profile on the Sepharose FF column, and both suggested the presence of a nucleic acid contaminant. The apparent dissociation constant of the N protein, purified by nickel-affinity and SP Sepharose FF chromatography, and the 5' end of the viral S-segment genome were measured using a filter binding assay. The N protein-vRNA complex had an apparent dissociation constant of 140 nM. 相似文献
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Rift Valley fever is considered to be one of the most important viral zoonoses in Africa. In 2000, the Rift valley fever virus spread to the Arabian Peninsula and caused two simultaneous outbreaks in Yemen and Saudi Arabia. It is transmitted to ruminants and to humans by mosquitoes. The viral agent is an arbovirus, which belongs to the Phlebovirus genus in the Bunyaviridae family. This family of viruses comprises more than 300 members grouped into five genera: Orthobunyavirus, Phlebovirus, Hantavirus, Nairovirus, and Tospovirus. Several members of the Bunyaviridae family are responsible for fatal hemorrhagic fevers: Rift Valley fever virus (Phlebovirus), Crimean-Congo hemorrhagic fever virus (Nairovirus), Hantaan, Sin Nombre and related viruses (Hantavirus), and recently Garissa, now identified as Ngari virus (Orthobunyavirus). Here are reviewed recent advances in Rift Valley fever virus, its epidemiology, molecular biology and focus on recent data on the interactions between viral and cellular proteins, which help to understand the molecular mechanisms utilized by the virus to circumvent the host cellular response. 相似文献
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The bunyavirus nucleocapsid protein is an RNA chaperone: possible roles in viral RNA panhandle formation and genome replication 总被引:1,自引:0,他引:1
Cellular RNA chaperones are crucial for the genesis of correctly folded functional RNAs. Using several complementary in vitro assays we find that the bunyavirus nucleocapsid protein (N) is an RNA chaperone. In the Bunyaviridae genomic RNA is in stable "panhandle" formation that arises through the hydrogen bonding of the terminal nucleotides of the RNA. The RNA chaperone function of N facilitates panhandle formation even though the termini are separated by >2 kb. RNA panhandle formation is likely driven by the exceptionally high base-pairing specificity of the terminal nucleotides as evidenced by P-num analysis. N protein can nonspecifically dissociate RNA duplexes. In addition, following panhandle formation, the RNA chaperone activity of N also appears to be involved in dissociation of the RNA panhandle and remains in association with the 5' terminus of the viral RNA following dissociation. Thus, N likely functions in the initiation of genome replication to allow efficient initiation of RNA synthesis by the viral polymerase. The RNA chaperone activity of N may be facilitated by an intrinsically disordered domain that catalyzes RNA unfolding driven by reciprocal entropy transfer. These observations highlight the essential features that are probably common to all RNA chaperones in which the role of the chaperone is to nonspecifically dissociate higher order structure and formation of functional higher order structure may often be predicted by RNA P-num value. The data also highlight features of N that are probably specifically important during replication of bunyavirus RNA. 相似文献
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Uukuniemi virus S RNA segment: ambisense coding strategy, packaging of complementary strands into virions, and homology to members of the genus Phlebovirus. 总被引:4,自引:3,他引:1 下载免费PDF全文
We determined the complete nucleotide sequence of the small (S) RNA segment of Uukuniemi virus, the prototype of the Uukuvirus genus within the Bunyaviridae family. The RNA, which is 1,720 nucleotides long, contains two nonoverlapping open reading frames. The 5' end of one strand (complementary to the viral strand) encodes the nonstructural protein NSs (273 residues; molecular weight, 32,019), whereas the 5' end of the viral-sense strand encodes the nucleocapsid protein N (254 residues; molecular weight, 28,508). Thus, the S RNA uses an ambisense coding strategy previously described for the S segment of two phleboviruses and the arenaviruses. The localization of the N protein within the S RNA sequence was confirmed by amino-terminal sequence analysis of all five possible cyanogen bromide fragments obtained from purified N protein. Northern (RNA) blot analyses with strand-specific probes showed that the N and NSs proteins are translated from subgenomic mRNAs about 800 and 850 nucleotides long, respectively. These mRNAs are apparently transcribed from full-length S RNAs of opposite polarities. The two mRNA species were also detected in virus-infected cells. Interestingly, highly purified virions contained full-length S RNA copies of both polarities at a ratio of about 10:1. In contrast, virions contained exclusively negative-strand copies of the M RNA segment. The possible significance of these results for viral infection is discussed. The amino acid sequence of the N protein showed 35 and 32% homology (identity) with the N protein of Punta Toro and sandfly fever Sicilian viruses, two members of the Phlebovirus genus. The NSs proteins were much less related (about 15% identity). In addition, the extreme 5' and 3' ends of the S RNA, which are complementary to each other, also showed a high degree of conservation with the two phleboviruses. These results indicate that the uukuviruses and phleboviruses are evolutionarily related and suggest that the two genera could be merged into a single genus within the Bunyaviridae family. 相似文献
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Tischler ND Fernández J Müller I Martínez R Galeno H Villagra E Mora J Ramírez E Rosemblatt M Valenzuela PD 《Biological research》2003,36(2):201-210
We report here the complete genomic sequence of the Chilean human isolate of Andes virus CHI-7913. The S, M, and L genome segment sequences of this isolate are 1,802, 3,641 and 6,466 bases in length, with an overall GC content of 38.7%. These genome segments code for a nucleocapsid protein of 428 amino acids, a glycoprotein precursor protein of 1,138 amino acids and a RNA-dependent RNA polymerase of 2,152 amino acids. In addition, the genome also has other ORFs coding for putative proteins of 34 to 103 amino acids. The encoded proteins have greater than 98% overall similarity with the proteins of Andes virus isolates AH-1 and Chile R123. Among other sequenced Hantavirus, CHI-7913 is more closely related to Sin Nombre virus, with an overall protein similarity of 92%. The characteristics of the encoded proteins of this isolate, such as hydrophobic domains, glycosylation sites, and conserved amino acid motifs shared with other Hantavirus and other members of the Bunyaviridae family, are identified and discussed. 相似文献
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Severson W Partin L Schmaljohn CS Jonsson CB 《The Journal of biological chemistry》1999,274(47):33732-33739
The nucleocapsid (N) protein functions in hantavirus replication through its interactions with the viral genomic and antigenomic RNAs. To address the biological functions of the N protein, it was critical to first define this binding interaction. The dissociation constant, K(d), for the interaction of the Hantaan virus (HTNV) N protein and its genomic S segment (vRNA) was measured under several solution conditions. Overall, increasing the NaCl and Mg(2+) in these binding reactions had little impact on the K(d). However, the HTNV N protein showed an enhanced specificity for HTNV vRNA as compared with the S segment open reading frame RNA or a nonviral RNA with increasing ionic strength and the presence of Mg(2+). In contrast, the assembly of Sin Nombre virus N protein-HTNV vRNA complexes was inhibited by the presence of Mg(2+) or an increase in the ionic strength. The K(d) values for HTNV and Sin Nombre virus N proteins were nearly identical for the S segment open reading frame RNA, showing weak affinity over several binding reaction conditions. Our data suggest a model in which specific recognition of the HTNV vRNA by the HTNV N protein resides in the noncoding regions of the HTNV vRNA. 相似文献
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A newly recognized virus associated with a fatal case of hantavirus pulmonary syndrome in Louisiana. 总被引:8,自引:4,他引:4 下载免费PDF全文
S P Morzunov H Feldmann C F Spiropoulou V A Semenova P E Rollin T G Ksiazek C J Peters S T Nichol 《Journal of virology》1995,69(3):1980-1983
Genetic analysis of virus detected in autopsy tissues of a fatal hantavirus pulmonary syndrome-like case in Louisiana revealed the presence of a previously unrecognized hantavirus. Nucleotide sequence analysis of PCR fragments of the complete S and M segments of the virus amplified from RNA extracted from the tissues showed the virus to be novel, differing from the closest related hantavirus, Sin Nombre virus, by approximately 30%. Both genome segments were unique, and there was no evidence of genetic reassortment with previously characterized hantaviruses. The primary rodent reservoir of Sin Nombre virus, the deer mouse Peromyscus maniculatus, is absent from Louisiana. Thus, the virus detected in Louisiana, referred to here as Bayou virus, must possess a different rodent reservoir. 相似文献
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Bunyamwera bunyavirus RNA synthesis requires cooperation of 3'- and 5'-terminal sequences 总被引:1,自引:0,他引:1 下载免费PDF全文
Bunyamwera virus (BUNV) is the prototype of both the Orthobunyavirus genus and the Bunyaviridae family of segmented negative-sense RNA viruses. The tripartite BUNV genome consists of small (S), medium (M), and large (L) segments that are each transcribed to yield a single mRNA and are replicated to generate an antigenome that acts as a template for synthesis of further genomic strands. As for all negative-sense RNA viruses, the 3'- and 5'-terminal nontranslated regions (NTRs) of the BUNV S, M, and L segments exhibit nucleotide complementarity and, except for one conserved U-G pairing, this complementarity extends for 15, 18, and 19 nucleotides, respectively. We investigated whether the complementarity of 3' and 5' NTRs reflected a functional requirement for terminal cooperation to promote BUNV RNA synthesis or, alternatively, was a consequence of genomic and antigenomic NTRs having similar functions requiring sequence conservation. We show that cooperation between 3'- and 5'-NTR sequences is required for BUNV RNA synthesis, and our results suggest that this cooperation is due to nucleotide complementarity allowing 3' and 5' NTRs to associate through base-pairing interactions. To examine the importance of complementarity in promoting BUNV RNA synthesis, we utilized a competitive replication assay able to examine the replication ability of all possible combinations of interacting nucleotides within a defined region of BUNV 3' and 5' NTRs. We show here that maximal RNA replication was signaled when sequences exhibiting perfect complementarity within 3' and 5' NTRs were selected. 相似文献
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Battisti AJ Chu YK Chipman PR Kaufmann B Jonsson CB Rossmann MG 《Journal of virology》2011,85(2):835-841
Hantaan virus is the prototypic member of the Hantavirus genus within the family Bunyaviridae and is a causative agent of the potentially fatal hemorrhagic fever with renal syndrome. The Bunyaviridae are a family of negative-sense RNA viruses with three-part segmented genomes. Virions are enveloped and decorated with spikes derived from a pair of glycoproteins (Gn and Gc). Here, we present cryo-electron tomography and single-particle cryo-electron microscopy studies of Hantaan virus virions. We have determined the structure of the tetrameric Gn-Gc spike complex to a resolution of 2.5 nm and show that spikes are ordered in lattices on the virion surface. Large cytoplasmic extensions associated with each Gn-Gc spike also form a lattice on the inner surface of the viral membrane. Rod-shaped ribonucleoprotein complexes are arranged into nearly parallel pairs and triplets within virions. Our results differ from the T=12 icosahedral organization found for some bunyaviruses. However, a comparison of our results with the previous tomographic studies of the nonpathogenic Tula hantavirus indicates a common structural organization for hantaviruses. 相似文献
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Hussein IT Cheng E Ganaie SS Werle MJ Sheema S Haque A Mir MA 《Journal of virology》2012,86(14):7520-7529
Hantavirus glycoprotein precursor (GPC) is posttranslationally cleaved into two glycoproteins, Gn and Gc. Cells transfected with plasmids expressing either GPC or both Gn and Gc revealed that Gn is posttranslationally degraded. Treatment of cells with the autophagy inhibitors 3-methyladenine, LY-294002, or Wortmanin rescued Gn degradation, suggesting that Gn is degraded by the host autophagy machinery. Confocal microscopic imaging showed that Gn is targeted to autophagosomes for degradation by an unknown mechanism. Examination of autophagy markers LC3-I and LC3-II demonstrated that both Gn expression and Sin Nombre hantavirus (SNV) infection induce autophagy in cells. To delineate whether induction of autophagy and clearance of Gn play a role in the virus replication cycle, we downregulated autophagy genes BCLN-1 and ATG7 using small interfering RNA (siRNA) and monitored virus replication over time. These studies revealed that inhibition of host autophagy machinery inhibits Sin Nombre virus replication in cells, suggesting that autophagic clearance of Gn is required for efficient virus replication. Our studies provide mechanistic insights into viral pathogenesis and reveal that SNV exploits the host autophagy machinery to decrease the intrinsic steady-state levels of an important viral component for efficient replication in host cells. 相似文献
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We report the first complete genome sequence of Maripa virus identified in 2009 from a patient with hantavirus pulmonary syndrome in French Guiana. Maripa virus corresponds to a new variant of the Rio Mamoré virus species in the Bunyaviridae family, genus Hantavirus. 相似文献
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Klempa B Witkowski PT Popugaeva E Auste B Koivogui L Fichet-Calvet E Strecker T Ter Meulen J Krüger DH 《Journal of virology》2012,86(7):3819-3827
We have discovered the first indigenous African hantavirus, Sangassou virus (SANGV). The virus was isolated from an African wood mouse (Hylomyscus simus), trapped in a forest habitat in Guinea, West Africa. Here, we report on the characterization of the genetic and functional properties of the virus. The complete genome of SANGV was determined and showed typical hantavirus organization. The small (S), medium (M), and large (L) genome segments containing genes encoding nucleocapsid protein, two envelope glycoproteins, and viral polymerase were found to be 1,746, 3,650, and 6,531 nucleotides long, respectively. The exact 5' and 3' termini for all three segments of the SANGV genome were determined and were predicted to form the panhandle structures typical of bunyaviruses. Phylogenetic analyses of all three segment sequences confirmed SANGV as a Murinae-associated hantavirus most closely related to the European Dobrava-Belgrade virus. We showed, however, that SANGV uses β(1) integrin rather than β(3) integrin and decay-accelerating factor (DAF)/CD55 as an entry receptor. In addition, we demonstrated a strong induction of type III lambda interferon (IFN-λ) expression in type I IFN-deficient Vero E6 cells by SANGV. These properties are unique within Murinae-associated hantaviruses and make the virus useful in comparative studies focusing on hantavirus pathogenesis. 相似文献
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Frederick SB Kibenge Marcos G Godoy Yingwei Wang Molly JT Kibenge Valentina Gherardelli Soledad Mansilla Angelica Lisperger Miguel Jarpa Geraldine Larroquete Fernando Avendaño Marcela Lara Alicia Gallardo 《Virology journal》2009,6(1):1-16