Interaction Between Tomato spotted wilt virus N Protein Monomers Involves Nonelectrostatic Forces Governed by Multiple Distinct Regions in the Primary Structure
Abstract:The ambisense RNA genome of Tomato spotted wilt virus (TSWV) isby interaction with numerous copies of the virus encoded nucleocapsid (N) protein to form a subvirion structure called a ribonucleo-protein (RNP). RNPs are central to the viral replication cycle because they, and not free viral RNA, serve as templates for viral gene expression and genome replication. N protein monomers bind to viral RNA molecules in a cooperative manner. We have examined regions of the N protein that are involved in the N-N interac… Show more
“…The N-N interaction interface of BUNV has been mapped to N-terminal region (residues 1-10), a small middle region (residues 94-158), and C-terminal region (residues 217-233) (Eifan and Elliott 2009 ;Leonard et al 2005 ) . Similar fi ndings have been reported for TSWV: both N-and C-termini (residues 1-39 and residues 233-248) together with some discrete middle regions are involved in the homotypic interaction or multimerization of the N protein (Kainz et al 2004 ;Uhrig et al 1999 ) . A "head-to-head-and-tail-to-tail" type of interaction and a "head-to-tail" model have been proposed for the BUNV and the TWSV N proteins, respectively.…”
Section: Protein N: the Viral Nucleoproteinsupporting
confidence: 85%
“…In addition to Phleboviruses, homotypic interactions of N were also noted for orthobunyaviruses, for example, BUNV (Eifan and Elliott 2009 ;Leonard et al 2005 ;Mohl and Barr 2009 ;Rodgers et al 2006 ) , and Tospoviruses, for example, TSWV (Kainz et al 2004 ;Snippe et al 2005 ;Uhrig et al 1999 ) . The N-N interaction interface of BUNV has been mapped to N-terminal region (residues 1-10), a small middle region (residues 94-158), and C-terminal region (residues 217-233) (Eifan and Elliott 2009 ;Leonard et al 2005 ) .…”
Section: Protein N: the Viral Nucleoproteinmentioning
The Bunyaviridae family is comprised of a large number of negative-sense, single-stranded RNA viruses that infect animals, insects, and plants. The tripartite genome of bunyaviruses, encapsidated in the form of individual ribonucleoprotein complexes, encodes four structural proteins, the glycoproteins Gc and Gn, the nucleoprotein N, and the viral polymerase L. Some bunyaviruses also use an ambi-sense strategy to encode the nonstructural proteins NSs and NSm. While some bunyaviruses have a T = 12 icosahedral symmetry, others only have locally ordered capsids, or capsids with no detectable symmetry. Bunyaviruses enter cells through clathrin-mediated endocytosis or phagocytosis. In endosome, viral glycoproteins facilitate membrane fusion at acidic pH, thus allowing bunyaviruses to uncoat and deliver their genomic RNA into host cytoplasm. Bunyaviruses replicate in cytoplasm where the viral polymerase L catalyzes both transcription and replication of the viral genome. While transcription requires a cap primer for initiation and ends at specific termination signals before the 3' end of the template is reached, replication copies the entire template and does not depend on any primer for initiation. This review will discuss some of the most interesting aspects of bunyavirus replication, including L protein/N protein-mediated cap snatching, prime-and-realign for transcription and replication initiation, translation-coupled transcription, sequence/secondary structure-dependent transcription termination, ribonucleoprotein encapsidation, and N protein-mediated initiation of viral protein translation. Recent developments on the structure and functional characterization of the bunyavirus capsid and the RNA synthesis machineries (including both protein L and N) will also be discussed.
“…The N-N interaction interface of BUNV has been mapped to N-terminal region (residues 1-10), a small middle region (residues 94-158), and C-terminal region (residues 217-233) (Eifan and Elliott 2009 ;Leonard et al 2005 ) . Similar fi ndings have been reported for TSWV: both N-and C-termini (residues 1-39 and residues 233-248) together with some discrete middle regions are involved in the homotypic interaction or multimerization of the N protein (Kainz et al 2004 ;Uhrig et al 1999 ) . A "head-to-head-and-tail-to-tail" type of interaction and a "head-to-tail" model have been proposed for the BUNV and the TWSV N proteins, respectively.…”
Section: Protein N: the Viral Nucleoproteinsupporting
confidence: 85%
“…In addition to Phleboviruses, homotypic interactions of N were also noted for orthobunyaviruses, for example, BUNV (Eifan and Elliott 2009 ;Leonard et al 2005 ;Mohl and Barr 2009 ;Rodgers et al 2006 ) , and Tospoviruses, for example, TSWV (Kainz et al 2004 ;Snippe et al 2005 ;Uhrig et al 1999 ) . The N-N interaction interface of BUNV has been mapped to N-terminal region (residues 1-10), a small middle region (residues 94-158), and C-terminal region (residues 217-233) (Eifan and Elliott 2009 ;Leonard et al 2005 ) .…”
Section: Protein N: the Viral Nucleoproteinmentioning
The Bunyaviridae family is comprised of a large number of negative-sense, single-stranded RNA viruses that infect animals, insects, and plants. The tripartite genome of bunyaviruses, encapsidated in the form of individual ribonucleoprotein complexes, encodes four structural proteins, the glycoproteins Gc and Gn, the nucleoprotein N, and the viral polymerase L. Some bunyaviruses also use an ambi-sense strategy to encode the nonstructural proteins NSs and NSm. While some bunyaviruses have a T = 12 icosahedral symmetry, others only have locally ordered capsids, or capsids with no detectable symmetry. Bunyaviruses enter cells through clathrin-mediated endocytosis or phagocytosis. In endosome, viral glycoproteins facilitate membrane fusion at acidic pH, thus allowing bunyaviruses to uncoat and deliver their genomic RNA into host cytoplasm. Bunyaviruses replicate in cytoplasm where the viral polymerase L catalyzes both transcription and replication of the viral genome. While transcription requires a cap primer for initiation and ends at specific termination signals before the 3' end of the template is reached, replication copies the entire template and does not depend on any primer for initiation. This review will discuss some of the most interesting aspects of bunyavirus replication, including L protein/N protein-mediated cap snatching, prime-and-realign for transcription and replication initiation, translation-coupled transcription, sequence/secondary structure-dependent transcription termination, ribonucleoprotein encapsidation, and N protein-mediated initiation of viral protein translation. Recent developments on the structure and functional characterization of the bunyavirus capsid and the RNA synthesis machineries (including both protein L and N) will also be discussed.
“…According to PISA, the intermolecular interactions were mainly hydrogen bonds, but van der Waals and hydrophobic interactions also contribute to hold the monomers together (data not shown). This interaction model is further corroborated by the available mutational studies on TSWV N [4, 17, 18]. Fig.…”
Section: Resultssupporting
confidence: 74%
“…Multiple studies have attempted to identify candidate N protein regions involved in RNA binding and protein multimerization for TSWV using yeast two-hybrid systems (Y2HS) and site-directed mutagenesis [4, 6, 17, 18], but the tospovirus RNPs remains largely uncharacterized at the molecular level and the lack of structural information prevents detailed insight into these interactions. The lack of a reverse genetics system, which is available for other bunyaviruses, has hampered tospovirus research.…”
BackgroundTospovirus is a plant-infecting genus within the family Bunyaviridae, which also includes four animal-infecting genera: Hantavirus, Nairovirus, Phlebovirus and Orthobunyavirus. Compared to these members, the structures of Tospovirus proteins still are poorly understood. Despite multiple studies have attempted to identify candidate N protein regions involved in RNA binding and protein multimerization for tospovirus using yeast two-hybrid systems (Y2HS) and site-directed mutagenesis, the tospovirus ribonucleocapsids (RNPs) remains largely uncharacterized at the molecular level and the lack of structural information prevents detailed insight into these interactions.ResultsHere we used the nucleoprotein structure of LACV (La Crosse virus-Orthobunyavirus) and molecular dynamics simulations to access the structure and dynamics of the nucleoprotein from tospovirus GRSV (Groundnut ringspot virus). The resulting model is a monomer composed by a flexible N-terminal and C-terminal arms and a globular domain with a positively charged groove in which RNA is deeply encompassed. This model allowed identifying the candidate amino acids residues involved in RNA interaction and N-N multimerization. Moreover, most residues predicted to be involved in these interactions are highly conserved among tospoviruses.ConclusionsCrucially, the interaction model proposed here for GRSV N is further corroborated by the all available mutational studies on TSWV (Tomato spotted wilt virus) N, so far. Our data will help designing further and more accurate mutational and functional studies of tospovirus N proteins. In addition, the proposed model may shed light on the mechanisms of RNP shaping and could allow the identification of essential amino acid residues as potential targets for tospovirus control strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1339-4) contains supplementary material, which is available to authorized users.
“…N may be essential for RNA synthesis together with the L RNA polyprotein [30]. Similar to the TSWV ortholog, the C-terminal motif KKDGKGKKSK 264-273 was predicted to bind RNA [40], whereas other discrete amino acids, including PSN 7-9 , RK 51-52 , RY 54-55 , and KK 73-74 may interact with the virus RNAs to prevent premature termination caused by base pairing of the newly synthesized RNA strands and to protect it from degradation [30]. The SVNaV is a distinct member of the genus Tospovirus, as it shares minimal similarity with any of the established species in the genus-all SVNaV proteins exhibit less than 40% aa identity to their orthologs.…”
A new, widespread disease was recently observed in soybean in the United States. The disease, named Soybean vein necrosis, is manifested by intraveinal chlorosis and necrosis, and has been found in almost all of the 50 fields visited over a period of 3 years in the midwest and midsouth part of the United States. A virus was isolated from symptomatic material, and detection protocols were developed. More than 150 symptomatic specimens collected from seven US States were tested, and all were found positive for the virus unlike 75 asymptomatic samples, revealing the absolute association between virus and disease. Protein pairwise comparisons coupled with phylogenetic analyses indicate that the virus is a new member of the genus Tospovirus.
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