scholarly journals Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein 2 Interacts with a Host Protein Complex Involved in Mitochondrial Biogenesis and Intracellular Signaling

2009 ◽  
Vol 83 (19) ◽  
pp. 10314-10318 ◽  
Author(s):  
Cromwell T. Cornillez-Ty ◽  
Lujian Liao ◽  
John R. Yates ◽  
Peter Kuhn ◽  
Michael J. Buchmeier
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Intracellular Signaling ◽  
Nonstructural Protein ◽  
Host Protein ◽  
Nonstructural Proteins ◽  
Host Proteins ◽  
Catalytic Activities ◽  
Nonstructural Protein 2 ◽  
Host Signaling ◽  
Prohibitin 1

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) generates 16 nonstructural proteins (nsp's) through proteolytic cleavage of a large precursor protein. Although several nsp's exhibit catalytic activities that are important for viral replication and transcription, other nsp's have less clearly defined roles during an infection. In order to gain a better understanding of their functions, we attempted to identify host proteins that interact with nsp's during SARS-CoV infections. For nsp2, we identified an interaction with two host proteins, prohibitin 1 (PHB1) and PHB2. Our results suggest that nsp2 may be involved in the disruption of intracellular host signaling during SARS-CoV infections.

scholarly journals Role for Conserved Residues of Sindbis Virus Nonstructural Protein 2 Methyltransferase-Like Domain in Regulation of Minus-Strand Synthesis and Development of Cytopathic Infection

2008 ◽  
Vol 82 (15) ◽  
pp. 7284-7297 ◽  
Author(s):  
Mayuri ◽  
Todd W. Geders ◽  
Janet L. Smith ◽  
Richard J. Kuhn
Keyword(s):  
Defense Mechanisms ◽  
Sindbis Virus ◽  
Nonstructural Protein ◽  
Temperature Sensitive ◽  
Minus Strand ◽  
Nonstructural Proteins ◽  
Equine Encephalitis Virus ◽  
Nonstructural Protein 2 ◽  
Transcriptional Inhibition ◽  
Lysine Residues

ABSTRACT The plus-strand RNA genome of Sindbis virus (SINV) encodes four nonstructural proteins (nsP1 to nsP4) that are involved in the replication of the viral RNA. The ∼800-amino-acid nsP2 consists of an N-terminal domain with nucleoside triphosphatase and helicase activities and a C-terminal protease domain. Recently, the structure determined for Venezuelan equine encephalitis virus nsP2 indicated the presence of a previously unrecognized methyltransferase (MTase)-like domain within the C-terminal ∼200 residues and raised a question about its functional importance. To assess the role of this MTase-like region in viral replication, highly conserved arginine and lysine residues were mutated to alanine. The plaque phenotypes of these mutants ranged from large/wild-type to small plaques with selected mutations demonstrating temperature sensitive lethality. The proteolytic polyprotein processing activity of nsP2 was unaffected in most of the mutants. Some of the temperature-sensitive mutants showed reduction in the minus-strand RNA synthesis, a function that has not yet been ascribed to nsP2. Mutation of SINV residue R615 rendered the virus noncytopathic and incapable of inhibiting the host cell translation but with no effects on the transcriptional inhibition. This property differentiated the mutation at R615 from previously described noncytopathic mutations. These results implicate nsP2 in regulation of minus-strand synthesis and suggest that different regions of the nsP2 MTase-like domain differentially modulate host defense mechanisms, independent of its role as the viral protease.

scholarly journals Ribosomal Protein S6 Associates with Alphavirus Nonstructural Protein 2 and Mediates Expression from Alphavirus Messages

2006 ◽  
Vol 80 (15) ◽  
pp. 7729-7739 ◽  
Author(s):  
Stephanie A. Montgomery ◽  
Peter Berglund ◽  
Clayton W. Beard ◽  
Robert E. Johnston
Keyword(s):  
Ribosomal Protein ◽  
Nonstructural Protein ◽  
Cellular Proteins ◽  
Nonstructural Proteins ◽  
Replication Complex ◽  
Equine Encephalitis Virus ◽  
Nonstructural Protein 2 ◽  
Ribosomal Protein S6 ◽  
Cellular Translation ◽  
Rps6 Phosphorylation

ABSTRACT Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.

scholarly journals Noroviruses Co-opt the Function of Host Proteins VAPA and VAPB for Replication via a Phenylalanine–Phenylalanine-Acidic-Tract-Motif Mimic in Nonstructural Viral Protein NS1/2

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Broc T. McCune ◽  
Wei Tang ◽  
Jia Lu ◽  
James B. Eaglesham ◽  
Lucy Thorne ◽  
...  
Keyword(s):  
Viral Replication ◽  
Function Analysis ◽  
Nonstructural Protein ◽  
Host Protein ◽  
Murine Norovirus ◽  
Human Pathogens ◽  
Host Proteins ◽  
Human Noroviruses ◽  
Murine Noroviruses

ABSTRACT The Norovirus genus contains important human pathogens, but the role of host pathways in norovirus replication is largely unknown. Murine noroviruses provide the opportunity to study norovirus replication in cell culture and in small animals. The human norovirus nonstructural protein NS1/2 interacts with the host protein VAMP-associated protein A (VAPA), but the significance of the NS1/2-VAPA interaction is unexplored. Here we report decreased murine norovirus replication in VAPA- and VAPB-deficient cells. We characterized the role of VAPA in detail. VAPA was required for the efficiency of a step(s) in the viral replication cycle after entry of viral RNA into the cytoplasm but before the synthesis of viral minus-sense RNA. The interaction of VAPA with viral NS1/2 proteins is conserved between murine and human noroviruses. Murine norovirus NS1/2 directly bound the major sperm protein (MSP) domain of VAPA through its NS1 domain. Mutations within NS1 that disrupted interaction with VAPA inhibited viral replication. Structural analysis revealed that the viral NS1 domain contains a mimic of the phenylalanine–phenylalanine-acidic-tract (FFAT) motif that enables host proteins to bind to the VAPA MSP domain. The NS1/2-FFAT mimic region interacted with the VAPA-MSP domain in a manner similar to that seen with bona fide host FFAT motifs. Amino acids in the FFAT mimic region of the NS1 domain that are important for viral replication are highly conserved across murine norovirus strains. Thus, VAPA interaction with a norovirus protein that functionally mimics host FFAT motifs is important for murine norovirus replication. IMPORTANCE Human noroviruses are a leading cause of gastroenteritis worldwide, but host factors involved in norovirus replication are incompletely understood. Murine noroviruses have been studied to define mechanisms of norovirus replication. Here we defined the importance of the interaction between the hitherto poorly studied NS1/2 norovirus protein and the VAPA host protein. The NS1/2-VAPA interaction is conserved between murine and human noroviruses and was important for early steps in murine norovirus replication. Using structure-function analysis, we found that NS1/2 contains a short sequence that molecularly mimics the FFAT motif that is found in multiple host proteins that bind VAPA. This represents to our knowledge the first example of functionally important mimicry of a host FFAT motif by a microbial protein.

scholarly journals A Genetically Engineered Rotavirus NSP2 Phosphorylation Mutant Impaired in Viroplasm Formation and Replication Shows an Early Interaction between vNSP2 and Cellular Lipid Droplets

2020 ◽  
Vol 94 (15) ◽  
Author(s):  
Jeanette M. Criglar ◽  
Sue E. Crawford ◽  
Boyang Zhao ◽  
Hunter G. Smith ◽  
Fabio Stossi ◽  
...  
Keyword(s):  
Virus Replication ◽  
Lipid Droplet ◽  
Lipid Droplets ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Genetically Engineered ◽  
Wild Type ◽  
Nonstructural Proteins ◽  
Nonstructural Protein 2 ◽  
Infected Cells

ABSTRACT Many RNA viruses replicate in cytoplasmic compartments (virus factories or viroplasms) composed of viral and cellular proteins, but the mechanisms required for their formation remain largely unknown. Rotavirus (RV) replication in viroplasms requires interactions between virus nonstructural proteins NSP2 and NSP5, which are associated with components of lipid droplets (LDs). We previously identified two forms of NSP2 in RV-infected cells, a cytoplasmically dispersed form (dNSP2) and a viroplasm-specific form (vNSP2), which interact with hypophosphorylated and hyperphosphorylated NSP5, respectively, indicating that a coordinated phosphorylation cascade controls viroplasm assembly. The cellular kinase CK1α phosphorylates NSP2 on serine 313, triggering the localization of vNSP2 to sites of viroplasm assembly and its association with hyperphosphorylated NSP5. Using reverse genetics, we generated a rotavirus with a phosphomimetic NSP2 (S313D) mutation to directly evaluate the role of CK1α NSP2 phosphorylation in viroplasm formation. Recombinant rotavirus NSP2 S313D (rRV NSP2 S313D) is significantly delayed in viroplasm formation and in virus replication and interferes with wild-type RV replication in coinfection. Taking advantage of the delay in viroplasm formation, the NSP2 phosphomimetic mutant was used as a tool to observe very early events in viroplasm assembly. We show that (i) viroplasm assembly correlates with NSP5 hyperphosphorylation and (ii) vNSP2 S313D colocalizes with RV-induced LDs without NSP5, suggesting that vNSP2 phospho-S313 is sufficient for interacting with LDs and may be the virus factor required for RV-induced LD formation. Further studies with the rRV NSP2 S313D virus are expected to reveal new aspects of viroplasm and LD initiation and assembly. IMPORTANCE Reverse genetics was used to generate a recombinant rotavirus with a single phosphomimetic mutation in nonstructural protein 2 (NSP2 S313D) that exhibits delayed viroplasm formation, delayed replication, and an interfering phenotype during coinfection with wild-type rotavirus, indicating the importance of this amino acid during virus replication. Exploiting the delay in viroplasm assembly, we found that viroplasm-associated NSP2 colocalizes with rotavirus-induced lipid droplets prior to the accumulation of other rotavirus proteins that are required for viroplasm formation and that NSP5 hyperphosphorylation is required for viroplasm assembly. These data suggest that NSP2 phospho-S313 is sufficient for interaction with lipid droplets and may be the virus factor that induces lipid droplet biogenesis in rotavirus-infected cells. Lipid droplets are cellular organelles critical for the replication of many viral and bacterial pathogens, and thus, understanding the mechanism of NSP2-mediated viroplasm/lipid droplet initiation and interaction will lead to new insights into this important host-pathogen interaction.

scholarly journals Structural Basis and Function of the N Terminus of SARS-CoV-2 Nonstructural Protein 1

2021 ◽  
Author(s):  
Kaitao Zhao ◽  
Zunhui Ke ◽  
Hongbing Hu ◽  
Yahui Liu ◽  
Aixin Li ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Nonstructural Protein ◽  
Protein Translation ◽  
Host Protein ◽  
Structural Basis ◽  
Pathogenic Factor ◽  
Nonstructural Protein 1 ◽  
C Terminus ◽  
N Terminus ◽  
And Function

Nonstructural protein 1 (Nsp1) of severe acute respiratory syndrome coronaviruses (SARS-CoVs) is an important pathogenic factor that inhibits host protein translation by means of its C terminus. However, its N-terminal function remains elusive.

scholarly journals Severe Acute Respiratory Syndrome Coronavirus nsp9 Dimerization Is Essential for Efficient Viral Growth

2009 ◽  
Vol 83 (7) ◽  
pp. 3007-3018 ◽  
Author(s):  
Zachary J. Miknis ◽  
Eric F. Donaldson ◽  
Timothy C. Umland ◽  
Ryan A. Rimmer ◽  
Ralph S. Baric ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Viral Replication ◽  
Reverse Genetics ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Binding Activity ◽  
Nonstructural Proteins ◽  
Protein Protein Interaction ◽  
Dna Binding Activity ◽  
Fold Reduction

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) devotes a significant portion of its genome to producing nonstructural proteins required for viral replication. SARS-CoV nonstructural protein 9 (nsp9) was identified as an essential protein with RNA/DNA-binding activity, and yet its biological function within the replication complex remains unknown. Nsp9 forms a dimer through the interaction of parallel α-helices containing the protein-protein interaction motif GXXXG. In order to study the role of the nsp9 dimer in viral reproduction, residues G100 and G104 at the helix interface were targeted for mutation. Multi-angle light scattering measurements indicated that G100E, G104E, and G104V mutants are monomeric in solution, thereby disrupting the dimer. However, electrophoretic mobility assays revealed that the mutants bound RNA with similar affinity. Further experiments using fluorescence anisotropy showed a 10-fold reduction in RNA binding in the G100E and G104E mutants, whereas the G104V mutant had only a 4-fold reduction. The structure of G104E nsp9 was determined to 2.6-Å resolution, revealing significant changes at the dimer interface. The nsp9 mutations were introduced into SARS-CoV using a reverse genetics approach, and the G100E and G104E mutations were found to be lethal to the virus. The G104V mutant produced highly debilitated virus and eventually reverted back to the wild-type protein sequence through a codon transversion. Together, these data indicate that dimerization of SARS-CoV nsp9 at the GXXXG motif is not critical for RNA binding but is necessary for viral replication.

scholarly journals Dodecamer Structure of Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein nsp10

2006 ◽  
Vol 80 (16) ◽  
pp. 7902-7908 ◽  
Author(s):  
Dan Su ◽  
Zhiyong Lou ◽  
Fei Sun ◽  
Yujia Zhai ◽  
Haitao Yang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Severe Acute Respiratory Syndrome ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Nonstructural Protein ◽  
Three Dimensional ◽  
Sequence Motifs ◽  
Nonstructural Proteins ◽  
New Class ◽  
Monomer Structure

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural proteins nsp1 to nsp16 have been implicated by genetic analysis in the assembly of a functional replication/transcription complex. We report the crystal structure of nsp10 from SARS-CoV at 2.1-Å resolution. The nsp10 structure has a novel fold, and 12 identical subunits assemble to form a unique spherical dodecameric architecture. Two zinc fingers have been identified from the nsp10 monomer structure with the sequence motifs C-(X)2-C-(X)5-H-(X)6-C and C-(X)2-C-(X)7-C-(X)-C. The nsp10 crystal structure is the first of a new class of zinc finger protein three-dimensional structures to be revealed experimentally. The zinc finger sequence motifs are conserved among all three coronavirus antigenic groups, implicating an essential function for nsp10 in all coronaviruses. Based on the structure, we propose that nsp10 is a transcription factor for coronavirus replication/transcription.

scholarly journals The Porcine Reproductive and Respiratory Syndrome Virus nsp2 Cysteine Protease Domain Possesses both trans- and cis-Cleavage Activities

2009 ◽  
Vol 83 (18) ◽  
pp. 9449-9463 ◽  
Author(s):  
Jun Han ◽  
Mark S. Rutherford ◽  
Kay S. Faaberg
Keyword(s):  
Cysteine Protease ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Point Mutations ◽  
Respiratory Syndrome Virus ◽  
Protease Domain ◽  
Nonstructural Protein 2 ◽  
Cleavage Activity ◽  
Cysteine Protease Domain

ABSTRACT The N terminus of the replicase nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a putative cysteine protease domain (PL2). Previously, we demonstrated that deletion of either the PL2 core domain (amino acids [aa] 47 to 180) or the immediate downstream region (aa 181 to 323) is lethal to the virus. In this study, the PL2 domain was found to encode an active enzyme that mediates efficient processing of nsp2-3 in CHO cells. The PL2 protease possessed both trans- and cis-cleavage activities, which were distinguished by individual point mutations in the protease domain. The minimal size required to maintain these two enzymatic activities included nsp2 aa 47 to 240 (Tyr47 to Cys240) and aa 47 to 323 (Tyr47 to Leu323), respectively. Introduction of targeted amino acid mutations in the protease domain confirmed the importance of the putative Cys55- His124 catalytic motif for nsp2/3 proteolysis in vitro, as were three additional conserved cysteine residues (Cys111, Cys142, and Cys147). The conserved aspartic acids (e.g., Asp89) were essential for the PL2 protease trans-cleavage activity. Reverse genetics revealed that the PL2 trans-cleavage activity played an important role in the PRRSV replication cycle in that mutations that impaired the PL2 protease trans function, but not the cis activity, were detrimental to viral viability. Lastly, the potential nsp2/3 cleavage site was probed. Mutations with the largest impact on in vitro cleavage were at or near the G1196|G1197 dipeptide.

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