An Attenuating Mutation in nsP1 of the Sindbis-Group Virus S.A.AR86 Accelerates Nonstructural Protein Processing and Up-Regulates Viral 26S RNA Synthesis

ABSTRACT The Sindbis-group alphavirus S.A.AR86 encodes a threonine at nonstructural protein 1 (nsP1) 538 that is associated with neurovirulence in adult mice. Mutation of the nsP1 538 Thr to the consensus Ile found in nonneurovirulent Sindbis-group alphaviruses attenuates S.A.AR86 for adult mouse neurovirulence, while introduction of Thr at position 538 in a nonneurovirulent Sindbis virus background confers increased neurovirulence (M. T. Heise et al., J. Virol. 74:4207-4213, 2000). Since changes in the viral nonstructural region are likely to affect viral replication, studies were performed to evaluate the effect of Thr or Ile at nsP1 538 on viral growth, nonstructural protein processing, and RNA synthesis. Multistep growth curves in Neuro2A and BHK-21 cells revealed that the attenuated s51 (nsP1 538 Ile) virus had a slight, but reproducible growth advantage over the wild-type s55 (nsP1 538 Thr) virus. nsP1 538 lies within the cleavage recognition domain between nsP1 and nsP2, and the presence of the attenuating Ile at nsP1 538 accelerated the processing of S.A.AR86 nonstructural proteins both in vitro and in infected cells. Since nonstructural protein processing is known to regulate alphavirus RNA synthesis, experiments were performed to evaluate the effect of Ile or Thr at nsP1 538 on viral RNA synthesis. A combination of S.A.AR86-derived reporter assays and RNase protection assays determined that the presence of Ile at nsP1 538 led to earlier expression from the viral 26S promoter without affecting viral minus- or plus-strand synthesis. These results suggest that slower nonstructural protein processing and delayed 26S RNA synthesis in wild-type S.A.AR86 infections may contribute to the adult mouse neurovirulence phenotype of S.A.AR86.

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Attenuation of Zika Virus by Passage in Human HeLa Cells

Vaccines ◽

10.3390/vaccines7030093 ◽

2019 ◽

Vol 7 (3) ◽

pp. 93 ◽

Cited By ~ 1

Author(s):

Li ◽

Collins ◽

Widen ◽

Davis ◽

Kaiser ◽

...

Keyword(s):

Hela Cell ◽

Hela Cells ◽

Zika Virus ◽

Infected Animal ◽

Vaccine Development ◽

Nonstructural Protein ◽

A549 Cells ◽

Wild Type ◽

Nonstructural Protein 1

Zika virus (ZIKV) is a mosquito-borne Flavivirus. Previous studies have shown that mosquito-transmitted flaviviruses, including yellow fever, Japanese encephalitis, and West Nile viruses, could be attenuated by serial passaging in human HeLa cells. Therefore, it was hypothesized that wild-type ZIKV would also be attenuated after HeLa cell passaging. A human isolate from the recent ZIKV epidemic was subjected to serial HeLa cell passaging, resulting in attenuated in vitro replication in both Vero and A549 cells. Additionally, infection of AG129 mice with 10 plaque forming units (pfu) of wild-type ZIKV led to viremia and mortality at 12 days, whereas infection with 103 pfu of HeLa-passage 6 (P6) ZIKV led to lower viremia, significant delay in mortality (median survival: 23 days), and increased cytokine and chemokine responses. Genomic sequencing of HeLa-passaged virus identified two amino acid substitutions as early as HeLa-P3: pre-membrane E87K and nonstructural protein 1 R103K. Furthermore, both substitutions were present in virus harvested from HeLa-P6-infected animal tissue. Together, these data show that, similarly to other mosquito-borne flaviviruses, ZIKV is attenuated following passaging in HeLa cells. This strategy can be used to improve understanding of substitutions that contribute to attenuation of ZIKV and be applied to vaccine development across multiple platforms.

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Requirements at the 3′ End of the Sindbis Virus Genome for Efficient Synthesis of Minus-Strand RNA

Journal of Virology ◽

10.1128/jvi.79.8.4630-4639.2005 ◽

2005 ◽

Vol 79 (8) ◽

pp. 4630-4639 ◽

Cited By ~ 44

Author(s):

Richard W. Hardy ◽

Charles M. Rice

Keyword(s):

Sindbis Virus ◽

Rna Synthesis ◽

Core Promoter ◽

Genome Replication ◽

Minus Strand ◽

Wild Type ◽

Sequence Element ◽

Conserved Sequence ◽

Type 3

ABSTRACT The 3′-untranslated region of the Sindbis virus genome is 0.3 kb in length with a 19-nucleotide conserved sequence element (3′ CSE) immediately preceding the 3′-poly(A) tail. The 3′ CSE and poly(A) tail have been assumed to constitute the core promoter for minus-strand RNA synthesis during genome replication; however, their involvement in this process has not been formally demonstrated. Utilizing both in vitro and in vivo analyses, we have examined the role of these elements in the initiation of minus-strand RNA synthesis. The major findings of this study with regard to efficient minus-strand RNA synthesis are the following: (i) the wild-type 3′ CSE and the poly(A) tail are required, (ii) the poly(A) tail must be a minimum of 11 to 12 residues in length and immediately follow the 3′ CSE, (iii) deletion or substitution of the 3′ 13 nucleotides of the 3′ CSE severely inhibits minus-strand RNA synthesis, (iv) templates possessing non-wild-type 3′ sequences previously demonstrated to support virus replication do not program efficient RNA synthesis, and (v) insertion of uridylate residues between the poly(A) tail and a non-wild-type 3′ sequence can restore promoter function to a limited extent. This study shows that the optimal structure of the 3′ component of the minus-strand promoter is the wild-type 3′ CSE followed a poly(A) tail of at least 11 residues. Our findings also show that insertion of nontemplated bases can restore function to an inactive promoter.

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DIFFERENTIATION OF DROSOPHILA CELLS LACKING RIBOSOMAL DNA, IN VITRO

Genetics ◽

10.1093/genetics/73.3.429 ◽

1973 ◽

Vol 73 (3) ◽

pp. 429-434

Author(s):

J James Donady ◽

R L Seecof ◽

M A Fox

Keyword(s):

Drosophila Melanogaster ◽

Ribosomal Rna ◽

Ribosomal Dna ◽

Rna Synthesis ◽

Wild Type ◽

Drosophila Cells

ABSTRACT Drosophila melanogaster embryos that lacked ribosomal DNA were obtained from appropriate crosses. Cells were taken from such embryos before overt differentiation took place and were cultured in vitro. These cells differentiated into neurons and myocytes with the same success as did wild-type controls. Therefore, ribosomal RNA synthesis is not necessary for the differentiation of neurons and myocytes in vitro.

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Production of Noncapped Genomic RNAs Is Critical to Sindbis Virus Disease and Pathogenicity

mBio ◽

10.1128/mbio.02675-20 ◽

2020 ◽

Vol 11 (6) ◽

Author(s):

Autumn T. LaPointe ◽

V Douglas Landers ◽

Claire E. Westcott ◽

Kevin J. Sokoloski

Keyword(s):

Sindbis Virus ◽

Virus Disease ◽

Severe Disease ◽

Wild Type Virus ◽

Wild Type ◽

Genomic Rnas ◽

Mortality And Morbidity ◽

The Impact

ABSTRACT Alphaviruses are positive-sense RNA viruses that utilize a 5′ cap structure to facilitate translation of viral proteins and to protect the viral RNA genome. Nonetheless, significant quantities of viral genomic RNAs that lack a canonical 5′ cap structure are produced during alphaviral replication and packaged into viral particles. However, the role/impact of the noncapped genomic RNA (ncgRNA) during alphaviral infection in vivo has yet to be characterized. To determine the importance of the ncgRNA in vivo, the previously described D355A and N376A nsP1 mutations, which increase or decrease nsP1 capping activity, respectively, were incorporated into the neurovirulent AR86 strain of Sindbis virus to enable characterization of the impact of altered capping efficiency in a murine model of infection. Mice infected with the N376A nsP1 mutant exhibited slightly decreased rates of mortality and delayed weight loss and neurological symptoms, although levels of inflammation in the brain were similar to those of wild-type infection. Although the D355A mutation resulted in decreased antiviral gene expression and increased resistance to interferon in vitro, mice infected with the D355A mutant showed significantly reduced mortality and morbidity compared to mice infected with wild-type virus. Interestingly, expression of proinflammatory cytokines was found to be significantly decreased in mice infected with the D355A mutant, suggesting that capping efficiency and the production of ncgRNA are vital to eliciting pathogenic levels of inflammation. Collectively, these data indicate that the ncgRNA have important roles during alphaviral infection and suggest a novel mechanism by which noncapped viral RNAs aid in viral pathogenesis. IMPORTANCE Mosquito-transmitted alphaviruses have been the cause of widespread outbreaks of disease that can range from mild illness to lethal encephalitis or severe polyarthritis. There are currently no safe and effective vaccines or therapeutics with which to prevent or treat alphaviral disease, highlighting the need to better understand alphaviral pathogenesis to develop novel antiviral strategies. This report reveals production of noncapped genomic RNAs (ncgRNAs) to be a novel determinant of alphaviral virulence and offers insight into the importance of inflammation to pathogenesis. Taken together, the findings reported here suggest that the ncgRNAs contribute to alphaviral pathogenesis through the sensing of the ncgRNAs during alphaviral infection and are necessary for the development of severe disease.

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Modification of the 5′ Terminus of Sindbis Virus Genomic RNA Allows nsP4 RNA Polymerases with Nonaromatic Amino Acids at the N Terminus To Function in RNA Replication

Journal of Virology ◽

10.1128/jvi.77.4.2301-2309.2003 ◽

2003 ◽

Vol 77 (4) ◽

pp. 2301-2309 ◽

Cited By ~ 12

Author(s):

Yukio Shirako ◽

Ellen G. Strauss ◽

James H. Strauss

Keyword(s):

Amino Acid ◽

Secondary Structure ◽

Sindbis Virus ◽

Rna Synthesis ◽

Wild Type Virus ◽

Progeny Virus ◽

Minus Strand ◽

Genomic Rna ◽

Wild Type ◽

N Terminus

ABSTRACT We have previously shown that Sindbis virus RNA polymerase requires an N-terminal aromatic amino acid or histidine for wild-type or pseudo-wild-type function; mutant viruses with a nonaromatic amino acid at the N terminus of the polymerase, but which are otherwise wild type, are unable to produce progeny viruses and will not form a plaque at any temperature tested. We now show that such mutant polymerases can function to produce progeny virus sufficient to form plaques at both 30 and 40°C upon addition of AU, AUA, or AUU to the 5′ terminus of the genomic RNA or upon substitution of A for U as the third nucleotide of the genome. These results are consistent with the hypothesis that (i) 3′-UA-5′ is required at the 3′ terminus of the minus-strand RNA for initiation of plus-strand genomic RNA synthesis; (ii) in the wild-type virus this sequence is present in a secondary structure that can be opened by the wild-type polymerase but not by the mutant polymerase; (iii) the addition of AU, AUA, or AUU to the 5′ end of the genomic RNA provides unpaired 3′-UA-5′ at the 3′ end of the minus strand that can be utilized by the mutant polymerase, and similarly, the effect of the U3A mutation is to destabilize the secondary structure, freeing 3′-terminal UA; and (iv) the N terminus of nsP4 may directly interact with the 3′ terminus of the minus-strand RNA for the initiation of the plus-strand genomic RNA synthesis. This hypothesis is discussed in light of our present results as well as of previous studies of alphavirus RNAs, including defective interfering RNAs.

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Mutations in the NS1 Protein of Swine Influenza Virus Impair Anti-Interferon Activity and Confer Attenuation in Pigs

Journal of Virology ◽

10.1128/jvi.79.12.7535-7543.2005 ◽

2005 ◽

Vol 79 (12) ◽

pp. 7535-7543 ◽

Cited By ~ 176

Author(s):

Alicia Solórzano ◽

Richard J. Webby ◽

Kelly M. Lager ◽

Bruce H. Janke ◽

Adolfo García-Sastre ◽

...

Keyword(s):

Influenza Virus ◽

Nonstructural Protein ◽

Swine Influenza Virus ◽

Swine Influenza ◽

Influenza Viruses ◽

Model Systems ◽

Ns1 Protein ◽

Wild Type ◽

Virulence Attenuation

ABSTRACT It has been shown previously that the nonstructural protein NS1 of influenza virus is an alpha/beta interferon (IFN-α/β) antagonist, both in vitro and in experimental animal model systems. However, evidence of this function in a natural host has not yet been obtained. Here we investigated the role of the NS1 protein in the virulence of a swine influenza virus (SIV) isolate in pigs by using reverse genetics. The virulent wild-type A/Swine/Texas/4199-2/98 (TX/98) virus and various mutants encoding carboxy-truncated NS1 proteins were rescued. Growth properties of TX/98 viruses with mutated NS1, induction of IFN in tissue culture, and virulence-attenuation in pigs were analyzed and compared to those of the recombinant wild-type TX/98 virus. Our results indicate that deletions in the NS1 protein decrease the ability of the TX/98 virus to prevent IFN-α/β synthesis in pig cells. Moreover, all NS1 mutant viruses were attenuated in pigs, and this correlated with the amount of IFN-α/β induced in vitro. These data suggest that the NS1 protein of SIV is a virulence factor. Due to their attenuation, NS1-mutated swine influenza viruses might have a great potential as live attenuated vaccine candidates against SIV infections of pigs.

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Cleavage between Replicase Proteins p28 and p65 of Mouse Hepatitis Virus Is Not Required for Virus Replication

Journal of Virology ◽

10.1128/jvi.78.11.5957-5965.2004 ◽

2004 ◽

Vol 78 (11) ◽

pp. 5957-5965 ◽

Cited By ~ 40

Author(s):

Mark R. Denison ◽

Boyd Yount ◽

Sarah M. Brockway ◽

Rachel L. Graham ◽

Amy C. Sims ◽

...

Keyword(s):

Rna Synthesis ◽

Hepatitis Virus ◽

Mouse Hepatitis Virus ◽

Wild Type ◽

Virus Growth ◽

Amino Terminal ◽

Normal Expression ◽

Infected Cells ◽

Critical Residues

ABSTRACT The p28 and p65 proteins of mouse hepatitis virus (MHV) are the most amino-terminal protein domains of the replicase polyprotein. Cleavage between p28 and p65 has been shown to occur in vitro at cleavage site 1 (CS1), 247Gly↓Val248, in the polyprotein. Although critical residues for CS1 cleavage have been mapped in vitro, the requirements for cleavage have not been studied in infected cells. To define the determinants of CS1 cleavage and the role of processing at this site during MHV replication, mutations and deletions were engineered in the replicase polyprotein at CS1. Mutations predicted to allow cleavage at CS1 yielded viable virus that grew to wild-type MHV titers and showed normal expression and processing of p28 and p65. Mutant viruses containing predicted noncleaving mutations or a CS1 deletion were also viable but demonstrated delayed growth kinetics, reduced peak titers, decreased RNA synthesis, and small plaques compared to wild-type controls. No p28 or p65 was detected in cells infected with predicted noncleaving CS1 mutants or the CS1 deletion mutant; however, a new protein of 93 kDa was detected. All introduced mutations and the deletion were retained during repeated virus passages in culture, and no phenotypic reversion was observed. The results of this study demonstrate that cleavage between p28 and p65 at CS1 is not required for MHV replication. However, proteolytic separation of p28 from p65 is necessary for optimal RNA synthesis and virus growth, suggesting important roles for these proteins in the formation or function of viral replication complexes.

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Identification of Determinants Involved in Initiation of Hepatitis C Virus RNA Synthesis by Using Intergenotypic Replicase Chimeras

Journal of Virology ◽

10.1128/jvi.00032-07 ◽

2007 ◽

Vol 81 (10) ◽

pp. 5270-5283 ◽

Cited By ~ 80

Author(s):

Marco Binder ◽

Doris Quinkert ◽

Olga Bochkarova ◽

Rahel Klein ◽

Nikolina Kezmic ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Synthesis ◽

Nonstructural Protein ◽

Negative Strand ◽

Virus Rna ◽

Nontranslated Region ◽

Genotype 2 ◽

Positive Strand Rna

ABSTRACT The 5′ nontranslated region (NTR) and the X tail in the 3′ NTR are the least variable parts of the hepatitis C virus (HCV) genome and play an important role in the initiation of RNA synthesis. By using subgenomic replicons of the HCV isolates Con1 (genotype 1) and JFH1 (genotype 2), we characterized the genotype specificities of the replication signals contained in the NTRs. The replacement of the JFH1 5′ NTR and X tail with the corresponding Con1 sequence resulted in a significant decrease in replication efficiency. Exchange of the X tail specifically reduced negative-strand synthesis, whereas substitution of the 5′ NTR impaired the generation of progeny positive strands. In search for the proteins involved in the recognition of genotype-specific initiation signals, we analyzed recombinant nonstructural protein 5B (NS5B) RNA polymerases of both isolates and found some genotype-specific template preference for the 3′ end of positive-strand RNA in vitro. To further address genotype specificity, we constructed a series of intergenotypic replicon chimeras. When combining NS3 to NS5A of Con1 with NS5B of JFH1, we observed more-efficient replication with the genotype 2a X tail, indicating that NS5B recognizes genotype-specific signals in this region. In contrast, a combination of the NS3 helicase with NS5A and NS5B was required to confer genotype specificity to the 5′ NTR. These results present the first genetic evidence for an interaction between helicase, NS5A, and NS5B required for the initiation of RNA synthesis and provide a system for the specific analysis of HCV positive- and negative-strand syntheses.

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Differential Rescue of Poliovirus RNA Replication Functions by Genetically Modified RNA Polymerase Precursors

Journal of Virology ◽

10.1128/jvi.78.23.13007-13018.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13007-13018 ◽

Cited By ~ 7

Author(s):

Christopher T. Cornell ◽

Jo Ellen Brunner ◽

Bert L. Semler

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Genetically Modified ◽

Rna Replication ◽

Amino Acid Sequences ◽

In Vitro Translation ◽

Wild Type ◽

Positive Strand ◽

Positive Strand Rna

ABSTRACT We have previously described the RNA replication properties of poliovirus transcripts harboring chimeric RNA polymerase sequences representing suballelic exchanges between poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3) utilizing an in vitro translation and RNA replication assay (C. Cornell, R. Perera, J. E. Brunner, and B. L. Semler, J. Virol. 78:4397-4407, 2004). We showed that three of the seven chimeras were capable of RNA replication in vitro, although replication levels were greatly reduced compared to that of wild-type transcripts. Interestingly, one of the replication-competent transcripts displayed a strand-specific RNA synthesis defect suggesting (i) a differential replication complex assembly mechanism involving 3D and/or precursor molecules (i.e., 3CD) required for negative- versus positive-strand RNA synthesis or (ii) effect(s) on the ability of the 3D polymerase to form higher-ordered structures required for positive-strand RNA synthesis. In this study, we have attempted to rescue defective RNA replication in vitro by cotranslating nonstructural proteins from a transcript encoding a large precursor polyprotein (P3) to complement 3D polymerase and/or precursor polypeptide functions altered in each of the chimeric constructs. Utilization of a wild-type P3 construct revealed that all transcripts containing chimeric PV1/CVB3 polymerase sequences can be complemented in trans for both negative- and positive-strand RNA synthesis. Furthermore, data from experiments utilizing genetically modified forms of the P3 polyprotein, containing mutations within 3C or 3D sequences, strongly suggest the existence of different protein-protein and protein-RNA interactions required for positive- versus negative-strand RNA synthesis. These results, combined with data from in vitro RNA elongation assays, indicate that the delivery of active 3D RNA polymerase to replication complexes requires a series of macromolecular interactions that rely on the presence of specific 3D amino acid sequences.

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Severe Acute Respiratory Syndrome Coronavirus ORF7a Inhibits Bone Marrow Stromal Antigen 2 Virion Tethering through a Novel Mechanism of Glycosylation Interference

Journal of Virology ◽

10.1128/jvi.02274-15 ◽

2015 ◽

Vol 89 (23) ◽

pp. 11820-11833 ◽

Cited By ~ 50

Author(s):

Justin K. Taylor ◽

Christopher M. Coleman ◽

Sandra Postel ◽

Jeanne M. Sisk ◽

John G. Bernbaum ◽

...

Keyword(s):

Bone Marrow ◽

Severe Acute Respiratory Syndrome ◽

Nonstructural Protein ◽

Atypical Pneumonia ◽

Growth Promoter ◽

Host Cell Membrane ◽

Nonstructural Protein 1 ◽

Antiviral Function ◽

Novel Coronavirus

ABSTRACTSevere acute respiratory syndrome (SARS) emerged in November 2002 as a case of atypical pneumonia in China, and the causative agent of SARS was identified to be a novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV). Bone marrow stromal antigen 2 (BST-2; also known as CD317 or tetherin) was initially identified to be a pre-B-cell growth promoter, but it also inhibits the release of virions of the retrovirus human immunodeficiency virus type 1 (HIV-1) by tethering budding virions to the host cell membrane. Further work has shown that BST-2 restricts the release of many other viruses, including the human coronavirus 229E (hCoV-229E), and the genomes of many of these viruses encode BST-2 antagonists to overcome BST-2 restriction. Given the previous studies on BST-2, we aimed to determine if BST-2 has the ability to restrict SARS-CoV and if the SARS-CoV genome encodes any proteins that modulate BST-2's antiviral function. Through anin vitroscreen, we identified four potential BST-2 modulators encoded by the SARS-CoV genome: the papain-like protease (PLPro), nonstructural protein 1 (nsp1), ORF6, and ORF7a. As the function of ORF7a in SARS-CoV replication was previously unknown, we focused our study on ORF7a. We found that BST-2 does restrict SARS-CoV, but the loss of ORF7a leads to a much greater restriction, confirming the role of ORF7a as an inhibitor of BST-2. We further characterized the mechanism of BST-2 inhibition by ORF7a and found that ORF7a localization changes when BST-2 is overexpressed and ORF7a binds directly to BST-2. Finally, we also show that SARS-CoV ORF7a blocks the restriction activity of BST-2 by blocking the glycosylation of BST-2.IMPORTANCEThe severe acute respiratory syndrome coronavirus (SARS-CoV) emerged from zoonotic sources in 2002 and caused over 8,000 infections and 800 deaths in 37 countries around the world. Identifying host factors that regulate SARS-CoV pathogenesis is critical to understanding how this lethal virus causes disease. We have found that BST-2 is capable of restricting SARS-CoV release from cells; however, we also identified a SARS-CoV protein that inhibits BST-2 function. We show that the SARS-CoV protein ORF7a inhibits BST-2 glycosylation, leading to a loss of BST-2's antiviral function.

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