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

mBio ◽  
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.

scholarly journals Noncapped Genomic RNA Are Critical for Alphaviral Infection and Pathogenicity

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 44
Author(s):  
Autumn T. LaPointe ◽  
Kevin J Sokoloski
Keyword(s):  
Growth Kinetics ◽  
Particle Production ◽  
Immune Cell ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Genomic Rnas ◽  
Viral Rnas ◽  
The Impact

Alphaviruses are positive-sense RNA arthropod-borne viruses that represent a significant threat to public health. During alphaviral replication, significant quantities of viral genomic RNAs that lack a canonical 5’ cap structure are produced and packaged into viral particles, despite the fact that the noncapped genomes cannot be translated and are essentially noninfectious. Previously, we have reported that the capping efficiency of nsP1, the alphaviral capping enzyme, of Sindbis virus (SINV) could be modulated via point mutation. It was found that increasing RNA capping efficiency led to decreased viral growth kinetics via decreased particle production, despite increased innate immune evasion, whereas decreasing capping efficiency led to wild-type growth kinetics and particle production. This led to the conclusion that the noncapped viral RNAs meaningfully contribute to the biology of alphaviral infections at the molecular level. To determine the importance of the noncapped viral RNAs in vivo, we characterized the impact of altered capping efficiency in a murine model of infection utilizing a neurovirulent strain of SINV. Mice infected with the nsP1 mutant with decreased capping exhibited wild-type rates of mortality, weight loss, and neurological symptoms. Conversely, the mice infected with the increased capping nsP1 mutant showed significantly reduced mortality and morbidity compared to mice infected with the wild-type virus. Interestingly, viral titers in the ankle, serum, and brain were equivalent between the wild-type virus and the two mutant viruses. Importantly, examination of the brain tissue revealed that mice infected with the increased capping mutant had significantly reduced immune cell infiltration and expression of proinflammatory cytokines compared to the decreased capping mutant and wild-type virus. Collectively, these data indicate that the noncapped viral RNAs have important roles during the early and late stages of alphaviral infection and suggest a novel mechanism by which noncapped viral RNA aids in viral pathogenesis.

scholarly journals Production of Noncapped Genomic RNAs is Critical to Sindbis Virus Disease and Pathogenicity

2020 ◽  
Author(s):  
Autumn T. LaPointe ◽  
V. Douglas Landers ◽  
Claire E. Westcott ◽  
Kevin J. Sokoloski
Keyword(s):  
Viral Replication ◽  
Sindbis Virus ◽  
Viral Rna ◽  
Wild Type Virus ◽  
Type Virus ◽  
Genomic Rna ◽  
Wild Type ◽  
Genomic Rnas ◽  
The Brain

ABSTRACTAlphaviruses 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 wild type infection. The mice infected with the D355A nsP1 mutant showed significantly reduced mortality and morbidity compared to mice infected with wild type virus. Interestingly, both capping mutants had roughly equivalent viral titer in the brain compared to wild type virus, illustrating that the changes in mortality were not due to deficits in viral replication or dissemination. Examination of the brain tissue revealed that mice infected with the D355A capping mutant had significantly reduced cell death and immune cell infiltration compared to the N376A mutant and wild type virus. Finally, 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 RNA aid in viral pathogenesis.AUTHOR SUMMARYMosquito transmitted alphaviruses have been the cause of widespread outbreaks of disease which can range from mild illness to lethal encephalitis or severe polyarthritis. In order to successfully replicate, the alphavirus RNA genome needs a 5’ cap structure so that the genome can be translated and produce the viral replication machinery. Despite this, a large number of viral genomes produced during infection do not have a 5’ cap structure, and their role during infection is unknown. Using mouse models of infection and point mutations in the nsP1 protein of Sindbis virus which alter the amount of noncapped genomic RNA (ncgRNA) produced, we found the decreasing the production of ncgRNA greatly reduced morbidity and mortality as well as proinflammatory cytokine expression, resulting in less tissue-damaging inflammation in the brain. These studies suggest that the ncgRNAs contribute to pathogenesis through the sensing of the ncgRNAs during alphaviral infection and are necessary for the development of severe disease.

scholarly journals Vaccinia Virus Envelope H3L Protein Binds to Cell Surface Heparan Sulfate and Is Important for Intracellular Mature Virion Morphogenesis and Virus Infection In Vitro and In Vivo

2000 ◽  
Vol 74 (7) ◽  
pp. 3353-3365 ◽  
Author(s):  
Chi-Long Lin ◽  
Che-Sheng Chung ◽  
Hans G. Heine ◽  
Wen Chang
Keyword(s):  
Cell Surface ◽  
Vaccinia Virus ◽  
Heparan Sulfate ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virion Morphogenesis

ABSTRACT An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L−) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L− mutant virus. IMV from the H3L− mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L− mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.

scholarly journals Inhibition of Cellular Proteasome Activities Enhances Hepadnavirus Replication in an HBX-Dependent Manner

2004 ◽  
Vol 78 (9) ◽  
pp. 4566-4572 ◽  
Author(s):  
Zhensheng Zhang ◽  
Ulrike Protzer ◽  
Zongyi Hu ◽  
James Jacob ◽  
T. Jake Liang
Keyword(s):  
Recombinant Adenovirus ◽  
Hepatitis Virus ◽  
Proteasome Inhibitors ◽  
Productive Infection ◽  
Wild Type Virus ◽  
Dependent Manner ◽  
Type Virus ◽  
Wild Type

ABSTRACT The X protein (HBX) of the hepatitis B virus (HBV) is not essential for the HBV life cycle in vitro but is important for productive infection in vivo. Our previous study suggests that interaction of HBX with the proteasome complex may underlie the pleiotropic functions of HBX. With the woodchuck model, we demonstrated that the X-deficient mutants of woodchuck hepatitis virus (WHV) are not completely replication defective, possibly behaving like attenuated viruses. In the present study, we analyzed the effects of the proteasome inhibitors on the replication of wild-type and X-negative HBV and WHV. Recombinant adenoviruses or baculoviruses expressing replicating HBV or WHV genomes have been developed as a robust and convenient system to study viral replication in tissue culture. In cells infected with either the recombinant adenovirus-HBV or baculovirus-WHV, the replication level of the X-negative construct was about 10% of that of the wild-type virus. In the presence of proteasome inhibitors, the replication of the wild-type virus was not affected, while the replication of the X-negative virus of either HBV or WHV was enhanced and restored to the wild-type level. Our data suggest that HBX affects hepadnavirus replication through a proteasome-dependent pathway.

scholarly journals TMC125 Displays a High Genetic Barrier to the Development of Resistance: Evidence from In Vitro Selection Experiments

2005 ◽  
Vol 79 (20) ◽  
pp. 12773-12782 ◽  
Author(s):  
Johan Vingerhoets ◽  
Hilde Azijn ◽  
Els Fransen ◽  
Inky De Baere ◽  
Liesbet Smeulders ◽  
...  
Keyword(s):  
Wild Type Virus ◽  
Cross Resistance ◽  
Resistant Virus ◽  
Wild Type ◽  
Genetic Barrier ◽  
Development Of Resistance ◽  
Selection Experiments ◽  
The Impact ◽  
Hiv 1

ABSTRACT TMC125 is a potent new investigational nonnucleoside reverse transcriptase inhibitor (NNRTI) that is active against human immunodeficiency virus type 1 (HIV-1) with resistance to currently licensed NNRTIs. Sequential passage experiments with both wild-type virus and NNRTI-resistant virus were performed to identify mutations selected by TMC125 in vitro. In addition to “classic” selection experiments at a low multiplicity of infection (MOI) with increasing concentrations of inhibitors, experiments at a high MOI with fixed concentrations of inhibitors were performed to ensure a standardized comparison between TMC125 and current NNRTIs. Both low- and high-MOI experiments demonstrated that the development of resistance to TMC125 required multiple mutations which frequently conferred cross-resistance to efavirenz and nevirapine. In high-MOI experiments, 1 μM TMC125 completely inhibited the breakthrough of resistant virus from wild-type and NNRTI-resistant HIV-1, in contrast to efavirenz and nevirapine. Furthermore, breakthrough of virus from site-directed mutant (SDM) SDM-K103N/Y181C occurred at the same time or later with TMC125 as breakthrough from wild-type HIV-1 with efavirenz or nevirapine. The selection experiments identified mutations selected by TMC125 that included known NNRTI-associated mutations L100I, Y181C, G190E, M230L, and Y318F and the novel mutations V179I and V179F. Testing the antiviral activity of TMC125 against a panel of SDMs indicated that the impact of these individual mutations on resistance was highly dependent upon the presence and identity of coexisting mutations. These results demonstrate that TMC125 has a unique profile of activity against NNRTI-resistant virus and possesses a high genetic barrier to the development of resistance in vitro.

scholarly journals Discordance between Bovine Leukemia Virus Tax Immortalization In Vitro and Oncogenicity In Vivo

2000 ◽  
Vol 74 (21) ◽  
pp. 9895-9902 ◽  
Author(s):  
Jean-Claude Twizere ◽  
Pierre Kerkhofs ◽  
Arsène Burny ◽  
Daniel Portetelle ◽  
Richard Kettmann ◽  
...  
Keyword(s):  
Viral Replication ◽  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Wild Type Virus ◽  
Target Cells ◽  
Phosphorylation Sites ◽  
Primary Cells ◽  
Wild Type

ABSTRACT Bovine leukemia virus (BLV) Tax protein, a transcriptional activator of viral expression, is essential for viral replication in vivo. Tax is believed to be involved in leukemogenesis because of its second function, immortalization of primary cells in vitro. These activities of Tax can be dissociated on the basis of point mutations within specific regions of the protein. For example, mutation of the phosphorylation sites at serines 106 and 293 abrogates immortalization potential in vitro but maintains transcriptional activity. This type of mutant is thus particularly useful for unraveling the role of Tax immortalization activity during leukemogenesis independently of viral replication. In this report, we describe the biological properties of BLV recombinant proviruses mutated in the Tax phosphorylation sites (BLVTax106+293). Titration of the proviral loads by semiquantitative PCR revealed that the BLV mutants propagated at wild-type levels in vivo. Furthermore, two animals (sheep 480 and 296) infected with BLVTax106+293 developed leukemia or lymphosarcoma after 16 and 36 months, respectively. These periods of time are within the normal range of latencies preceding the onset of pathogenesis induced by wild-type viruses. The phenotype of the mutant-infected cells was characteristic of a B lymphocyte (immunoglobulin M positive) expressing CD11b and CD5 (except at the final stage for the latter marker), a pattern that is typical of wild-type virus-infected target cells. Interestingly, the transformed B lymphocytes from sheep 480 also coexpressed the CD8 marker, a phenotype rarely observed in tumor biopsies from chronic lymphocytic leukemia patients. Finally, direct sequencing of the tax gene demonstrated that the leukemic cells did not harbor revertant proviruses. We conclude that viruses expressing a Tax mutant unable to transform primary cells in culture are still pathogenic in the sheep animal model. Our data thus provide a clear example of the discordant conclusions that can be drawn from in vitro immortalization assays and in vivo experiments. These observations could be of interest for other systems, such as the related human T-cell leukemia virus type 1, which currently lack animal models allowing the study of the leukemogenic process.

scholarly journals Targeting Human Immunodeficiency Virus (HIV) Type 2 Integrase Protein into HIV Type 1

1999 ◽  
Vol 73 (10) ◽  
pp. 8831-8836 ◽  
Author(s):  
Hongmei Liu ◽  
Xiaoyun Wu ◽  
Hongling Xiao ◽  
John C. Kappes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcription ◽  
Wild Type Virus ◽  
Strand Transfer ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Integrase (IN) is the only retroviral enzyme necessary for the integration of retroviral cDNA into the host cell’s chromosomes. The structure and function of IN is highly conserved. The human immunodeficiency virus type 2 (HIV-2) IN has been shown to efficiently support 3′ processing and strand transfer of HIV-1 DNA substrate in vitro. To determine whether HIV-2 IN protein (IN2) could substitute for HIV-1 IN function in vivo, we used HIV-1 Vpr to deliver the IN2 into IN mutant HIV-1 virions by expression intrans as a Vpr-IN fusion protein.Trans-complementation with IN2 markedly increased the infectivity of IN-minus HIV-1. Compared with the homologous trans-IN protein, infectivity was increased to a level of 16%. Since IN has been found to play a role in reverse transcription (Wu et al., J. Virol. 73:2126–2135, 1999), cells infected with IN2-complemented HIV-1 were analyzed for DNA products of reverse transcription. DNA levels of approximately 18% of that of wild type were detected. The homologous trans-IN protein restored the synthesis of viral cDNA to approximately 86% of that of wild-type virus. By complementing integration-defective HIV-1 IN mutant viruses, which were not impaired in cDNA synthesis, thetrans-IN2 protein was shown to support integration up to a level of 55% compared with that of the homologoustrans-IN protein. The delivery of heterologous IN protein into HIV-1 particles in trans offers a novel approach to understand IN protein function in vivo.

scholarly journals Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection

2018 ◽  
Author(s):  
Autumn T. LaPointe ◽  
Joaquín Moreno-Contreras ◽  
Kevin J. Sokoloski
Keyword(s):  
Viral Infection ◽  
Growth Kinetics ◽  
Sindbis Virus ◽  
Nonstructural Protein ◽  
Severe Disease ◽  
Protein Accumulation ◽  
Genomic Rnas ◽  
Viral Growth ◽  
Viral Packaging ◽  
The Impact

AbstractAlphaviruses are arthropod-borne RNA viruses that are capable of causing severe disease and are a significant burden to public health. Alphaviral replication results in the production of both capped and noncapped viral genomic RNAs, which are packaged into virions during the infections of vertebrate and invertebrate cells. However, the roles that the noncapped genomic RNAs (ncgRNAs) play during alphaviral infection have yet to be exhaustively characterized. Here, the importance of the ncgRNAs to alphaviral infection was assessed by using mutants of the nsP1 protein of Sindbis virus (SINV), which altered the synthesis of the ncgRNAs during infection by modulating the protein’s capping efficiency. Specifically, point mutants at residues Y286A and N376A decreased capping efficiency, while a point mutant at D355A increased the capping efficiency of the SINV genomic RNA during genuine viral infection. Viral growth kinetics were significantly reduced for the D355A mutant relative to wild type infection, whereas the Y286A and N376A mutants showed modest decreases in growth kinetics. Overall genomic translation and nonstructural protein accumulation was found to correlate with increases and decreases in capping efficiency. However, genomic, minus strand, and subgenomic viral RNA synthesis was largely unaffected by the modulation of alphaviral capping activity. In addition, translation of the subgenomic vRNA was found to be unimpacted by changes in capping efficiency. The mechanism by which decreased presence of ncgRNAs reduced viral growth kinetics was through the impaired production of viral particles. Collectively, these data illustrate the importance of ncgRNAs to viral infection and suggests that they play in integral role in the production of viral progeny.ImportanceAlphaviruses have been the cause of both localized outbreaks and large epidemics of severe disease. Currently, there are no strategies or vaccines which are either safe or effective for preventing alphaviral infection or treating alphaviral disease. This deficit of viable therapeutics highlights the need to better understand the mechanisms behind alphaviral infection in order to develop novel antiviral strategies for alphaviral disease. In particular, this study details a previously uncharacterized aspect of the alphaviral life cycle, the importance of noncapped genomic viral RNAs to alphaviral infection. This offers new insights into the mechanisms of alphaviral replication and the impact of the noncapped genomic RNAs on viral packaging.

scholarly journals Residues in the Heptad Repeat A Region of the Fusion Protein Modulate the Virulence of Sendai Virus in Mice

2009 ◽  
Vol 84 (2) ◽  
pp. 810-821 ◽  
Author(s):  
Laura E. Luque ◽  
Olga A. Bridges ◽  
John N. Mason ◽  
Kelli L. Boyd ◽  
Allen Portner ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Sendai Virus ◽  
Syncytium Formation ◽  
Heptad Repeat ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
F Protein

ABSTRACT While the molecular basis of fusion (F) protein refolding during membrane fusion has been studied extensively in vitro, little is known about the biological significance of membrane fusion activity in parainfluenza virus replication and pathogenesis in vivo. Two recombinant Sendai viruses, F-L179V and F-K180Q, were generated that contain F protein mutations in the heptad repeat A region of the ectodomain, a region of the protein known to regulate F protein activation. In vitro, the F-L179V virus caused increased syncytium formation (cell-cell membrane fusion) yet had a rate of replication and levels of F protein expression and cleavage similar to wild-type virus. The F-K180Q virus had a reduced replication rate along with reduced levels of F protein expression, cleavage, and fusogenicity. In DBA/2 mice, the hyperfusogenic F-L179V virus induced greater morbidity and mortality than wild-type virus, while the attenuated F-K180Q virus was much less pathogenic. During the first week of infection, virus replication and inflammation in the lungs were similar for wild-type and F-L179V viruses. After approximately 1 week of infection, the clearance of F-L179V virus was delayed, and more extensive interstitial inflammation and necrosis were observed in the lungs, affecting entire lobes of the lungs and having significantly greater numbers of syncytial cell masses in alveolar spaces on day 10. On the other hand, the slower-growing F-K180Q virus caused much less extensive inflammation than wild-type virus, presumably due to its reduced replication rate, and did not cause observable syncytium formation in the lungs. Overall, the results show that residues in the heptad repeat A region of the F protein modulate the virulence of Sendai virus in mice by influencing both the spread and clearance of the virus and the extent and severity of inflammation. An understanding of how the F protein contributes to infection and inflammation in vivo may assist in the development of antiviral therapies against respiratory paramyxoviruses.

scholarly journals Drug Susceptibility and Replicative Capacity of Multidrug-Resistant Recombinant Human Cytomegalovirus Harboring Mutations in UL56 and UL54 Genes

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Jocelyne Piret ◽  
Nathalie Goyette ◽  
Guy Boivin
Keyword(s):  
Human Cytomegalovirus ◽  
Double Mutant ◽  
Antiviral Agent ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Wild Type Virus ◽  
Replicative Capacity ◽  
Wild Type ◽  
The Impact

ABSTRACT Letermovir is an investigational antiviral agent with a novel mechanism of action involving the viral terminase (pUL56). We evaluated the impact of the V236M mutation in the UL56 gene alone and in combination with the E756K mutation in the UL54 gene on drug susceptibility and viral replicative capacity of recombinant human cytomegalovirus. The double mutant exhibited at least borderline resistance to all antivirals tested (ganciclovir, foscarnet, cidofovir, brincidofovir, and letermovir) and replicated less efficiently than the wild-type virus in vitro.

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