Independent Structural Domains in Paramyxovirus Polymerase Protein

Dochow, Melanie; Krumm, Stefanie A.; Crowe, James E.; Moore, Martin L.; Plemper, Richard K.

doi:10.1074/jbc.m111.325258

Cited by 50 publications

(64 citation statements)

References 87 publications

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“…We observed the same phenomenon for the VSV L protein (27), while other groups of investigators extended these findings to additional paramyxoviruses (28,29) as well Ebola virus (30). Although it seems likely that other regions of the L protein appendage also function as hinges to allow appendage domain movement, these may not be tolerant of large insertions and have yet to be identified.…”

supporting

confidence: 80%

“…Notably, a recent study showed that paramyxovirus L protein constructs encoding the region upstream and downstream of the hinge region could not reconstitute polymerase activity when they were mixed together unless first fused to dimerization tags (29). No reconstitution of activity was observed when the two L fragments originated from different paramyxoviruses.…”

mentioning

confidence: 99%

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Putative Domain-Domain Interactions in the Vesicular Stomatitis Virus L Polymerase Protein Appendage Region

Ruedas

Perrault

2014

J Virol

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The multidomain polymerase protein (L) of nonsegmented negative-strand (NNS) RNA viruses catalyzes transcription and replication of the virus genome. The N-terminal half of the protein forms a ring-like polymerase structure, while the C-terminal half encoding viral mRNA transcript modifications consists of a flexible appendage with three distinct globular domains. To gain insight into putative transient interactions between L domains during viral RNA synthesis, we exchanged each of the four distinct regions encompassing the appendage region of vesicular stomatitis virus (VSV) Indiana serotype L protein with their counterparts from VSV New Jersey and analyzed effects on virus polymerase activity in a minigenome system. The methyltransferase domain exchange yielded a fully active polymerase protein, which functioned as well as wild-type L in the context of a recombinant virus. Exchange of the downstream C-terminal nonconserved region abolished activity, but coexchanging it with the methyltransferase domain generated a polymerase favoring replicase over transcriptase activity, providing strong evidence of interaction between these two regions. Exchange of the capping enzyme domain or the adjacent nonconserved region thought to function as an "unstructured" linker also abrogated polymerase activity even when either domain was coexchanged with other appendage domains. Further probing of the putative linker segment using in-frame enhanced green fluorescent protein (EGFP) insertions similarly abrogated activity. We discuss the implications of these findings with regard to L protein appendage domain structure and putative domain-domain interactions required for polymerase function. IMPORTANCE NNS viruses include many well-known human pathogens (e.g., rabies, measles, and Ebola viruses), as well as emerging viral threats (e.g., Nipah and Hendra viruses). These viruses all encode a large L polymerase protein similarly organized into multiple domains that work in concert to enable virus genome transcription and replication. But how the unique L protein carries out the multiplicity of individual steps in these two distinct processes is poorly understood. Using two different approaches, i.e., exchanging individual domains in the C-terminal appendage region of the protein between two closely related VSV serotypes and inserting unrelated protein domains, we shed light on requirements for domain-domain interactions and domain contiguity in polymerase function. These findings further our understanding of the conformational dynamics of NNS L polymerase proteins, which play an essential role in the pathogenic properties of these viruses and represent attractive targets for the development of antiviral measures.

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supporting

confidence: 80%

mentioning

confidence: 99%

Putative Domain-Domain Interactions in the Vesicular Stomatitis Virus L Polymerase Protein Appendage Region

Ruedas

Perrault

2014

J Virol

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“…Importantly, the data we have obtained suggest that, in the context of RSV infection, YM-53403 suppresses overall RSV RNA synthesis but likely does not result in an increase in the levels of abortive RNAs. The location of YM-53403 resistance mutations in variable region 2 of the L gene, combined with structural domain studies of the RSV RNA polymerase indicating a dimerization function in this area (16,28,29), suggests that the compound may allosterically inhibit a tertiary complex essential for transcription or replication. Our findings are consistent with a recent publication showing the ability of a closely related compound, AZ-27, to inhibit a common step in the initiation of RSV transcription and replication (30).…”

Section: Discussionmentioning

confidence: 99%

The Interferon Type I/III Response to Respiratory Syncytial Virus Infection in Airway Epithelial Cells Can Be Attenuated or Amplified by Antiviral Treatment

McCutcheon

Jordan

Mawhorter

et al. 2016

J Virol

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Human respiratory syncytial virus (RSV) is a single-stranded RNA virus that causes acute, and occasionally fatal, lower respiratory illness in young infants, the elderly, and immunocompromised patients. Therapeutic interventions able to cut short viral replication and quickly return the airways to normal function are needed. An understanding of antiviral activities and their effects on host defense mechanisms is important for the design of safe and effective therapy. We targeted functionally and temporally distinct steps within the viral life cycle using small-molecule RSV inhibitors and studied their antiviral activities and their effects on innate interferon responses of airway epithelial cells in vitro. H uman respiratory syncytial virus (RSV) is a single-stranded, negative-sense RNA virus belonging to the family Paramyxoviridae. Premature and very young infants are at the highest risk of having severe lower respiratory tract infections and, later, a higher incidence of chronic asthma (1). Older adults with chronic lung or heart disease and individuals with suppressed immune systems also often require medical care after RSV infection. The lack of long-lasting protection after primary infection contributes to the capacity of RSV to cause yearly outbreaks and has challenged vaccine development (2). Synagis, a monoclonal antibody (MAb) directed against the RSV fusion protein, has shown prophylactic utility, but its cost and intramuscular route of administration have limited its use to hospitalized, high-risk infants Ͻ2 years of age (2). The only therapeutic intervention for RSV infection currently available to patients is the use of ribavirin (3), but its use is also limited because of poor efficacy and teratogenicity and the requirement of an aerosol and/or intravenous (i.v.) mode of administration in hospital settings (4). New therapeutic interventions able to lower the viral load, decrease transmission, and prevent lower respiratory complications are needed.RSV infection is initiated by attachment of the viral G protein to the surface of airway epithelial (AE) cells (5,6). Following attachment, the viral F protein mediates fusion of the viral and cellular membranes, allowing the RSV ribonucleic protein (RNP) complex, comprised of the viral RNA genome encapsulated with nucleoprotein (N) and associated with the phosphoprotein (P) and RNA-dependent RNA polymerase (L), to enter the cytoplasm. The RNP complex performs viral transcription to produce capped and polyadenylated mRNAs and genome replication. Detailed understanding of RSV biology is made difficult by the intimate coupling of transcription and replication activities. Furthermore, the RSV polymerase is large and complex, containing multiple domains and enzymatic activities that allow it to function and be

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“…Based on a described pT7-RSV-luciferase minigenome reporter (36), an RSV minigenome construct was generated under the control of the constitutive RNA pol I promoter (pHH-RSV-repl-firefly). Huh-7 cells were cotransfected with this plasmid and plasmids pRSV-L, pRSV-M2-1, pRSV-N, and pRSV-P under CMV promoter control.…”

Section: Methodsmentioning

confidence: 99%

Cross-resistance mechanism of respiratory syncytial virus against structurally diverse entry inhibitors

Yan

Lee

Thakkar

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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109

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Respiratory syncytial virus (RSV) is a leading pediatric pathogen that is responsible for a majority of infant hospitalizations due to viral disease. Despite its clinical importance, no vaccine prophylaxis against RSV disease or effective antiviral therapeutic is available. In this study, we established a robust high-throughput drug screening protocol by using a recombinant RSV reporter virus to expand the pool of RSV inhibitor candidates. Mechanistic characterization revealed that a potent newly identified inhibitor class blocks viral entry through specific targeting of the RSV fusion (F) protein. Resistance against this class was induced and revealed overlapping hotspots with diverse, previously identified RSV entry blockers at different stages of preclinical and clinical development. A structural and biochemical assessment of the mechanism of unique, broad RSV cross-resistance against structurally distinct entry inhibitors demonstrated that individual escape hotspots are located in immediate physical proximity in the metastable conformation of RSV F and that the resistance mutations lower the barrier for prefusion F triggering, resulting in an accelerated RSV entry kinetics. One resistant RSV recombinant remained fully pathogenic in a mouse model of RSV infection. By identifying molecular determinants governing the RSV entry machinery, this study spotlights a molecular mechanism of broad RSV resistance against entry inhibition that may affect the impact of diverse viral entry inhibitors presently considered for clinical use and outlines a proactive design for future RSV drug discovery campaigns.antiviral drugs | drug resistance

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Independent Structural Domains in Paramyxovirus Polymerase Protein

Cited by 50 publications

References 87 publications

Putative Domain-Domain Interactions in the Vesicular Stomatitis Virus L Polymerase Protein Appendage Region

Putative Domain-Domain Interactions in the Vesicular Stomatitis Virus L Polymerase Protein Appendage Region

The Interferon Type I/III Response to Respiratory Syncytial Virus Infection in Airway Epithelial Cells Can Be Attenuated or Amplified by Antiviral Treatment

Cross-resistance mechanism of respiratory syncytial virus against structurally diverse entry inhibitors

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