Faculty Opinions recommendation of West Nile virus nonstructural protein NS1 inhibits complement activation by binding the regulatory protein factor H.

ABSTRACT We demonstrate the presence of nonstructural protein 1 (NS1)-specific antibodies in a significant proportion of convalescent-phase human serum samples obtained from a cohort in an area where Japanese encephalitis virus (JEV) is endemic. Sera containing antibodies to NS1 but not those with antibodies to other JEV proteins, such as envelope, brought about complement-mediated lysis of JEV-infected BHK-21 cells. Target cells infected with a recombinant poxvirus expressing JEV NS1 on the cell surface confirmed the NS1 specificity of cytolytic antibodies. Mouse anti-NS1 cytolytic sera caused a complement-dependent reduction in virus output from infected human cells, demonstrating their important role in viral control. Antibodies elicited by JEV NS1 did not cross lyse West Nile virus- or dengue virus-infected cells despite immunoprecipitating the NS1 proteins of these related flaviviruses. Additionally, JEV NS1 failed to bind complement factor H, in contrast to NS1 of West Nile virus, suggesting that the NS1 proteins of different flaviviruses have distinctly different mechanisms for interacting with the host. Our results also point to an important role for JEV NS1-specific human immune responses in protection against JE and provide a strong case for inclusion of the NS1 protein in next generation of JEV vaccines.

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Potential Dual Role of West Nile Virus NS2B in Orchestrating NS3 Enzymatic Activity in Viral Replication

Viruses ◽

10.3390/v13020216 ◽

2021 ◽

Vol 13 (2) ◽

pp. 216

Author(s):

Alanna C. Tseng ◽

Vivek R. Nerurkar ◽

Kabi R. Neupane ◽

Helmut Kae ◽

Pakieli H. Kaufusi

Keyword(s):

West Nile Virus ◽

Enzymatic Activity ◽

Nonstructural Protein ◽

Antiviral Drug ◽

West Nile ◽

Biochemical Assays ◽

In Trans ◽

Viral Polyprotein ◽

Ns3 Protease

West Nile virus (WNV) nonstructural protein 3 (NS3) harbors the viral triphosphatase and helicase for viral RNA synthesis and, together with NS2B, constitutes the protease responsible for polyprotein processing. NS3 is a soluble protein, but it is localized to specialized compartments at the rough endoplasmic reticulum (RER), where its enzymatic functions are essential for virus replication. However, the mechanistic details behind the recruitment of NS3 from the cytoplasm to the RER have not yet been fully elucidated. In this study, we employed immunofluorescence and biochemical assays to demonstrate that NS3, when expressed individually and when cleaved from the viral polyprotein, is localized exclusively to the cytoplasm. Furthermore, NS3 appeared to be peripherally recruited to the RER and proteolytically active when NS2B was provided in trans. Thus, we provide evidence for a potential additional role for NS2B in not only serving as the cofactor for the NS3 protease, but also in recruiting NS3 from the cytoplasm to the RER for proper enzymatic activity. Results from our study suggest that targeting the interaction between NS2B and NS3 in disrupting the NS3 ER localization may be an attractive avenue for antiviral drug discovery.

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Complement Regulatory Protein Factor H Is a Soluble Prion Receptor That Potentiates Peripheral Prion Pathogenesis

The Journal of Immunology ◽

10.4049/jimmunol.1701100 ◽

2017 ◽

Vol 199 (11) ◽

pp. 3821-3827 ◽

Cited By ~ 4

Author(s):

Sarah J. Kane ◽

Taylor K. Farley ◽

Elizabeth O. Gordon ◽

Joshua Estep ◽

Heather R. Bender ◽

...

Keyword(s):

Regulatory Protein ◽

Factor H ◽

Complement Regulatory Protein ◽

Protein Factor

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Arg127 of complement regulatory protein Factor H is important for its secretion from human fibroblasts

Molecular Immunology ◽

10.1016/j.molimm.2009.05.328 ◽

2009 ◽

Vol 46 (14) ◽

pp. 2869

Author(s):

José Antonio Tavares Albuquerque ◽

Dayseanne Araújo Falcão ◽

Lourdes Isaac

Keyword(s):

Human Fibroblasts ◽

Regulatory Protein ◽

Factor H ◽

Complement Regulatory Protein ◽

Protein Factor

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Functional properties of the allotypes of mouse complement regulatory protein, factor H: Difference of compatibility of each allotype with human factor I

Molecular Immunology ◽

10.1016/0161-5890(93)90007-x ◽

1993 ◽

Vol 30 (9) ◽

pp. 841-848 ◽

Cited By ~ 3

Author(s):

Okada Michiyo ◽

Kojima Ayako ◽

Takano Hiromi ◽

Harada Yoshinobu ◽

Nonaka Mayumi ◽

...

Keyword(s):

Functional Properties ◽

Human Factor ◽

Regulatory Protein ◽

Factor H ◽

Complement Regulatory Protein ◽

Factor I ◽

Protein Factor

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Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans

Journal of Clinical Microbiology ◽

10.1128/jcm.02735-14 ◽

2014 ◽

Vol 53 (2) ◽

pp. 557-566 ◽

Cited By ~ 17

Author(s):

Day-Yu Chao ◽

Jedhan Ucat Galula ◽

Wen-Fan Shen ◽

Brent S. Davis ◽

Gwong-Jen J. Chang

Keyword(s):

West Nile Virus ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Nonstructural Protein ◽

Encephalitis Virus ◽

Igg Antibody ◽

West Nile ◽

Dengue Viruses ◽

Nonstructural Protein 1 ◽

Antibody Capture

IgM antibody- and IgG antibody-capture enzyme-linked immunosorbent assays (MAC/GAC-ELISAs) targeted at envelope protein (E) of dengue viruses (DENV), West Nile virus, and Japanese encephalitis virus (JEV) are widely used as serodiagnostic tests for presumptive confirmation of viral infection. Antibodies directed against the flavivirus nonstructural protein 1 (NS1) have been proposed as serological markers of natural infections among vaccinated populations. The aim of the current study is to optimize an IgM and IgG antibody-capture ELISA (MAC/GAC-ELISA) to detect anti-NS1 antibodies and compare it with anti-E MAC/GAC-ELISA. Plasmids to express premembrane/envelope (prM/E) or NS1 proteins of six medically important flaviviruses, including dengue viruses (DENV-1 to DENV-4), West Nile virus (WNV), and Japanese encephalitis virus (JEV), were constructed. These plasmids were used for the production of prM/E-containing virus-like particles (VLPs) and secreted NS1 (sNS1) from COS-1 cells. Archived clinical specimens from patients with confirmed DENV, JEV, and WNV infections, along with naive sera, were subjected to NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by preabsorption with or without VLPs. Human serum specimens from previously confirmed DENV infections showed significantly enhanced positive-to-negative (P/N) ratios for NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical differences in sensitivities and specificities were found between the newly developed NS1- and VLP-MAC/GAC-ELISAs. Further application of the assays to WNV- and JEV-infected serum panels showed similar results. A novel approach to perform MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with great potential to differentiate antibodies elicited by the tetravalent chimeric yellow fever-17D/dengue vaccine or DENV infection.

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Sequence analysis of a cDNA clone encoding the C-terminal end of human complement factor H

Bioscience Reports ◽

10.1007/bf01124790 ◽

1987 ◽

Vol 7 (3) ◽

pp. 201-207 ◽

Cited By ~ 2

Author(s):

A. J. Day ◽

J. Ripoche ◽

A. Lyons ◽

B. McIntosh ◽

T. J. R. Harris ◽

...

Keyword(s):

Amino Acids ◽

Tertiary Structure ◽

Regulatory Protein ◽

Peptide Sequencing ◽

Factor H ◽

Cdna Libraries ◽

Complement Factor ◽

Protein Factor ◽

The Complement System ◽

Full Length Clone

Peptide sequencing of the complement system regulatory protein, factor H, permitted the synthesis of a mixed sequence oligonucleotide probe. Human liver cDNA libraries were screened and factor H-specific clones selected. No full-length clone was obtained, but the largest available clone, R2a, was found to encode the C-terminal 657 amino acids of factor H. The derived amino acid sequence consists of 10 contiguous internally homologous segments, each about 60 amino acids long. Sequences homologous to these are found in several other complement and non-complement proteins. Such sequences are likely to represent a particular type of tertiary structure subunit.

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An interaction between the methyltransferase and RNA dependent RNA polymerase domains of the West Nile virus NS5 protein

Journal of General Virology ◽

10.1099/vir.0.054395-0 ◽

2013 ◽

Vol 94 (9) ◽

pp. 1961-1971 ◽

Cited By ~ 14

Author(s):

Cindy S. E. Tan ◽

Jody M. Hobson-Peters ◽

Martin J. Stoermer ◽

David P. Fairlie ◽

Alexander A. Khromykh ◽

...

Keyword(s):

West Nile Virus ◽

Rna Polymerase ◽

Genetic Interaction ◽

Nonstructural Protein ◽

Specific Interaction ◽

Simian Virus 40 ◽

T Antigen ◽

Rna Dependent Rna Polymerase ◽

West Nile

The flavivirus nonstructural protein 5 (NS5) is a large protein that is structurally conserved among members of the genus, making it an attractive target for antiviral drug development. The protein contains a methyltransferase (MTase) domain and an RNA dependent RNA polymerase (POL) domain. Previous studies with dengue viruses have identified a genetic interaction between residues 46–49 in the αA3-motif in the MTase and residue 512 in POL. These genetic interactions are consistent with structural modelling of these domains in West Nile virus (WNV) NS5 that predict close proximity of these regions of the two domains, and potentially a functional interaction mediated via the αA3-motif. To demonstrate an interaction between the MTase and POL domains of the WNV NS5 protein, we co-expressed affinity-tagged recombinant MTase and POL proteins in human embryonic kidney cells with simian virus 40 large T antigen (HEK293T cells) and performed pulldown assays using an antibody to the flag tag on POL. Western blot analysis with an anti-MTase mAb revealed that the MTase protein was specifically co-immunoprecipitated with POL, providing the first evidence of a specific interaction between these domains. To further assess the role of the αA3 helix in this interaction, selected residues in this motif were mutated in the recombinant MTase and the effect on POL interaction determined by the pulldown assay. These mutations were also introduced into a WNV infectious clone (FLSDX) and the replication properties of these mutant viruses assessed. While none of the αA3 mutations had a significant effect on the MTase–POL association in pulldown assays, suggesting that these residues were not specific to the interaction, an E46L mutation completely abolished virus viability indicating a critical requirement of this residue in replication. Failure to generate compensatory mutations in POL to rescue replication, even after several passages of the transfection supernatant in Vero cells, precluded further conclusion of the role of this residue in the context of MTase–POL interactions.

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