Use of a tandem affinity purification assay to detect interactions between West Nile and dengue viral proteins and proteins of the mosquito vector

Colpitts, Tonya M.; Cox, James C.; Nguyen, Annie; Feitosa, Fabiana; Krishnan, Manoj N.; Fikrig, Erol

doi:10.1016/j.virol.2011.06.002

Cited by 40 publications

(39 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Accordingly, mutation of the basic residues might prevent interaction partners from entering the complex necessary for particle formation without affecting the NS2A-NS3 interaction itself or might interfere with RNA binding. For NS2A of DENV and WNV, several host proteins of mosquito origin, like translation factors or proteins involved in the cytoskeleton and cellular trafficking, have been found as binding partners (35). Further studies are necessary to determine whether these and analogous vertebrate proteins are involved in the formation of a complex necessary for assembly and release of infectious flavivirus particles.…”

Section: Discussionmentioning

confidence: 99%

A Basic Cluster in the N Terminus of Yellow Fever Virus NS2A Contributes to Infectious Particle Production

Voßmann

Wieseler

Kerber

et al. 2015

J Virol

View full text Add to dashboard Cite

The flavivirus NS2A protein is involved in the assembly of infectious particles. To further understand its role in this process, a charged-to-alanine scanning analysis was performed on NS2A encoded by an infectious cDNA clone of yellow fever virus (YFV). Fifteen mutants containing single, double, or triple charged-to-alanine changes were tested. Five of them did not produce infectious particles, whereas efficient RNA replication was detectable for two of the five NS2A mutants (R22A-K23A-R24A and R99A-E100A-R101A mutants). Prolonged cultivation of transfected cells resulted in the recovery of pseudorevertants. Besides suppressor mutants in NS2A, a compensating second-site mutation in NS3 (D343G) arose for the NS2A R22A-K23A-R24A mutant. We found this NS3 mutation previously to be suppressive for the NS2A␣ cleavage site Q189S mutant, also deficient in virion assembly. In this study, the subsequently suggested interaction between NS2A and NS3 was proven by coimmunoprecipitation analyses. Using selectively permeabilized cells, we could demonstrate that the regions encompassing R22A-K23A-R24A and Q189S in NS2A are localized to the cytoplasm, where NS3 is also known to reside. However, the defect in particle production observed for the NS2A R22A-K23A-R24A and Q189S mutants was not due to a defect in physical interaction between NS2A and NS3, as the NS2A mutations did not interrupt NS3 interaction. In fact, a region just upstream of R22-K23-R24 was mapped to be critical for NS2A-NS3 interaction. Taken together, these data support a complex interplay between YFV NS2A and NS3 in virion assembly and identify a basic cluster in the NS2A N terminus to be critical in this process. IMPORTANCEDespite an available vaccine, yellow fever remains endemic in tropical areas of South America and Africa. To control the disease, antiviral drugs are required, and an understanding of the determinants of virion assembly is central to their development. In this study, we identified a basic cluster of amino acids in the N terminus of YFV NS2A which inhibited virion assembly upon mutation. The defect was rescued by a spontaneously occurring mutation in NS3. Our study proves an interaction between NS2A and NS3, which, remarkably, was maintained for the NS2A mutant in the presence and absence of the NS3 mutation. This suggests a role for other viral and/or cellular proteins in virion assembly. Residues important for YFV virion production reported here only partially coincided with those reported for other flaviviruses, suggesting that the determinants for particle production are virus specific. Reconstruction of a YFV encoding tagged NS2A paves the way to identify further NS2A interaction partners. Y ellow fever virus (YFV) belongs to the genus flavivirus within the family Flaviviridae. Clinical disease associated with this prototype flavivirus can result in acute viral hemorrhagic fever (1). Other important human flaviviruses include dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus, and tick-borne encephalitis v...

show abstract

Section: Discussionmentioning

confidence: 99%

A Basic Cluster in the N Terminus of Yellow Fever Virus NS2A Contributes to Infectious Particle Production

Voßmann

Wieseler

Kerber

et al. 2015

J Virol

View full text Add to dashboard Cite

show abstract

“…To date, very few studies have been conducted to determine the interactome between DENV proteins and mosquito cellular proteins using sensitive and reliable protein interaction assays [22,23], with some predictions reported through a computational approach [24]. Hence, the aim of this study was to identify adult A. aegypti mosquito midgut proteins interacting with DENV-2 viral proteins, using an improved yeast twohybrid (Y2H) screening system, a high-throughput screening assay that allows identification of genuine protein interacting partners in vivo.…”

Section: Introductionmentioning

confidence: 99%

Protein-protein interactions between A. aegypti midgut and dengue virus 2: two-hybrid screens using the midgut cDNA library

Tham

Balasubramaniam

Chew

et al. 2015

J Infect Dev Ctries

View full text Add to dashboard Cite

Introduction: Dengue virus (DENV) is principally transmitted by the Aedes aegypti mosquito. To date, mosquito population control remains the key strategy for reducing the continuing spread of DENV. The focus on the development of new vector control strategies through an understanding of the mosquito-virus relationship is essential, especially targeting the midgut, which is the first mosquito organ exposed to DENV infection. Methodology: A cDNA library derived from female adult A. aegypti mosquito midgut cells was established using the switching mechanism at the 5' end of the RNA transcript (SMART), in combination with a highly potent recombination machinery of Saccharomyces cerevisiae. Gal4-based yeast two-hybrid (Y2H) assays were performed against DENV-2 proteins (E, prM, M, and NS1). Mammalian two-hybrid (M2H) and double immunofluorescence assays (IFA) were conducted to validate the authenticity of the three selected interactions.Results: The cDNA library was of good quality based on its transformation efficiency, cell density, titer, and the percentage of insert size. A total of 36 midgut proteins interacting with DENV-2 proteins were identified, some involved in nucleic acid transcription, oxidoreductase activity, peptidase activity, and ion binding. Positive outcomes were obtained from the three selected interactions validated using M2H and double IFA assays. Conclusions: The identified proteins have different biological activities that may aid in the virus replication pathway. Therefore, the midgut cDNA library is a valuable tool for identifying DENV-2 interacting proteins. The positive outcomes of the three selected proteins validated supported the quality of the cDNA library and the robustness of the Y2H mechanisms.

show abstract

“…Involvement of host-factors in this particular region of the WNV RdRp could explain the host-dependent effects seen in this study. Thus far studies have shown the recruitment of host proteases in both the insect and vertebrate are required for viral polyprotein processing and a recent study demonstrated at least 18 mosquito proteins can interact with the WNV and DENV envelope, capsid, NS2a, and NS4b viral proteins in mosquito cell culture (Colpitts, Cox et al, 2011). However, little is known about host-factors that may interact with the viral replicase complex.…”

Section: Discussionmentioning

confidence: 99%

Point mutations in the West Nile virus (Flaviviridae; Flavivirus) RNA-dependent RNA polymerase alter viral fitness in a host-dependent manner in vitro and in vivo

et al. 2012

View full text Add to dashboard Cite

The West Nile virus (WNV) genome contains a single RNA-dependent RNA polymerase (RdRp) gene, which is responsible for replication of the viral genome and, as such, is an important target for antiviral therapy. Viral RdRps are known to lack proofreading capabilities and as a result viruses such as WNV exist as a mixture of viral genotypes within an infection, enabling the virus to readily emerge and adapt to new host environments. To test the consequences of subtle structural alterations remote from the RdRp active-site, the following single point mutations were engineered in the WNV NS5 RdRp coding region: T363N, A365N, and T537I; these mutations were selected in an effort to stabilize the secondary structural elements near the rNTP binding pocket of the RdRp. Mutant viruses were tested in vitro on Vero, C6/36, Culex tarsalis and DF-1 cell types and in vivo in one day old chickens and Culex pipiens mosquitoes. Plaque morphology was affected by each mutation and growth and RNA replication kinetics were altered as well. Our results demonstrate that subtle alteration of the RdRp protein away from the active site can have a significant overall biological effect on WNV fitness, and that this effect can be host-dependent. Key Words: RNA viruses, Arbovirus, Flavivirus, Mutagenesis, RNA relpication

show abstract

Use of a tandem affinity purification assay to detect interactions between West Nile and dengue viral proteins and proteins of the mosquito vector

Cited by 40 publications

References 53 publications

A Basic Cluster in the N Terminus of Yellow Fever Virus NS2A Contributes to Infectious Particle Production

A Basic Cluster in the N Terminus of Yellow Fever Virus NS2A Contributes to Infectious Particle Production

Protein-protein interactions between A. aegypti midgut and dengue virus 2: two-hybrid screens using the midgut cDNA library

Point mutations in the West Nile virus (Flaviviridae; Flavivirus) RNA-dependent RNA polymerase alter viral fitness in a host-dependent manner in vitro and in vivo

Contact Info

Product

Resources

About