Progress 01/01/17 to 06/30/19
Outputs
Target Audience:Veterinarians and research scientists are informed of the results from this project through scientific meeting presentations and through the publication of data and results in peer-reviewed journal articles. Changes/Problems:Since we could not identify a role for the PRRSV NSP2 putative PDL motif in modulating transcriptional/translational processes within host cells, or identify NSP1a interacting proteins, we decided to study the mechanistic roles of stress granules (SGs) during PRRSV infection. SGs have been shown to play significant roles in regulating immune responses, at both the transcriptional and translational levels to viral infection. As such, we were among the first to establish PRRSV induces SG formation at late times post infection, and that these complexes are associated with viral replication complexes (VRCs). Furthermore, we also determined that SGs are dispensible for viral replication, as the small interferring RNA (siRNA)-mediated knockdown of critical SG components (G3BP1 and G3BP2) did not affect the ability of the virus to replicate. Additional research also revealed that the PRRSV-induced SGs form in a PERK-dependent manner, suggesting that the virus was also able to induce additional cellular stress responses. This finding led us to investigate the role of the unfolded protein response (UPR), a key response to adverse conditions, such as overloaded ER resulting from viral infection. This line of research led us to establish an antiviral role for the UPR during PRRSV infection, as chemical induction of the UPR significantly suppressed PRRSV replication. Collectively, these results suggest that SGs may be involved in viral replication, but are not crucial to the virus life cycle. However, our studies clearly show that the UPR negatively regulates PRRSV infection. Ultimately, we were able to achieve our goal of further elucidating the molecular mechanisms of PRRSV replication. The studies supported by this fellowship significantly enhance our understanding of PRRSV replication and pathogenesis. What opportunities for training and professional development has the project provided?Nicholas Catanzaro was afforded the opportunity to complete Virginia Tech's Graduate School, "Preparing the Future Professoriate" teaching certificate in which he extensively learned about topics pertaining to higher education and contemporary pedagogy. This opportunity significantly enhanced his professional development such that one day he may achieve his goal of becoming a tenure track faculty member at a RO1 research institute. The fellowship has also led him to present his research findings at a major PRRSV conference, as well as publish his results in the journal, Virus Research. How have the results been disseminated to communities of interest?We have presented the results of this project at scientific meetings. A major manuscript reporting the results from this project have now been published in the journal, Virus Research. Another manuscript has also been submitted to the same journal and is currently under peer review. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The orginial specific objectives were (1) Define the molecular basis for NSP2-mediated immune suppression at the transcriptional level. (2) Identify NSP1a interacting proteins and the underlying mechanisms involved in suppression of cap-dependent translation. The data generated from preliminary experiments does not support our initial hypothesis forumulated in Objective 1 that NSP2 contains a PDL-motif to modulate NFkB signaling during PRRSV infection. Therefore, we pursued another line of investigation examining how PRRSV modulates the host immune response via manipulation of the host's RNA stress granule pathway. Stress granules are dynamic regulators of host transcription and translation and have been shown to be important for the regulation of host antiviral defense mechanisms. Through our work, we have clearly established induction of stress granules during PRRSV infection and their underlying molecular pathways. Although we have modified the original proposed approaches to acheive the ultimate goal, the ultimate goal of the project remained the same. Thus, we have successfully accomplished the ultimate goal of the project which was to determine additional molecular mechanisms of PRRSV replication and pathogenesis. Specifically, for the original objectives I & II (100% completed) we clearly established the induction of stress granules during PRRSV infection. As such, we delineated the molecular mechanisms regulating the SG response to PRRSV infection. Using confocal microscopy, we first demonstrated that infection with PRRSV strain VR2385 induces an accumulation of the SG markers G3BP1, G3BP2, TIAR, eIF3b, and USP10 as well as mRNAs into punctate structures in the cytoplasm of infected host cells. Subsequently, we demonstrated that the PRRSV-induced SGs were in close proximity to viral replication complexes (VRCs) and processing bodies (P-bodies), and that SG formation was coordinated with inhibition of host cellular translation. Treatment of infected cells with cycloheximide disrupted the PRRSV-induced SGs. Furthermore, impairment of SG assembly by the shRNA-mediated knockdown of G3BP1, G3BP2 and USP10 did not affect viral replication. Overall impact:Collectively, these results demonstrate that PRRSV infection induces formation of SGs associated with VRCs, which is coordinated with the suppression of host cell protein synthesis. This is the first study to extensively characterize the formation and underlying mechanism ofbona fideSGs during PRRSV infection. Our findings have important implications in understanding the mechanism of PRRSV-host interactions.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Catanzaro N, Meng XJ. Porcine reproductive and respiratory syndrome virus (PRRSV)-induced stress granules are associated with viral replication complexes and suppression of host translation. Virus Research. 2019 Feb 28; 265:47-56.
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Progress 01/01/17 to 12/31/17
Outputs
Target Audience:The information generated by the work described in this proposal is expected to advance the knowledge of other researchers in the fields of molecular virology and antiviral vaccine development, especially swine veterinarians and researchers. Changes/Problems:The data generated thus far does not support our initial hypothesis formulated in Objective 1 that NSP2 contains a PDL-motif to modulate NFkB signaling during PRRSV infection. Thus, we are pursuing another line of investigation in looking at how PRRSV modulates the host immune response via manipulation of the host's RNA stress granule pathway. Stress granules are dynamic regulators of host transcription and translation and have been shown to be important for the regulation of host antiviral defense mechanisms. Thus far, we have demonstrated that PRRSV induces a unique class of stress granules that are compositionally and functionally distinct from canonical stress granules. We will continue this line of investigation. What opportunities for training and professional development has the project provided?I have successfully completed the Future Professoriate Teaching Certificate offered through the Graduate School at Virginia Tech. Completion of the certificate involved taking 3, 3 credit courses including, Preparing the Future Professoriate, Contemporary Pedagogy and Citizen Scholar. Completion of the certificate and these courses guided my professional development and has helped me prepare for my goal of obtaining a job as a tenure track faculty member at a reasearch institute. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?The data generated thus far does not support our initial hypothesis that NSP2 contains a putative PDL-motif involved in modulating the transcription of antiviral cytokines through the NFkB signaling pathway. In light of these results, we have started to pursue other lines of investigation looking into other mechanisms regulating the transciption and translation of antiviral host factors during PRRSV infection. Specifically, we are examining the antiviral properties of RNA stress granules during PRRSV infection. Stress granules are dynamic regulators of host transcription and translation and have been shown to be important for the regulation of host antiviral defense mechanisms. Using various molecular biology techniques, such as immunofluorescence assays, western blotting and RNA interference assays, we will continue this investigation during the next reporting period to accomplish this goal of further understanding modulation of the host immune response during PRRSV infection.
Impacts
What was accomplished under these goals?
Objective 1: Define the molecular basis for NSP2- mediated immune suppression at the transcriptional level. Reverse genetics approaches have been utilized to investigate the potential role of the NSP2-mediated immune suppression observed during PRRSV infection. Point mutations were generated in the putative PDL-motif speculated to be involved in the suppression of the host innate immune response, and the ability of the protein to suppress NF-KB signaling pathways was investigated using transient transfection and luciferase reporter assays in cell culture. No significant changes were observed between the wild-type and mutant proteins. Thus, we conclude the PRRSV NSP2 PDL-motif is not involved in mediating immune suppression. Objective 2: Identify NSP1a interacting proteins and the underlying mechanisms involved in suppression of cap-dependent translation. We are still investigating the role of NSP1a in suppression of cap-dependent translation and have nothing to report on this objective.
Publications
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