In a recent study published in PLOS Pathogens, the researchers illustrated a novel strategy that viruses likely employ to promote infection.
Study: Targeting Nup358/RanBP2 by a viral protein disrupts stress granule formation. Image credit: Rost9/Shutterstock
Background
Viruses likely inhibit a host cell stress response by blocking the accumulation of stress granules (SG). SGs are ribonucleic acid (mRNA) “sinks” and protein condensates in host cells that take advantage of viral proteins and RNA to likely delay virus infection. Although many viruses employ this strategy, studies have not been able to explain how it benefits different viruses.
Previous studies have shown that arthropod-infecting dicistroviruses, cricket paralysis virus (CrPV) and Drosophila C virus (DCV) could evade the antiviral RNA interference (RNAi) response and SG inhibition in insects. to shut down its transcription and translation machinery. Arthropod-borne viruses, including dengue and chikungunya viruses, are a worldwide threat. Therefore, understanding fundamental virus-insect interactions, such as SG inhibition, could help design new antiviral strategies.
About the study
In one of their earlier studies, the researchers observed that CrPV-1A blocked SG in human cells. In the present study, they set out to determine whether there are common mechanisms for SG inhibition by CrPV-1A in different host species.
Furthermore, they systematically delineated the functions of CrPV-1A through specific single or combinatorial mutations within R146A, a CrPV-1A reporter RNA, to demonstrate that its ability to block SG formation was independent of its ability to bind Argonaute. -2 (Aug-2).
They expressed mutant CrPV-1A protein containing F114A or F114A/R146A mutations (double mutants). They monitored the formation of Ras-in-neurons-like protein (RIN) foci in Drosophila S2 cells against double-stranded Nup358 (dsRNA)-treated cells under arsenite or patheamine treatment using fluorescence in situ hybridization (FISH).
Study findings
The 166 amino acid (AA) long protein CrPV-1A modulates SG functions in multiple ways, for example, by modulating nuclear events, including messenger RNA (mRNA) export and transcription via polyadenylation or poly(A) tail enrichment ) in the nucleus. Since mRNAs are a scaffold for SG assembly, enrichment of the poly(A)+ tail in the nucleus of cells expressing CrPV-1A could block host mRNA translation and antiviral responses or deplete mRNA. cytoplasmic, leading to SG inhibition. This strategy is somewhat similar to how the influenza virus protein polymerase-acid protein-X (PA-X) inhibits SG formation in parallel with depletion of poly(A) RNA in the cytoplasm and nuclear accumulation of the protein. binding to poly(A) (PABP) ).
Regarding CrPV-1A domains that mediate specific cellular functions, the researchers noted that CrPV-1A bound Ago-2 using the argonaut domain for loss of silencing (TALOS). However, Ago-2 degradation was dependent on the recruitment of the Cul2-Rbx1-Elongin B and C ubiquitin ligase complex (EloBC). Interestingly, not the TALOS domain but mutations in the BC Box domain contributed to the enrichment of nuclear poly(A)+ mRNA, resulting in poorer SG inhibition by CrPV-1A. Clearly, recruitment of the ubiquitin ligase complex was mandated for CrPV-1A-mediated effects on SG inhibition.
Insects, such as Drosophila, employ a ‘Swiss army knife’ approach to blocking the antiviral response, including RNA metabolism and SG formation, all of which facilitate infection.
Recent studies have shown that Nup358, a unit of the cytoplasmic strand of the nuclear pore complex (NPC), localizes to SG and facilitates mRNA and protein transport. In this way, it plays a prominent role in viral infections. Nup358 levels were decreased in CrPV-1A-infected cells and its depletion resulted in SG inhibition.
The researchers also noted that CrPV infection also required proteasome activity. Furthermore, these effects were R146-dependent AA residues nested within the C-terminal tail of CrPV-1A. The study model also showed that this C-terminal tail of CrPV-1A directly or indirectly interacted with Nup358 to mediate proteasome-dependent degradation by the Cul2-Rbx1-EloBC complex.
Therefore, the R146A mutation, 20 AA stretches upstream of the stop-go cleavage, likely disrupted CrPV-1A/Nup358 interactions, the stop-go activity of the CrPV-2A peptide, ultimately altering subcellular localization. of CrPV-1A and the protein conformations that mediated these effects. .
Immunoblotting for Nup358 in S2 cells showed a protein band at >245 kDa, not detected in cells treated with Nup358 dsRNA, confirming RNAi-mediated depletion of Nup358. Nup358 depletion had many other effects. For example, pateamine A treatment of Nup358-depleted cells decreased the number of RIN foci per cell.
Similarly, arsenite treatment resulted in a ~40% decrease in RIN foci per cell compared to control dsRNA-treated cells. Depletion of NXF1, a critical RNA transport factor, resulted in loss of SG. Together, these results showed that blocking mRNA export through Nup358, or in general, could lead to SG inhibition.
conclusions
Overall, the current study provided mechanistic insights into viral protein-mediated SG inhibition, a viral strategy that has yet to be fully explored but promotes viral infections.
Sequencing studies have not revealed any conservation between CrPV-1A and other dicistrovirus 1A proteins; however, some may have similar functions. It would be interesting if future studies determine how these two viral proteins act similarly at the domain-specific level to establish a productive infection in the host. Furthermore, future studies should comprehensively investigate CrPV-1A/Nup358 interactions and whether Nup358 ubiquitination is essential for SG degradation.
Source: news.google.com