Abstract
We have previously shown that poliovirus (PV) infection induces stress granule (SG) formation early in infection and then inhibits the formation of SG and disperses processing bodies (PBs) by the mid-phase of infection. Loss of SG was linked to cleavage of G3BP1 by viral 3C proteinase (3Cpro), however dispersal of PBs was not strongly linked to cleavage of specific factors by viral proteinases, suggesting other viral proteins may play roles in inhibition of SG or PB formation. Here we have screened all viral proteins for roles in inducing or inhibiting the formation of RNA granules by creating fusions with mCherry and expressing them individually in cells. Expression of viral proteins separately revealed that the capsid region P1, 2Apro, 3A, 3Cpro, the protease precursor 3CD and 3D polymerase all affect RNA granules to varying extents, whereas 2BC does not. 2Apro, which cleaves eIF4GI, induced SGs as expected, and entered novel foci containing the SG nucleating protein G3BP1. Of the two forms of G3BP, only G3BP1 is cleaved by a virus proteinase, 3Cpro, whereas G3BP2 is not cleaved by 3Cpro or 2Apro. Surprisingly, 3CD, which contains proteinase activity, differentially repressed PBs but not SGs. Further, both 2Apro and 3Cpro expression dispersed PBs, however molecular targets were different since PB dispersal due to 2Apro and heat shock protein (Hsp)90 inhibition but not 3Cpro, could be rescued by application of oxidative stress to cells. The data indicate that PV repression of SGs and PBs is multifactorial, though protease function is dominant.
Highlights
Somatic cells form two major classes of cytoplasmic RNA granules known as stress granules (SGs) and processing bodies (PBs) that transiently store silenced messenger ribonucleoproteins
Exogenous Stress Rescues PBs Disrupted by PV 2A proteinase (2Apro) but not 3C proteinase (3Cpro) Since both 2Apro and 3Cpro expression reduced the levels of endogenous PBs and we have no known targets of 2Apro that associate with PBs, we explored if the proteases used a similar mechanism or targeted a common assembly pathway
Our results confirmed that PV 2Apro was a strong inducer of SG assembly, similar to a previous report for CVB3 2Apro [26] and that no other viral proteins induced SGs
Summary
Somatic cells form two major classes of cytoplasmic RNA granules known as stress granules (SGs) and processing bodies (PBs) that transiently store silenced messenger ribonucleoproteins (mRNPs). Unlike SGs, PBs contain mRNAs in association with 5I and 3I-mediated RNA decay factors, mRNA decapping, deadenylation, micro RNA (miRNA)-mediated mRNA silencing, and mRNA storage and are devoid of most translation initiation factors and ribosome subunits [1,4,5]. Both SGs and PBs are very dynamic foci, with protein and mRNA components rapidly exchanging with the surrounding cytosol. PBs are identified by containing RNA helicase Rck/p54 ( known as DDX6), the 5I-3I exoribonuclease Xrn, mRNA-decapping enzymes 1a and 2, (Dcp1a and Dcp2), and PABP1-dependent poly(A) nucleases 2 and 3, (Pan2/Pan3) [6]
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