Molecular Mechanism Of Inhibition Of Stress Granule Formation By Human Parainfluenza Virus Type 3 Infection

Human parainfluenza virus type 3(HPIV3),a member of paramyxoviridae family,belongs to the nonsegmented negative-strand(NNS)RNA virus family,which contains the well-known vesicular stomatitis virus,respiratory syncytial virus,ebola virus,rabies virus and measles virus.HPIV3 is the major cause of acute respiratory tract diseases such as pneumonia and bronchitis in infants and children.However,there is no effective therapy or any useful vaccine available so far,so it is meaningful to figure out the molecular mechanism and the pathogenic mechanism of HPIV3.Virus infection usually activates host cellular stress response,such as innate immune signal pathway and formation of cellular stress granules(SGs).Here,we found that HPIV3 infection activates PKR auto-phosphorylation and subsequently induces eIF2a phosphorylation.Phosphorylated eIF2a leads mRNA to stall at the initial stage of translation,which induces the formation of SGs.HPIV3 infection leads to the production of various viral proteins,but also a large amount of viral RNAs.Here,we found HPIV3 viral RNAs instead of its viral proteins is the major cause inducing cellular SGs formation.Upon detecting HPIV3 viral RNAs,PKR phosphorylates eIF2a,leading to the inhibition of cellular and viral translation and inducing SGs formation.Knockdown of the expression of G3BP disturbs the formation of SGs and significantly enhances the viral proteins expression and replication,which indicates the antiviral function of SGs.In general,most virus have evolved mechanisms to escape the host antiviral stress response to facilitate themselves replication.Many NNS RNA virus,including HPIV3,develop cytoplasmic inclusion bodies(IBs)as their viral replication factory during infection.IBs are mainly regarded as the viral RNA synthesis center,but may also manipulate host antiviral response.Here,we found a new function that HPIV3 IBs participate in the regulation of SGs formation.Over expression of HPIV3 viral protein N and P supports its IBs formation in cytoplasm.Although the IBs have little affection on the SGs formation induced by Na2ASO2 stimulation,pIC or viral RNA transfection,but they can significantly inhibit the SGs formation induced by HPIV3 infection.We concluded that HPIV3 IBs specially inhibit its viral infection induced SGs formation.Our previous work has proved the 478 Leu is important for N to form IBs with P,co-expression of a N mutant,NL478A,with P could not form IBs,and no longer disturb the SGs formation induced by HPIV3 infection.It indicates the IBs structure is critical for its antagonism effect on SGs formation.Furthermore,we figure out another functional defective truncated mutant,NΔN10,which lacks the 10 amino acids at the N terminal of N protein.NΔAN10 could formation IBs with P as well as wt N,but it has little activity in the minigenome report system.In our RNA Fish experiment,we found that HPIV3 viral RNA obviously co-localized within its wt IBs instead of the functional defective IBs formed by NΔN10 and P.What’s more,the functional defective IBs didn’t inhibit HPIV3 infection induced SGs formation any more.It indicates that the viral RNA-holding capacity of HPIV3 IBs affects its antagonism effect on its viral infection induced SGs formation.In conclusion,we illuminated the mechanism how HPIV3 infection induces host cellular SGs formation.Furthermore,we found for the first time that HPIV3 IBs participate in inhibition of SGs formation and manipulate host anti-viral stress response.