Autophagy Promotes Replication of Influenza A VirusIn Vitro

ABSTRACTInfluenza A virus (IAV) infection could induce autophagosome accumulation. However, the impact of the autophagy machinery on IAV infection remains controversial. Here, we showed that induction of cellular autophagy by starvation or rapamycin treatment increases progeny virus production, while disruption of autophagy using a small interfering RNA (siRNA) and pharmacological inhibitor reduces progeny virus production. Further studies revealed that alteration of autophagy significantly affects the early stages of the virus life cycle or viral RNA synthesis. Importantly, we demonstrated that overexpression of both the IAV M2 and NP proteins alone leads to the lipidation of LC3 to LC3-II and a redistribution of LC3 from the cytosol to punctate vesicles indicative of authentic autophagosomes. Intriguingly, both M2 and NP colocalize and interact with LC3 puncta during M2 or NP transfection alone and IAV infection, leading to an increase in viral ribonucleoprotein (vRNP) export and infectious viral particle formation, which indicates that the IAV-host autophagy interaction plays a critical role in regulating IAV replication. We showed that NP and M2 induce the AKT-mTOR-dependent autophagy pathway and an increase in HSP90AA1 expression. Finally, our studies provided evidence that IAV replication needs an autophagy pathway to enhance viral RNA synthesis via the interaction of PB2 and HSP90AA1 by modulating HSP90AA1 expression and the AKT-mTOR signaling pathway in host cells. Collectively, our studies uncover a new mechanism that NP- and M2-mediated autophagy functions in different stages of virus replication in the pathogenicity of influenza A virus.IMPORTANCEAutophagy impacts the replication cycle of many viruses. However, the role of the autophagy machinery in IAV replication remains unclear. Therefore, we explored the detailed mechanisms utilized by IAV to promote its replication. We demonstrated that IAV NP- and M2-mediated autophagy promotes IAV replication by regulating the AKT-mTOR signaling pathway and HSP90AA1 expression. The interaction of PB2 and HSP90AA1 results in the increase of viral RNA synthesis first; subsequently the binding of NP to LC3 favors vRNP export, and later the interaction of M2 and LC3 leads to an increase in the production of infectious viral particles, thus accelerating viral progeny production. These findings improve our understanding of IAV pathogenicity in host cells.

The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

ABSTRACT The capsid mRNA transcripts of human bocavirus 1 (HBoV1) can be generated by alternative splicing from the mRNA precursor transcribed from the P5 promoter. However, the alternative translation regulation mechanism of capsid mRNA transcripts is largely unknown. Here we report that the polycistronic capsid mRNA transcripts encode VP1, VP2, and VP3 in vitro and in vivo. The 5′ untranslated regions (UTRs) of capsid mRNA transcripts, which consist of exons, affected not only the abundance of mRNA but also the translation pattern of capsid proteins. Further study showed that exons 2 and 3 were critical for the abundance of mRNA, while exon 4 regulated capsid translation. Alternative translation of capsid mRNA involved a leaky scan mechanism. Mutating the upstream ATGs (uATGs) located in exon 4 resulted in more mRNA transcripts polyadenylated at the proximal polyadenylation [(pA)p] site, leading to increased capsid mRNA transcripts. Moreover, uATG mutations induced more VP1 expression, while VP3 expression was decreased, which resulted in less progeny virus production. Our data show that the 5′ UTR of HBoV1 plays a critical role in the modulation of mRNA abundance, alternative RNA processing, alternative translation, and progeny virus production. IMPORTANCE Alternative translation of HBoV1 capsid mRNAs is vital for the viral life cycle, as capsid proteins perform essential functions in genome packaging, assembly, and antigenicity. The 5′ untranslated regions (UTRs) of capsid mRNAs are generated by alternative splicing, and they contain different exons. Our study shows that the 5′ UTR not only modulates mRNA abundance but also regulates capsid expression. Two upstream ATGs (uATGs) that were upstream of the capsid translation initiation site in the 5′ UTR were found to affect viral capsid mRNA polyadenylation, alternative translation, and progeny virus production. The results reveal that uATGs play an important role in the viral life cycle and represent a new layer to regulate HBoV1 RNA processing, which could be a target for gene therapy.

Murine Cytomegalovirus Protein pM91 Interacts with pM79 and Is Critical for Viral Late Gene Expression

ABSTRACTViral gene expression is tightly regulated during cytomegalovirus (CMV) lytic replication, but the detailed mechanism of late gene transcription remains to be fully understood. Previous studies reported that six viral proteins (named viral transactivation factors [vTFs]) supporting late gene expression were conserved in beta- and gammaherpesviruses but not in alphaherpesviruses. Here, we performed coimmunoprecipitation experiments to elucidate the organization of these six proteins in murine CMV. Our results showed that these proteins formed a complex by both direct and indirect interactions. Specifically, pM91 strongly bound to pM79 even in the absence of other vTFs. Similar to pM79, pM91 exhibited early-late expression kinetics and localized within nuclear viral replication compartments during infection. Functional analysis was also performed using the pM91-deficient virus. Real-time PCR results revealed that abrogation of M91 expression markedly reduced viral late gene expression and progeny virus production without affecting viral DNA synthesis. Using mutagenesis, we found that residues E61, D62, D89, and D96 in pM91 were required for the pM91-pM79 interaction. Disruption of the interaction via E61A/D62A or D89A/D96A double mutation in the context of virus infection inhibited progeny virus production. Our data indicate that pM91 is a component of the viral late gene transcription factor complex and that the pM91-pM79 interaction is essential for viral late gene expression.IMPORTANCECytomegalovirus (CMV) infection is the leading cause of birth defects and causes morbidity and mortality in immunocompromised patients. The regulation of viral late gene transcription is not well elucidated, and understanding of this process benefits the development of novel therapeutics against CMV infection. This study (i) identified that six viral transactivation factors encoded by murine CMV form a complex, (ii) demonstrated that pM91 interacts with pM79 and that pM91 and pM79 colocalize in the nuclear viral replication compartments, (iii) confirmed that pM91 is critical for viral late gene expression but dispensable for viral DNA replication, and (iv) revealed that the pM91-pM79 interaction is required for progeny virus production. These findings give an explanation of how CMV regulates late gene expression and have important implications for the design of antiviral strategies.

Expression of Ac-PK2 protein from AcMNPV improved the progeny virus production via regulation of energy metabolism and protein synthesis

Baculovirus encoded PK2 protein can increase viral fitness through inhibition of the eIF2α family kinases activity.