Manipulation of autophagy by (+) RNA viruses.

Autophagy is an evolutionarily conserved process central to host metabolism. Among its major functions are conservation of energy during starvation, recycling organelles, and turnover of long-lived proteins. Besides, autophagy plays a critical role in removing intracellular pathogens and very likely represents a primordial intrinsic cellular defence mechanism. More recent findings indicate that it has not only retained its ability to degrade intracellular pathogens, but also functions to augment and fine tune antiviral immune responses. Interestingly, viruses have also co-evolved strategies to manipulate this pathway and use it to their advantage. Particularly intriguing is infection-dependent activation of autophagy with positive stranded (+)RNA virus infections, which benefit from the pathway without succumbing to lysosomal degradation. In this review we summarise recent data on viral manipulation of autophagy, with a particular emphasis on +RNA viruses and highlight key unanswered questions in the field that we believe merit further attention.

Viral subversion of selective autophagy is critical for biogenesis of virus replication organelles.

Infection by many (+)RNA viruses is accompanied by ER-expansion and membrane remodelling to form viral replication organelles, followed by assembly and secretion of viral progenies. We previously identified that virus-triggered lipophagy was critical for flaviviral assembly, and is driven by the lipid droplet associated protein Ancient ubiquitin protein 1 (Aup1). A ubiquitin conjugating protein Ube2g2 that functions as a co-factor for Aup1 was identified as a host dependency factor in our study. Here we characterized its function: Ube2g2-deficient cells displayed a dramatic reduction in virus production, which could be rescued by reconstituting the wild-type but not the catalytically deficient (C89K) mutant of Ube2g2, suggesting that its enzymatic activity is necessary. Ube2g2 deficiency did not affect entry of virus particles but resulted in a profound loss in formation of replication organelles, and production of infectious progenies. This phenomenon resulted from its dual activity in (i) triggering lipophagy in conjunction with Aup1, and (ii) degradation of ER chaperones such as Herpud1, SEL1L, Hrd1, along with Sec62 to restrict ER-phagy upon Xbp1-IRE1 triggered ER expansion. Our results therefore underscore an exquisite fine-tuning of selective autophagy by flaviviruses that drive host membrane reorganization during infection to enable biogenesis of viral replication organelles.

Usp25-Erlin1/2 activity limits cholesterol flux to restrict virus infection.

Reprogramming lipid metabolic pathways is a critical feature of activating immune responses to infection. However, how these reconfigurations occur is poorly understood. Our previous screen to identify cellular deubiquitylases (DUBs) activated during influenza virus infection revealed Usp25 as a prominent hit. Here, we show that Usp25-deleted human lung epithelial A549 cells display a >10-fold increase in pathogenic influenza virus production, which was rescued upon reconstitution with the wild type but not the catalytically deficient (C178S) variant. Proteomic analysis of Usp25 interactors revealed a strong association with Erlin1/2, which we confirmed as its substrate. Newly synthesized Erlin1/2 were degraded in Usp25-/- or Usp25C178S cells, activating Srebp2, with increased cholesterol flux and attenuated TLR3-dependent responses. Our study therefore defines the function of a deubiquitylase that serves to restrict a range of viruses by reprogramming lipid biosynthetic flux to install appropriate inflammatory responses.

OTUB1 Is a Key Regulator of RIG-I-Dependent Immune Signaling and Is Targeted for Proteasomal Degradation by Influenza A NS1.

Deubiquitylases (DUBs) regulate critical signaling pathways at the intersection of host immunity and viral pathogenesis. Although RIG-I activation is heavily dependent on ubiquitylation, systematic analyses of DUBs that regulate this pathway have not been performed. Using a ubiquitin C-terminal electrophile, we profile DUBs that function during influenza A virus (IAV) infection and isolate OTUB1 as a key regulator of RIG-I-dependent antiviral responses. Upon infection, OTUB1 relocalizes from the nucleus to mitochondrial membranes together with RIG-I, viral PB2, and NS1. Its expression depends on competing effects of interferon stimulation and IAV-triggered degradation. OTUB1 activates RIG-I via a dual mechanism of K48 polyubiquitin hydrolysis and formation of an E2-repressive complex with UBCH5c. We reconstitute this mechanism in a cell-free system comprising [35S]IRF3, purified RIG-I, mitochondrial membranes, and cytosol expressing OTUB1 variants. A range of IAV NS1 proteins trigger proteasomal degradation of OTUB1, antagonizing the RIG-I signaling cascade and antiviral responses.

MDCK-B4GalNT2 cells disclose a α2,3-sialic acid requirement for the 2009 pandemic H1N1 A/California/04/2009 and NA aid entry of A/WSN/33.

Switching of receptor binding preference has been widely considered as one of the necessary mutations for avian influenza viruses, enabling efficient transmissions between human hosts. By stably overexpressing B4GalNT2 gene in MDCK cells, surface α2,3-siallylactose receptors were modified without affecting α2,6-receptor expression. The cell line MDCK-B4GalNT2 was used as a tool to screen for α2,3-receptor requirements in a panel of influenza viruses with previously characterized glycan array data. Infection of viruses with α2,3-receptor binding capability was inhibited in MDCK-B4GalNT2 cells, with the exception of A/WSN/33 (WSN). Infection with the 2009 pandemic H1N1 strains, A/California/04/2009 (Cal04) and A/Hong Kong/415742/2009 (HK09), despite showing α2,6-receptor binding, was also found to be inhibited. Further investigation showed that viral inhibition was due to a reduction in viral entry rate and viral attachment. Recombinant WSN virus with the neuraminidase (NA) gene swapped to A/Puerto Rico/8/1934 (PR8) and Cal04 resulted in a significant viral inhibition in MDCK-B4GalNT2 cells. With oseltamivir, the NA active site was found to be important for the replication results of WSN, but not Cal04.

Neuraminidase activity and specificity of influenza A virus are influenced by haemagglutinin-receptor binding.

Influenza virus haemagglutinin (HA) and neuraminidase (NA) are involved in the recognition and modulation of sialic acids on the cell surface as the virus receptor. Although the balance between two proteins functions has been found to be crucial for viral fitness, the interplay between the proteins has not been well established. Herein we present evidence for interplay between influenza HA and NA, which may affect the balance between two glycoprotein functions. NA enzymatic activities against sialoglycans were promoted by the presence of HA, which is in accordance with the level of co-existing HA. Such activity enhancement was lost when the HA-receptor binding properties were abolished by low-pH treatment or by mutations at the HA receptor binding domain. Sialidase activities of NA-containing virus-like particles and native influenza viruses were detected using different NA-assays and sialic acid substrates. Most pronounced HA-mediated NA enhancement was found when intact virions were confronted with multivalent surface-anchored substrates, which mimics the physiological conditions on cell membranes. Using recombinant viruses with altered HA bindings preference between α2,3- and α2,6-linked sialic acids, we also found that NA function against different substrates is correlated with the HA-receptor specificity. The effect of HA-receptor specificities on NA functions, together with the HA-mediated NA enhancement, may play a role in virus evasion of the mucus barrier, as well as in cross-species adaptation. Our data also indicate the importance of using multivalent substrates in future studies of NA functions.