Abstract
The capsid precursor P1 constitutes the N-terminal part of the enterovirus polyprotein. It is processed into VP0, VP3, and VP1 by the viral proteases, and VP0 is cleaved autocatalytically into VP4 and VP2. We observed that poliovirus VP0 is recognized by an antibody against a cellular autophagy protein, LC3A. The LC3A-like epitope overlapped the VP4/VP2 cleavage site. Individually expressed VP0-EGFP and P1 strongly colocalized with a marker of selective autophagy, p62/SQSTM1. To assess the role of capsid proteins in autophagy development we infected different cells with poliovirus or encapsidated polio replicon coding for only the replication proteins. We analyzed the processing of LC3B and p62/SQSTM1, markers of the initiation and completion of the autophagy pathway and investigated the association of the viral antigens with these autophagy proteins in infected cells. We observed cell-type-specific development of autophagy upon infection and found that only the virion signal strongly colocalized with p62/SQSTM1 early in infection. Collectively, our data suggest that activation of autophagy is not required for replication, and that capsid proteins contain determinants targeting them to p62/SQSTM1-dependent sequestration. Such a strategy may control the level of capsid proteins so that viral RNAs are not removed from the replication/translation pool prematurely.
Highlights
Enteroviruses are ubiquitous human pathogens whose infection may be associated with a variety of pathological conditions, including the development of type I diabetes, temporary or permanent paralysis, fatal encephalitis, and many others
The N-terminal part of the polyprotein contains the precursor of structural proteins P1, which is separated in cis from the rest of the polyprotein by the viral protease 2A and is further processed in trans by the proteases 3CD and/or 3C into VP0, VP3, and VP1 (Figure 1)
Plasmids pVP0-EGFP and pVP012A-EGFP coding for wt VP0 or VP0 with alanines substituting the amino-acids at the VP4/VP2 autocatalytic site, respectively, were made using pEGFP-N1 vector (Clontech, Mountain View, CA, USA) and the corresponding constructs coding for poliovirus type I Mahoney VP0 with a codon sequence optimized for translation in human cells synthesized by GeneArt (Invitrogen, Waltham, MA, USA)
Summary
Enteroviruses are ubiquitous human pathogens whose infection may be associated with a variety of pathological conditions, including the development of type I diabetes, temporary or permanent paralysis, fatal encephalitis, and many others. It was recently established that sequestration of the progeny enterovirus virions in the autophagosome-like vesicles that have escaped fusion with lysosomes promotes their maturation and a non-lytic release from infected cells [34,35,36,37] This scenario suggests that enterovirus capsid proteins may have evolved specific features enabling interaction with the elements of the cellular autophagy machinery to promote nascent virions recruitment into autophagosome-like structures that support the final steps of the virion maturation and dissemination. Our data suggest that activation of autophagy is not directly required to support the viral replication, and that capsid proteins contain determinants mediating their sequestration in a p62/SQSTM1-dependent manner Such sequestration would provide enteroviruses with a mechanism of regulating the amount of accessible capsid proteins, preventing premature encapsidation of the replicating RNA early in infection
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