Membrane Interactions and Uncoating of Aichi Virus, a Picornavirus That Lacks a VP4.

Kobuviruses are an unusual and poorly characterized genus within the picornavirus family and can cause gastrointestinal enteric disease in humans, livestock, and pets. The human kobuvirus Aichi virus (AiV) can cause severe gastroenteritis and deaths in children below the age of 5 years; however, this is a very rare occurrence. During the assembly of most picornaviruses (e.g., poliovirus, rhinovirus, and foot-and-mouth disease virus), the capsid precursor protein VP0 is cleaved into VP4 and VP2. However, kobuviruses retain an uncleaved VP0. From studies with other picornaviruses, it is known that VP4 performs the essential function of pore formation in membranes, which facilitates transfer of the viral genome across the endosomal membrane and into the cytoplasm for replication. Here, we employ genome exposure and membrane interaction assays to demonstrate that pH plays a critical role in AiV uncoating and membrane interactions. We demonstrate that incubation at low pH alters the exposure of hydrophobic residues within the capsid, enhances genome exposure, and enhances permeabilization of model membranes. Furthermore, using peptides we demonstrate that the N terminus of VP0 mediates membrane pore formation in model membranes, indicating that this plays an analogous function to VP4. IMPORTANCE To initiate infection, viruses must enter a host cell and deliver their genome into the appropriate location. The picornavirus family of small nonenveloped RNA viruses includes significant human and animal pathogens and is also a model to understand the process of cell entry. Most picornavirus capsids contain the internal protein VP4, generated from cleavage of a VP0 precursor. During entry, VP4 is released from the capsid. In enteroviruses this forms a membrane pore, which facilitates genome release into the cytoplasm. Due to high levels of sequence similarity, it is expected to play the same role for other picornaviruses. Some picornaviruses, such as Aichi virus, retain an intact VP0, and it is unknown how these viruses rearrange their capsids and induce membrane permeability in the absence of VP4. Here, we have used Aichi virus as a model VP0 virus to test for conservation of function between VP0 and VP4. This could enhance understanding of pore function and lead to development of novel therapeutic agents that block entry.

Aichi virus 3C protease modulates LC3- and SQSTM1/p62-involved antiviral response.

Rationale: Autophagy is an essential, homeostatic process by which cells break down their own components, it also contributes to restricting bacterial infection in host defense systems; yet, how autophagy regulates viral infection remains inconclusive. Aichi virus (AiV), belonging to the genus Kobuvirus in the Picornaviridae family, causes acute gastroenteritis in human. The role of autophagy-mediated anti-viral activity on AiV infection was investigated in this study. Methods: The effect of autophagy-associated molecules in retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) antiviral signal axis was analyzed in AiV infected cells by using biochemistry and pharmacologic approaches. In addition, the AiV viral protein regulating autophagy-associated RLR activity was also evaluated. Results: In AiV-infected cells, autophagic flux including the formation of autophagic vacuoles, as well as degradation of microtubule-associated protein light chain 3 (LC3) and sequestosome-1 (SQSTM1/p62) were observed. Ectopic overexpression of LC3 and p62, but not Atg proteins, contributed to RLR antiviral signal axis, shRNA knockdown of LC3 and p62 led to a downregulation of antiviral inflammation. Moreover, AiV infection inhibited double-stranded RNA (dsRNA)-activated RLR activity by the viral protein 3C protease but not H42D, C143S protease dead mutants. AiV 3C protease caused the degradation of LC3 and p62, and also RLR signal proteins. Conclusion: This study reveals a possible mechanism of autophagy-associated proteins regulating virus replication. Maintaining a cellular level of LC3 and p62 during the viral infection period might help restrict virus replication. Although, AiV 3C protease dampens the LC3 and p62-mediated host antiviral machinery for AiV replication. Results obtained provide a better understanding of the molecular pathogenesis of AiV for developing methods of prevention and treatment.

Kobuviral Non-structural 3A Proteins Act as Molecular Harnesses to Hijack the Host ACBD3 Protein.

Picornaviruses are small positive-sense single-stranded RNA viruses that include many important human pathogens. Within the host cell, they replicate at specific replication sites called replication organelles. To create this membrane platform, they hijack several host factors including the acyl-CoA-binding domain-containing protein-3 (ACBD3). Here, we present a structural characterization of the molecular complexes formed by the non-structural 3A proteins from two species of the Kobuvirus genus of the Picornaviridae family and the 3A-binding domain of the host ACBD3 protein. Specifically, we present a series of crystal structures as well as a molecular dynamics simulation of the 3A:ACBD3 complex at the membrane, which reveals that the viral 3A proteins act as molecular harnesses to enslave the ACBD3 protein leading to its stabilization at target membranes. Our data provide a structural rationale for understanding how these viral-host protein complexes assemble at the atomic level and identify new potential targets for antiviral therapies.

The Molecular Basis of Aichi Virus 3A Protein Activation of Phosphatidylinositol 4 Kinase IIIß, PI4KB, through ACBD3.

Phosphatidylinositol 4-kinase III beta (PI4KIIIß) is an essential enzyme in mediating membrane transport, and plays key roles in facilitating viral infection. Many pathogenic positive-sense single-stranded RNA viruses activate PI4KIIIß to generate phosphatidylinositol 4-phosphate (PI4P)-enriched organelles for viral replication. The molecular basis for PI4KIIIß activation during viral infection has remained largely unclear. We describe the biochemical reconstitution and characterization of the complex of PI4KIIIß with the Golgi protein Acyl-coenzyme A binding domain containing protein 3 (ACBD3) and Aichi virus 3A protein on membranes. We find that 3A directly activates PI4KIIIß, and this activation is sensitized by ACBD3. The interfaces between PI4KIIIß-ACBD3 and ACBD3-3A were mapped with hydrogen-deuterium exchange mass spectrometry (HDX-MS). Determination of the crystal structure of the ACBD3 GOLD domain revealed a unique N terminus that mediates the interaction with 3A. Rationally designed complex-disrupting mutations in both ACBD3 and PI4KIIIß completely abrogated the sensitization of 3A activation by ACBD3.

The 3A protein from multiple picornaviruses utilizes the golgi adaptor protein ACBD3 to recruit PI4KIIIß.

The activity of phosphatidylinositol 4-kinase class III beta (PI4KIIIß) has been shown to be required for the replication of multiple picornaviruses; however, it is unclear whether a physical association between PI4KIIIß and the viral replication machinery exists and, if it does, whether association is necessary. We examined the ability of the 3A protein from 18 different picornaviruses to form a complex with PI4KIIIß by affinity purification of Strep-Tagged transiently transfected constructs followed by mass spectrometry and Western blotting for putative interacting targets. We found that the 3A proteins of Aichi virus, bovine kobuvirus, poliovirus, coxsackievirus B3, and human rhinovirus 14 all copurify with PI4KIIIß. Furthermore, we found that multiple picornavirus 3A proteins copurify with the Golgi adaptor protein acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GPC60), including those from Aichi virus, bovine kobuvirus, human rhinovirus 14, poliovirus, and coxsackievirus B2, B3, and B5. Affinity purification of ACBD3 confirmed interaction with multiple picornaviral 3A proteins and revealed the ability to bind PI4KIIIß in the absence of 3A. Mass-spectrometric analysis of transiently expressed Aichi virus, bovine kobuvirus, and human klassevirus 3A proteins demonstrated that the N-terminal glycines of these 3A proteins are myristoylated. Alanine-scanning mutagenesis along the entire length of Aichi virus 3A followed by transient expression and affinity purification revealed that copurification of PI4KIIIß could be eliminated by mutation of specific residues, with little or no effect on recruitment of ACBD3. One mutation at the N terminus, I5A, significantly reduced copurification of both ACBD3 and PI4KIIIß. The dependence of Aichi virus replication on the activity of PI4KIIIß was confirmed by both chemical and genetic inhibition. Knockdown of ACBD3 by small interfering RNA (siRNA) also prevented replication of both Aichi virus and poliovirus. Point mutations in 3A that eliminate PI4KIIIß association sensitized Aichi virus to PIK93, suggesting that disruption of the 3A/ACBD3/PI4KIIIß complex may represent a novel target for therapeutic intervention that would be complementary to the inhibition of the kinase activity itself.