Coxsackievirus A9 Infects Cells via Nonacidic Multivesicular Bodies

Abstract

Coxsackievirus A9 (CVA9) is a member of the human enterovirus B species in the Enterovirus genus of the family Picornaviridae. According to earlier studies, CVA9 binds to ␣V␤3 and ␣V␤6 integrins on the cell surface and utilizes ␤2-microglobulin, dynamin, and Arf6 for internalization. However, the structures utilized by the virus for internalization and uncoating are less well understood. We show here, based on electron microscopy, that CVA9 is found in multivesicular structures 2 h postinfection (p.i.). A neutral red labeling assay revealed that uncoating occurs mainly around 2 h p.i., while double-stranded RNA is found in the cytoplasm after 3 h p.i. The biogenesis of multivesicular bodies (MVBs) is crucial for promoting infection, as judged by the strong inhibitory effect of the wild-type form of Hrs and dominant negative form of VPS4 in CVA9 infection. CVA9 infection is dependent on phospholipase C at the start of infection, whereas Rac1 is especially important between 1 and 3 h p.i., when the virus is in endosomes. Several lines of evidence implicate that low pH does not play a role in CVA9 infection. The infection is not affected by Bafilomycin A1. In addition, CVA9 is not targeted to acidic late endosomes or lysosomes, and the MVBs accumulating CVA9 have a neutral pH. Thus, CVA9 is the second enterovirus demonstrated so far, after echovirus 1, that can trigger neutral MVBs, which are important for virus infection. IMPORTANCEWe demonstrate here that the enterovirus coxsackievirus A9 (CVA9) uses a nonclathrin and nonacidic pathway to infect cells. CVA9 does not accumulate in conventional late endosomes or lysosomes. We found that inhibitors of phospholipase C (PLC), Rac1, and the Na ؉ /H ؉ exchanger decreased CVA9 infection. The PLC inhibitor acts on early entry, the Rac1 inhibitor acts between 1 and 3 h, when the virus is in endosomes, and the Na ؉ /H ؉ exchange inhibitor acts during various steps during the virus life cycle. The infection depends on the formation of novel neutral multivesicular bodies (MVBs), which accumulate CVA9 during the first hours of entry. Thus, CVA9 is the second enterovirus demonstrated so far, after echovirus 1, that can trigger formation of neutral MVBs. The data show that these enteroviruses favor nonacidic conditions and complex MVBs to promote virus infection.

Hydrophobic pocket targeting probes for enteroviruses

“…39,40 For both native viruses and virus-fluorescent probe conjugates, the number of infected cells increased rapidly after 6, 12 and 24 h. However, as observed with the other infectivity methods, the virus-fluorescent probe conjugates remained for a longer time in the vesicles before the start of replication. and later, virus replication is observed as the resulting high cytoplasmic accumulation of newly made capsid proteins.…”

HCMV Induces Macropinocytosis for Host Cell Entry in Fibroblasts

“…The results indicated that HCMV belongs to those viruses that can infect cells through an endocytic mechanism but do not require low pH to trigger penetration from the endocytic vacuoles. Others include HSV‐1, respiratory syncytial virus, Echovirus 1 and Coxsackievirus A9 . HCMV is unusual because low pH is required in endothelial and epithelial cells whereas no such requirement is observed in dendritic cells and fibroblasts .…”

Viral exploitation of actin: force-generation and scaffolding functions in viral infection

“…Although, as reviewed by Moss (Moss B, 2012), the poxvirus entry fusion complex—composed of 11–12 glycosylated proteins—is abnormally complex, and other entry routes exist. Huttunem et al (2014) also implicate a Rac1-dependent entry mechanism, involving neutral multivesicular bodies, in Coxsackievirus A9 entry (Huttunen M, et al, 2014). Similarly, HSV-1 has been shown to utilize a RhoA, tyrosine kinase, and actin-dependent phagocytosis-like route for entry (Clement C, et al, 2006; Zheng K, et al, 2014 b ).…”