Abstract
Together, the three human rhinovirus (RV) species are the most frequent cause of the common cold. Because of their high similarity with other viral species of the genus Enterovirus, within the large family Picornaviridae, studies on RV infectious activities often offer a less pathogenic model for more aggressive enteroviruses, e.g. poliovirus or EV71. Picornaviruses enter via receptor mediated endocytosis and replicate in the cytosol. Most of them depend on functional F-actin, Rab proteins, and probably motor proteins. To assess the latter, we evaluated the role of myosin light chain kinase (MLCK) and two myosin V isoforms (Va and Vb) in RV-B14 infection. We report that ML-9, a very specific MLCK inhibitor, dramatically reduced RV-B14 entry. We also demonstrate that RV-B14 infection in cells expressing dominant-negative forms of myosin Va and Vb was impaired after virus entry. Using immunofluorescent localization and immunoprecipitation, we show that myosin Va co-localized with RV-B14 exclusively after viral entry (15 min post infection) and that myosin Vb was present in the clusters of newly synthesized RNA in infected cells. These clusters, observed at 180 min post infection, are reminiscent of replication sites. Taken together, these results identify myosin light chain kinase, myosin Va and myosin Vb as new players in RV-B14 infection that participate directly or indirectly in different stages of the viral cycle.
Highlights
The common cold is normally limited to the upper respiratory tract[1]
Internalization of RV-A2 and RV-B14 were reduced in cells expressing dominant-negative dynamin II (K44A)[24,25,26,27] probably due to disturbance of proper clathrin-coated pit maturation[28]
The current paper reports that RV-B14 infection requires myosin light chain kinase (MLCK) for cell entry while myosin Va and Vb are required for intracellular events prior to replication
Summary
The MLCK inhibitor, ML9, affected RV-B14 entry (Fig. 1) to a greater degree than that observed previously with wortmannin during an RV-A2 infection[36]. This approach allowed the incorporation of multiple BrU labels into each genome that appeared to provide high sensitivity in the identification of the RNA genome, from the internalized virus after release from the capsid and arrival in the cytoplasm This advancement was important to successfully observe the colocalization of myosin Va with the RV-B14 genome and the capsid proteins that occurred within 15 min PI (Fig. 3A–F). These groups of events could help to explain the differences observed in the patterns of de novo synthesized RNA (Fig. 5) between mock and infected cells, which we propose to represent RV replication sites in infected cells. More tests are needed to identify the adaptor proteins that, together with MLCK, myosin Va, and Vb, support viral entry, genome release and interaction with the RNA clusters observed here that resemble viral replication sites
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