Manipulating cell cycle is one of the common strategies used by viruses to generate favorable cellular environment to facilitate viral replication. Coxsackievirus B (CVB) is one of the major viral pathogens of human myocarditis and cardiomyopathy. Because of its small genome, CVB depends on cellular machineries for productive replication. However, how the structural and non-structural components of CVB would manipulate cell cycle is not clearly understood. In this study, we demonstrated that the capsid protein VP1 of CVB type 3 (CVB3) induced cell cycle arrest at G1 phase. G1 arrest was the result of the decrease level of cyclin E and the accumulation of p27^Kip1. Study on the gene expression profile of the cells expressing VP1 showed that the expression of both heat shock protein 70-1 (Hsp70-1) and Hsp70-2 was significantly up-regulated. Knockdown of Hsp70 resulted in the increased level of cyclin E and the reduction of p27^Kip1. We further demonstrated that the phosphorylation of the heat shock factor 1, which directly promotes the expression of Hsp70, was also increased in the cell expressing VP1. Moreover, we show that CVB3 infection also induced G1 arrest, likely due to dysregulating Hsp70, cyclin E, and p27, while knockdown of Hsp70 dramatically inhibited viral replication. Cell cycle arrest at G1 phase facilitated CVB3 infection, since viral replication in the cells synchronized at G1 phase dramatically increased. Taken together, this study demonstrates that the VP1 of CVB3 induces cell cycle arrest at G1 phase through up-regulating Hsp70. Our findings suggest that the capsid protein VP1 of CVB is capable of manipulating cellular activities during viral infection.

Myocarditis is an inflammatory disease of the cardiac muscle and one of the primary causes of dilated cardiomyopathy. Group B coxsackievirus (CVB) is one of the leading causative pathogens of viral myocarditis, which primarily affects children and young adults. Due to the lack of vaccines, the development of antiviral medicines is crucial to controlling CVB infection and the progression of myocarditis. In this study, we investigated the antiviral effect of baicalein, a flavonoid extracted from Scutellaria baicaleinsis. Our results demonstrated that baicalein treatment significantly reduced cytopathic effect and increased cell viability in CVB3-infected cells. In addition, significant reductions in viral protein 3D, viral RNA, and viral particles were observed in CVB3-infected cells treated with baicalein. We found that baicalein exerted its inhibitory effect in the early stages of CVB3 infection. Baicalein also suppressed viral replication in the myocardium and effectively alleviated myocarditis induced by CVB3 infection. Our study revealed that baicalein exerts its antiviral effect by inhibiting the activity of caspase-1 and viral protease 2A. Taken together, our findings demonstrate that baicalein has antiviral activity against CVB3 infection and may serve as a potential therapeutic option for the myocarditis caused by enterovirus infection.

Coxsackievirus B (CVB) is the major causative pathogen for severe diseases such as viral myocarditis, meningitis, and pancreatitis. There is no effective antiviral therapy currently available for CVB infection primarily due to that the pathogenesis of CVB has not been completely understood. Viruses are obligate intracellular pathogens which subvert cellular processes to ensure viral replication. Dysregulation of ubiquitination has been implicated in CVB infection. However, how ubiquitination is involved in CVB infection remains unclear. Here we found that the 3D protein of CVB3, the RNA-dependent RNA polymerase, was modified at K220 by K48-linked polyubiquitination which promoted its degradation through proteasome. Proteomic analysis showed that the E3 ligase TRIM56 was upregulated in CVB3-infected cells, while the majority of TRIMs remained unchanged. Pull-down and immunoprecipitation analyses showed that TRIM56 interacted with CVB3 3D. Immunofluorescence observation showed that viral 3D protein was colocalized with TRIM56. TRIM56 overexpression resulted in enhanced ubiquitination of CVB3 3D and decreased virus yield. Moreover, TRIM56 was cleaved by viral 3C protease in CVB3-infected cells. Taken together, this study demonstrated that TRIM56 mediates the ubiquitination and proteasomal degradation of the CVB3 3D protein. These findings demonstrate that TRIM56 is an intrinsic cellular restriction factor against CVB infection, and enhancing viral protein degradation could be a potential strategy to control CVB infection.