Role of the myristoylation site in expressing exogenous functional proteins in coxsackieviral vector.

We generated a cardiotropic replication-competent chimeric coxsackievirus B3 (CVB3) to express alcohol dehydrogenase (ADH). Although exogenously expressed ADH was found by Western blot analysis, its enzyme function was repressed. To define the factor that inhibits the enzymatic function of ADH, we introduced a site-directed mutation at the second amino acid (MGAQEF···) of the CVB3 VP0 capsid protein, effectively changing glycine to alanine. This glycine is known to be a myristoylation site during viral capsid protein maturation in infected cells. In contrast to the unmodified virus, ADH expression and enzymatic function were readily detectable in the mutated rCVB3-ADH (G2A) virus. While expression of ADH required mutation of the CVB3 VP0 myristoylation site for proper function, another chimeric virus that expresses green fluorescent protein (rCVB3-GFP (G or A)) worked independently of the myristoylation site. Indeed, infected HeLa cells displayed GFP under a fluorescent microscope. These results indicate that the myristoylation site in the VP0 capsid protein inhibited the expression of enzymatically active ADH but not GFP. VP0 myristoylation is dispensable for chimeric CVB3 virus replication.

Foreign gene transfer to cardiomyocyte using a replication-defective recombinant coxsackievirus B3 without cytotoxicity.

BACKGROUND: Replication-competent coxsackievirus B3 (CVB3) has been used as a gene transfer vector for cultured cardiomyocytes and hearts in vivo. However, CVB3 induces cell lysis when it replicates in infected cells. In this study, we investigated whether a replication-defective rCVB3 vector could be generated and used as a noncytotoxic gene transfer vector for cardiomyocytes. METHODS: We generated a replication-defective luciferase-expressing CVB3 plasmid. This recombinant cDNA and pCMV-P1 plasmids were amplified and cotransfected into Hek293 cells using transfection reagents. Replication-defective rLuCVB3 virus was recovered from the cells and cell culture supernatants for 3 days after transfection. The generated rLuCVB3 viruses were concentrated on a 30% sucrose cushion and semiquantified using a luciferase assay. In addition, foreign gene delivery by the rLuCVB3 was tested in cultured cardiomyocytes and intact mouse hearts after rLuCVB3 infection. RESULTS: Luciferase was expressed in Hek293, HeLa cells and cardiomyocytes after rLuCVB3 infection. In addition, these cells did not show a significant cytopathic effect after 72 h. Luciferase protein expression or activity were detected for 3 days in the myocardium of rLuCVB3-infected mouse hearts without producing cytotoxicity or inflammation. CONCLUSION: As a proof-of-concept, these data indicate that a replication-defective rCVB3 vector can be generated and used as a novel gene transfer system to transfect exogenous genes into cardiomyocytes without generating cytotoxicity.

Change in the cells that express connective tissue growth factor in acute Coxsackievirus-induced myocardial fibrosis in mouse.

Cardiac fibrosis and inflammation are major pathologic conditions that result from viral myocarditis. Connective tissue growth factor (CTGF) stimulates fibroblast proliferation and induces production of extracellular matrix molecules. We studied the correlation between CTGF and cardiac fibrosis in an acute Coxsackievirus B3 (CVB3) myocarditis animal model. Eight-week-old BALB/c mice were infected intraperitoneally with 10(4) plaque forming units (PFU) of CVB3. Myocardial inflammation peaked on day 7 and decreased markedly by day 14 post-infection (pi); cardiac fibrosis was noted from day 7 and peaked on day 14. By contrast, CTGF was weakly expressed by the interstitial cells in uninfected control hearts and also in the hearts of day 3 pi. CTGF expression measured by real-time PCR was elevated on day 3 and peaked on day 7 pi. TGF-beta expression peaked at day 7 pi. The cell type of CTGF expression changed from interstitial cells to myocytes after virus infection. On day 7, CTGF was strongly expressed by myocytes and inflammatory cells surrounding calcified necrotic areas. In addition, cardiac myocytes expressed CTGF on day 14. Our results, based on an acute CVB3 model of myocarditis, provide evidence that CTGF may mediate the development of fibrosis after viral myocarditis, and that the cells expressed CTGF changes during the course of viral myocarditis.

Virus receptor trap neutralizes coxsackievirus in experimental murine viral myocarditis.

OBJECTIVE: The coxsackie and adenovirus receptor (CAR) and the decay-accelerating factor (DAF) are receptors for coxsackievirus B3 (CVB3), which is known as the major cause of human viral myocarditis. We investigated the potential for therapeutic use of soluble virus receptor fusion proteins. METHODS: We designed and generated a novel virus receptor trap (hCAR-hDAF:Fc) consisting of both CVB3 receptors and the Fc portion of human IgG1 and evaluated its antiviral effects in experimental CVB3 myocarditis. RESULTS: Among four soluble virus receptor fusion proteins (hCAR:Fc, hDAF:Fc, hCAR-hDAF:Fc and hDAF-hCAR:Fc), hCAR:Fc and hCAR-hDAF:Fc in the supernatant of transfected cells neutralized echovirus, adenovirus, and various serotypes of CVB in a dose-dependent manner. Both soluble viral receptor proteins bound to the VP0 and VP1 capsid proteins of CVB3. The in vivo efficacy of viral receptor proteins was evaluated by intramuscular injection of plasmid (hCAR:Fc or hCAR-hDAF:Fc) followed by electroporation in a murine model of CVB3 myocarditis. Serum levels of the virus receptor proteins increased relative to baseline values from day 3 and peaked on day 14 at 12.9-fold for hCAR:Fc and 7.1-fold for hCAR-hDAF:Fc. The 3-week survival rate was significantly higher in hCAR-hDAF:Fc-treated mice (61%) than in hCAR:Fc-treated mice (29%) and in controls (15%; p<0.05). Myocardial inflammation, fibrosis, and myocardial virus titers were all significantly reduced in the hCAR:Fc and hCAR-hDAF:Fc groups compared to the controls. CONCLUSION: Our soluble virus receptor trap, hCAR-hDAF:Fc, attenuated viral infection, myocardial inflammation, and fibrosis, resulting in higher survival rates in mice with coxsackieviral myocarditis. Furthermore, it consists exclusively of human components, and we demonstrated that this soluble virus receptor trap may be used as a potential candidate for a novel therapeutic agent for the treatment of acute viral myocarditis during the viremic phase.

Coxsackievirus B3 replication is related to activation of the late extracellular signal-regulated kinase (ERK) signal.

MAP kinase signaling has been implicated in coxsackievirus B3 (CVB3) pathogenesis and as necessary in the virus lifecycle. We studied the correlation with extracellular signal-regulated kinase 1/2 (ERK1/2) signaling and virus replication in the presence of coxsackievirus and adenovirus receptor (CAR). In CHO cells that do not expressed CAR, specific ERK1/2 phosphorylation (pERK1/2) was not detected, and progeny virus was not produced after infection. By contrast, in HeLa and CHO-CAR cells, which expressed CAR, the specific early and late pERK1/2 at 0.5 and 8 h were induced, and progeny viruses were produced progressively through 24 h after infection. However, when CHO-CAR cells were infected with replication-defective CVB3, specific pERK1/2 was not detected. In addition, when late pERK1/2 is inhibited by the MEK1 inhibitor PD98059, at 4 h after infection, virus replication significantly decreased. Therefore, our findings suggest that early pERK1/2 is a response to virus binding to CAR, whereas late pERK1/2 is related to the viral replication.