Post-translational Modification of the Hepatitis C Virus Core Protein by Tissue Transglutaminase

Abstract

The hepatitis C virus (HCV) core protein is a structural protein that packages the viral genomic RNA. In this study, we demonstrate that a stable core protein dimer could be produced in liver cells. The production of this protein could be enhanced by calphostin C and serum deprivation. This protein was determined to be the core protein dimer because of its reactivity with the anti-core antibody, its similar electrophoretic mobility compared with that of the core protein dimer generated by cross-linking with glutaraldehyde, and its increase in size by a hemagglutinin tag fused to the core protein sequence. This core protein dimer was highly stable and resistant to SDS and ␤-mercaptoethanol. The enzyme that mediated the formation of this stable core protein dimer was determined to be the tissue transglutaminase (tTG) because, first, tTG could be activated by calphostin C and serum deprivation; second, the formation of this dimer was suppressed by monodansylcadaverine, a tTG inhibitor; and third, the core protein could be crosslinked by tTG in vitro. Thus, the HCV core protein represents the first known viral structural protein substrate of tTG. The post-translational modification by tTG reduced the RNA binding activity of the core protein, raising the possibility that tTG may regulate the biological functions of the HCV core protein.

Tissue transglutaminase triggers oligomerization and activation of dual leucine zipper-bearing kinase in calphostin C-treated cells to facilitate apoptosis

“…26,27 Since recent studies have shown that calphostin C stimulates the transamidating activity of tTG in a time-dependent manner, 18,33 we began this study by monitoring the in situ transamidating activity of tTG in mouse NIH 3T3 fibroblasts that were exposed to calphostin C for various lengths of time (Figure 1a). In agreement with the previous findings, 18,28 we detected a gradual and dramatic increase in tTG activity as early as 30 min following treatment with 250 nM calphostin C, a dose known to decrease survival in several cell types. 29,30 Immunoblots processed in parallel with lysates prepared from these cells and an antibody specifically targeted to tTG confirmed that the increase in transamidating activity was not attributable to variations in tTG protein levels (Figure 1b).…”

Role and predictive strength of transglutaminase type 2 expression in premalignant lesions of the cervix

“…Except for normal tissues (20 cases), which were negative for HPV infection, all dysplastic lesions (60 cases) showed HPV genotype positivity (Table 1). Cervical samples were assayed for 25 HPV types, 14 types were of high oncogenic risk (HR) (16,18,31,33,34,35,39,45, 51, 52, 53, 56, 58, and 59) and 11 were of low oncogenic risk (LR) (6, 11, 40, 43, 44, 61, 70, 72, 81, 83, and 84). In the CIN I, HR-HPV genotypes were found in nine of the 37 lesions tested (24.3%), whereas 28 lesions were infected by LR types (75.6%).…”

“…15,16 A function for TG2 in viral pathogenesis has also been recently suggested by the discovery of a growing number of viral proteins, and the cellular proteins with which they interact, found to be modified by TG2. [17][18][19] The study of Jeon and coworkers 20 have established that the catalytic activity of TG2 can incorporate polyamine into HPV18 E7, and thereby inhibit its binding to pRb. It is of interest to note that TG2 can translocate from the cytosol into the nucleus incorporating polyamines into the retinoblastoma protein (Rb) to protect it from caspase-mediated degradation.…”

Hepatitis C Virus F Protein Is a Short-Lived Protein Associated with the Endoplasmic Reticulum

“…pCDEF-9aCore is identical to pCDEF-HAF with the exception that it does not have the C-to-T mutation at nt 57 (28). At 48 h after transfection, cells were treated with DMSO (lane 1), 1 g of MG132/ml (lanes 2 and 4), or 20 M of lactacystin (LC) (lanes 3 and 5) for 6 h. Cells were then lysed for Western blot analysis with the anti-HA antibody by using our previous procedures (13). The protein signals were analyzed by the enhanced chemiluminescence kit (Pierce).…”

“…The identity of this protein was confirmed by Western blotting with the anti-core antibody (data not shown). (13) were double stained with rabbit anti-HCV core and mouse anti-HA primary antibodies and FITCconjugated goat anti-rabbit and RITC-conjugated goat anti-mouse secondary antibodies. (E and F) Huh7 cells containing the HCV subgenomic RNA replicon (unpublished data) were transfected with pCDEF-HAF and double stained with mouse anti-NS5A and rat anti-HA primary antibodies and FITC-conjugated goat anti-mouse and RITC-conjugated goat anti-rat secondary antibodies.…”