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Human guanylate binding protein-1 (hGBP-1) is a member of the GTPase family which is induced by interferon gamma (IFN-γ) and interferon alpha/beta (IFN-α/β). It is not clear that whether hGBP-1 mediates the antiviral biologic activity of interferons. Anderson et al report that hGBP-1 inhibited Viscular stomatitis virus (VSV) and Encephalomyocarditis virus (EMCV) replication in Hela cells (1). To further investigate the antiviral effects of hGBP-1, we cloned the hGBP-1 gene and inserted it into the pcDNA3.1(-) eukayotic expression vector and analyzed the inhibition effect against Hepatitis B virus (HBV) and Coxsackie virus B3 (CVB3).
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A 1790-bp fragment was obtained by long chain RT-PCR. This fragment was cloned into pCR2.1 vector by T/A method, and the inserted sequence was identified by restriction endonuclease cleavage (Fig. 1) and sequence analysis. After digestion with EcoRⅠor Hind Ⅲ, the fragments were 1.8/3.9kb and 1.11/ 4.60kb respectively, indicating that the hGBP-1 gene was inserted into pCR2.1 in the reverse direction. Sequence alignment indicated that the cloned hGBP-1 gene had 100% homology with the hGBP-1gene published in GenBank (Accession number: M55542). The hGBP-1 gene fragment was then subcloned into the pcDNA3.1(-) vector by digestion with BamHⅠ and NotⅠ, the recombinant plasmid was named as pGBP-1.
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HepG2 and Hela cells were transfected with the pGBP-1 plasmid, the cell lysates were analyzed by Western blot. The hGBP-1 (about 67 kDa) protein was high level expression in pGBP-1 transfected cells, but was low level expression in the cells transfected with monk plasmid (Fig. 2).
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HBV replication in HepG2 cells contransfected with pGBP-1 and pHBV1.3 was studied by ELISA and Southern blot. The levels of HBsAg and HBeAg in supernatant were similar to that of the cells contransfected with pHBV1.3 and monk plasmid (Table 1). Furthermore, the levels of intracellular HBV replicative intermediates had no significant difference between the two groups (Fig. 3).
Table 1. The levels of HBsAg and HBeAg in supernatant after transfection (x±s)
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CVB3 replication in Hela cells transiently transfected with hGBP-1 was studied by TCID50 assay. In brief, the viral products in the supernatant and cells were assessed by measuring the cytopathic effect (CPE) on Hela cells incubated with 0.1mL culture lysates. TCID50 of 0.1mL culture lysates was markedly reduced when compared with that of the Hela cells without hGBP-1 transfection, especially in Hela cells chal-lenged with lower doses of CVB3 (Table 2).
Table 2. Virus production in cultures after Hela cells challenged with CVB3 (TCID50/0.1mL)