Vitamin B12 and hepatitis C: molecular biology and human pathology

Abstract

Cobalamins are stored in high concentrations in the human liver and thus are available to participate in the regulation of hepatotropic virus functions. We show that cyanocobalamin (vitamin B12) inhibited the HCV internal ribosome entry site (IRES)-dependent translation of a reporter gene in vitro in a dose-dependent manner without significantly affecting the cap-dependent mechanism. Vitamin B12 failed to inhibit translation by IRES elements from encephalomyocarditis virus (EMCV) or classical swine fever virus (CSFV). We also demonstrate a relationship between the total cobalamin concentration in human sera and HCV viral load (a measure of viral replication in the host). The mean viral load was two orders of magnitude greater when the serum cobalamin concentration was above 200 pM (P < 0.003), suggesting that the total cobalamin concentration in an HCV-infected liver is biologically significant in HCV replication.

Figure 1

The proposed secondary structure of the genotype 1b HCV IRES, adapted from Honda et al. (6). The authentic initiation codon is boxed. (Inset) Comparison between HCV and classical swine fever virus (CSFV) IRES structures in domains IIIe, IIIf, and IV.

Figure 2

Plasmids used for in vitro translation inhibition assays. (a) The bicistronic constructs under the control of a T7 transcriptional promoter contained the Renilla luciferase (R-Luc) reporter gene, a viral IRES element, and the chloramphenicol acetyltransferase (CAT) reporter gene. The viral sequences incorporated in these constructs consisted of the 5′ UTRs of EMCV (586 nucleotides), CSFV (373 nucleotides), and HCV (341 nucleotides), respectively. (b) The monocistronic construct (T368-CAT) contained the HCV IRES sequence, consisting of the entire 5′ UTR (341 nucleotides) and 27 nucleotides of the polyprotein coding sequence, and the CAT reporter gene cloned into the pGEMT-Easy (Promega) expression vector.

Figure 3

Selective inhibition of the HCV IRES in vitro. Lane 1 shows the expression of both the HCV IRES-dependent CAT reporter gene and the cap-dependent firefly (FF)-Luc reporter gene in the lysate in the absence of added vitamin B12. Lane 2 shows the selective inhibition when the mRNA was preincubated with vitamin B12 (2.5 mM final concentration on the lysate) before translation. The unidentified minor bands in the gel represent peptide fragments that are characteristic products of the FF-Luc control RNA.

Figure 4

Vitamin B12 concentration dependence of the observed HCV IRES-dependent translational inhibition. The HCV IRES efficiency was plotted as a function of final vitamin B12 concentration from a bicistronic mRNA (Fig. 2a, ▴) or from a monocistronic mRNA (Fig. 2b, ●). The IRES efficiency, defined as [CAT]/([CAT] + [Luc]), was calculated for each reaction and normalized to the reaction with no added vitamin B12. The curve fit a regular hyperbolic binding function (r = 0.98). The minimum IRES efficiency was ≈0.40, suggesting a maximum of 60% inhibition relative to the uninhibited control.

Figure 5

Specificity for the HCV IRES. Lanes 1–3 show the in vitro translation products from bicistronic mRNA containing EMCV, CSFV, and HCV IRES elements, respectively. Lanes 4–6 show the in vitro translation products formed when vitamin B12 was incorporated in the reaction mixture to a final concentration of 3 mM. The faster-migrating species is the HCV IRES-dependent CAT protein and the slower-migrating species is the cap-dependent R-Luc protein. The IRES efficiency for each reaction, defined as in Fig. 4 but not normalized, is listed below the corresponding lane. Lane 6 shows the characteristic 60% inhibition relative to lane 3.

Figure 6

Trapping by the HCV IRES–vitamin B12 complex. (a) Nonspecific inhibition of translation was observed when the full-length HCV IRES RNA (368 nucleotides) was added to a standard translation assay in the presence of 250 μM vitamin B12 (lane 4). This inhibition was not observed when either the HCV IRES RNA or vitamin B12 was omitted from the reaction (lanes 2 and 3, respectively). The gel was deliberately overloaded to convincingly illustrate the absence of protein in lane 4. (b) The suggested mechanism of the observed translational inactivation. Vitamin B12 binds specifically to the HCV IRES, trapping the preinitiation complex [minimally the 40S ribosomal subunit and the eIF2/Met-tRNA/GTP ternary complex (11)] on the ribosomal subunit loading site.

Figure 7

Relationship between HCV viral load and total serum cobalamin concentration. The HCV viral load was plotted against the total serum cobalamin concentration. The mean of the data below 200 pM differed significantly from the mean of the data above 200 pM (Mann–Whitney U test, P < 0.003).