Chang S. Hahn

Hepatitis C Virus Core Selectively Suppresses Interleukin-12 Synthesis in Human Macrophages by Interfering with AP-1 Activation

Hepatitis C virus (HCV) is remarkably efficient at establishing persistent infection, suggesting that it has evolved one or more strategies aimed at evading the host immune response. T cell responses, including interferon-γ production, are severely suppressed in chronic HCV patients. The HCV core protein has been previously shown to circulate in the bloodstream of HCV-infected patients and inhibit host immunity through an interaction with gC1qR. To determine the role of the HCV core-gC1qR interaction in modulation of inflammatory cytokine production, we examined interleukin (IL)-12 production, which is critical for the induction of interferon-γ synthesis, in lipopolysaccharide-stimulated human monocyte/macrophages. We found that core protein binds the gC1qR displayed on the cell surface of monocyte/macrophages and inhibits the production of IL-12p70 upon lipopolysaccharide stimulation. This inhibition was found to be selective in that HCV core failed to affect the production of IL-6, IL-8, IL-1β, and tumor necrosis factor α. In addition, suppression of IL-12 production by core protein occurred at the transcriptional level by inhibition of IL-12p40 mRNA synthesis. Importantly, core-induced inhibition of IL-12p40 mRNA synthesis resulted from impaired activation of AP-1 rather than enhanced IL-10 production. These results suggest that the HCV core-gC1qR interaction may play a pivotal role in establishing persistent infection by dampening TH1 responses.

Dideoxy sequencing of RNA using reverse transcriptase.

Shortly after the dideoxy (dd) chain-termination method for DNA sequencing was described by Sanger et al., Zimmern and Kaesberg modified this method to sequence RNA directly, using purified encephalomyocarditis viral RNA as a template, pdT_(7)rC as a specific primer, and reverse transcriptase as the elongation enzyme. Bina-Stein et al. further developed the method to use total cellular mRNA as a template and complementary DNA restriction fragments, which anneal to a particular mRNA at specific locations, as primers for reverse transcriptase. Recently, sequence-specific oligonucleotides, synthesized with an automated DNA synthesizer, have been used as primers on both DNA and RNA templates.

Low pH-dependent sindbis virus-induced fusion of bhk cells: Differences between strains correlate with amino acid changes in the E1 glycoprotein

Expression of alphavirus glycoproteins on the surface of infected cells leads to cell fusion after exposure to acidic pH. Two strains of Sindbis virus, AR339 (SV) and neuroadapted Sindbis virus (NSV), which differ in virulence for weanling mice, were found to differ in pH-dependent fusion. BHK-21 cells infected with SV fused maximally after shifting to pH 5.4, whereas cells infected with NSV required a lower pH, pH 4.8, for maximal fusion. No difference was noted in the optimal pH for agglutination of goose erythrocytes (5.75 for both viruses). To determine the molecular basis for the difference in fusion a series of recombinant viruses was constructed using a cDNA clone of Sindbis virus from which infectious RNA can be transcribed in vitro. Cells infected with a recombinant virus that had the SV E1 and NSV E2 genes had a fusion response curve as a function of pH like SV, while cells infected with recombinant virus with the NSV E1 and SV E2 genes fused like NSV. The E1 glycoproteins of SV and NSV differ at two positions: Val-72 in SV is Ala in NSV (a change near the putative fusion site), and Gly-313 in SV is Asp in NSV. Recombinant viruses which had Val-72 (SV) and Asp-313 (NSV) or Ala-72 (NSV) and Gly-313 (SV) had a lowered pH of fusion like NSV suggesting that both positions participate in determining some aspect of the conformational change in the E1-E2 heterodimer associated with pH-dependent fusion.

Endogenous presentation of a nascent antigenic epitope to CD8+ CTL is more efficient than exogenous presentation.

Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides in association with class I MHC molecules at the cell surface. Newly synthesized viral polypeptides are processed in the cytoplasm and the fragments of antigen are transported into the endoplasmic reticulum (ER) via a peptide transporter where they complex with nascent class I molecules. The peptide‐MHC complex is transported to the cell surface and presented to CTL. Sequence analysis of endogenously expressed, MHC‐associated self or viral antigens indicates that the naturally processed peptides bound to class I MHC molecules are in general 9 ± 1 residues long. Peptides bound to specific class I MHC molecules have in common allele‐specific motifs of conserved residues. The motif for the class I Kd molecules has been shown to be nine or 10 residues with the sequence X‐Tyr‐(X)6‐I/L or X‐Tyr‐(X)7‐I/L. The Tyr residue at the second position and the I/L residue at the ninth position are allele‐specific anchor residues which appear to be required for binding of the peptide to Kd. To examine the stringency of the requirement for Tyr at the second position, we have performed saturation mutagenesis of a minigene encoding the class I Kd‐restricted influenza HA210‐219 site at the Tyr residue 211. A series of 10 mutants was tested for effects on target‐cell sensitization. Most amino acid substitutions for the Tyr residue resulted in a loss of endogenous peptide recognition by HA210‐219 reactive CTL, consistent with the critical role of the Tyr at the second position for interaction with Kd molecules. One mutant gene‐product encoding a His substitution for the Tyr residue was recognized by CTL. However, the corresponding synthetic peptide containing a His substitution at the dominant anchor position bound only weakly to Kd, and target cells treated with the peptide were poorly recognized by CTL. The endogenous His‐containing peptide was also less stably associated with class I MHC Kd molecules at the cell surface than the wild‐type Tyr peptide. These data indicate that endogenous antigenic peptides may bind newly‐synthesized class I MHC molecules in the ER more efficiently than fully formed class I molecules at the cell surface and that endogenous peptides may dissociate from class I MHC molecules at different rates. The implications of these findings for CTL recognition and epitope mapping are discussed.