Liver-specific expression of the transgene encoding NS5A. a, schematic structure of the transgene encoding HCV NS5A genotype 1b. To direct the expression of the transgene to the liver, the albumin promoter was instrumental. A sufficient expression level was ensured by including the β-globin intron and for stabilization of the mRNA a poly(A) site was introduced at the 3′-end. For an efficient enrichment from tissue lysate and simple detection, NS5A was produced as a fusion protein with an N-terminal His₆ tag and a C-terminal V5 epitope. b, Western blot analysis of liver-derived lysates derived from two different male mice per the two founder lineages 01 and 03. A V5-specific antiserum served for detection of NS5A. c, immunofluorescence microscopy of liver sections derived from 3-month-old male NS5A-transgenic mice. Sections derived from sex- and age-matched wild-type littermates served as control. A V5-specific antiserum was used for detection. The staining was performed using a Cy3-conjugated secondary antibody. The weak staining in the negative control is due to autofluorescence and background staining of the secondary antibody. Merge was obtained by overlay of the immunofluorescence and of the phase contrast. The micrographs were taken at 400× magnification, the inlet at 630× magnification. d, Western blot analysis of lysates derived from liver, heart, spleen, lung, and kidney of a NS5A-transgenic mouse. A V5-specific antiserum served for detection of NS5A, an Erk2-specific serum was used to control equal loading. e, Western blot analysis of lysates derived from liver of 3-, 5-, 9-, or 12-month-old male or female mice. A V5-specific antiserum served for detection of NS5A, an actin-specific serum was used to control equal loading.

Impaired virus elimination in NS5A transgenic mice. a, NS5A-transgenic mice and the corresponding wild-type littermates were infected with LCMV (WE strain). The virus titer in the liver and spleen was determined 3, 6, 9, and 12 days after infection (22). The data are mean values from 6 animals per group. (**, p ≤ 0.01; ***, p ≤ 0.001). b, GPT activity in 6 NS5A and 6 wild-type mice at days 3, 6, 9, and 12 after LCMV infection. The values are given in units/liter (**, p ≤ 0.01). c, number of T cells was determined by immunohistochemistry of liver tissue using a CD3-specific serum. d, HE staining of paraffin sections derived from wild-type or NS5A transgenic mice sacrificed 3, 6, 9, and 12 days after LCMV infection (200× magnification).

NS5A interferes with the CTL response. Wild-type and NS5A-transgenic mice were immunized with coNS3/4A-pVAX1 or empty pVAX1 vector, indicated as not immunized, 2 weeks prior to hydrodynamic injection of coNS3/4A-pVAX1 into the tail vein. Control mice without injection were used as a negative controls as indicated. Three days after the hydrodynamic injection, mice were sacrificed and analyzed for NS3 expression in the liver. a, by immunohistochemistry of paraffin sections using a NS3-specific serum. b and c, by IP/Western blot of liver lysates. d, presence of T cells was visualized by immunohistochemistry staining of liver tissue using a CD3-specific antiserum. e, CTL activity was analyzed by ⁵¹chromium release assay using spleenic CTLs. Mice no. 7 and 8 have not been immunized. f, Western blot analysis using a NS3-specific serum of (left panel) liver lysate derived from wt and NS5A transgenic mice after hydrodynamic injection of pVaxNS3/4A or (right panel) lysate derived from HuH7.5 cells that were cotransfected with a constant amount of pVaxNS3/4A and variable amounts of palbNS5A.

Summary representation of genes affected by NS5A. According to their functions, genes that were affected by NS5A are summarized in different gene groups. The numbers reflect their quantity, respectively. a summarizes NS5A-dependent induced genes; b, the repressed genes.

NS5A impairs interferon immune response. a, co-immunoprecipitation of liver lysates derived from wild-type and NS5A transgenic mice using rabbit-derived NS5A- and PKR-specific antisera. For detection a PKR-specific monoclonal was used. b, RT-PCR for quantification of IFNβ-, 2′,5′-OAS-, and PKR-specific transcripts. RT-PCR using actin-specific primers served as control. Serial dilutions of the corresponding cDNA served in every PCR as a standard. Three wild-type and three transgenic mice were analyzed. One representative experiment is shown. c, Western blot analysis of liver-derived lysates of LCMV-infected NS5A-transgenic or wild-type mice using PKR- or 2′,5′-OAS-specific antisera. An actin-specific serum was used to control equal loading. Three wild-type and three transgenic mice were analyzed in duplicate. One representative experiment is shown. d, Lightcycler PCR for quantification of the 2′,5′-OAS- and the IFNβ-specific transcripts. Lightcycler RT-PCR was performed as described (43). The data are given as fold inductions and are mean values from three animals per group.