Viperin (cig5), an IFN-inducible antiviral protein directly induced by human cytomegalovirus

Abstract

Little is known about the mechanism by which IFNs inhibit human cytomegalovirus (HCMV) replication. Indeed, infection of fibroblasts with HCMV initiates the expression of a subset of type I IFN-inducible genes whose role in the infectious process is unclear. We describe here the identification of a cytoplasmic antiviral protein that is induced by IFNs, by HCMV infection, and by the HCMV envelope protein, glycoprotein B (gB). Stable expression of the protein in fibroblasts inhibits productive HCMV infection, down-regulating several HCMV structural proteins (gB, pp28, and pp65) known to be indispensable for viral assembly and maturation. We have named the protein viperin (for virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible). HCMV infection causes the redistribution of the induced viperin from its normal endoplasmic reticulum association, first to the Golgi apparatus and then to cytoplasmic vacuoles containing gB and pp28. Expression before HCMV infection reduces viperin redistribution from the endoplasmic reticulum to the Golgi apparatus and prevents vacuolar localization, perhaps reflecting the mechanism used by HCMV to evade the antiviral function.

Figure 1

Sequences of human viperin and comparison with homologues from other species. (A) The human cDNA sequence encodes an ORF with 361 amino acids. (B) Comparison of viperin amino acid sequence with mouse, rat (Best 5; GenBank accession no. YO7704), and trout (Vig 1; GenBank accession no. AF076620) sequences. The leucine residues in bold type indicate the leucine zipper motif, and boxed residues are those differing from the human sequence. cDNA sequences for human and mouse viperin are available through GenBank accession nos. AF442151 and AF442152, respectively.

Figure 2

Cell-type-specific induction of viperin expression by type I and II IFNs. (A) Viperin is induced in macrophages by IFN-γ. Extracts of untreated and IFN-γ-treated primary human macrophages were subjected to SDS/PAGE and viperin was detected by immunoblotting with a specific rabbit antiserum. (B) Induction of viperin in macrophages by type I and II IFNs. (C) Viperin expression is stimulated preferentially by type I IFNs in a variety of other cell types.

Figure 3

Induction of viperin expression by HCMV. (A) HCMV infection of primary human fibroblasts induces viperin expression detected by immunoblotting. (B) Kinetics of viperin induction by IFN-α and by HCMV infection. (C) Direct induction of viperin by gB, an HCMV envelope glycoprotein. Purified gB was added to cells, and viperin expression was assayed by immunoblotting at 8 and 24 h.

Figure 4

Viperin inhibits HCMV replication. (A) Human fibroblasts, transduced with either a retrovirus encoding viperin or with a control retrovirus, were infected with HCMV, and supernatants were harvested 7 days after infection and analyzed by a standard plaque assay. (B) Viperin expression inhibits the synthesis of late viral proteins. Fibroblasts expressing viperin or control fibroblasts were infected with HCMV and after 3 days extracts were subjected to SDS/PAGE and immunoblotted with Abs to immediate-early protein, pp65, gB, and pp28 of HCMV.

Figure 5

HCMV infection alters the normal cellular distribution of viperin. (A) Viperin localizes to the ER. Fibroblasts treated with IFN-α for 24 h were fixed, permeabilized, and analyzed by intracellular immunofluorescence, using a rabbit anti-viperin antiserum and a mAb to calnexin, an ER marker. (B) HCMV infection induces viperin redistribution. Fibroblasts infected with HCMV were harvested and stained with the rabbit anti-viperin serum on days 1–4 after infection. “N” indicates the location of the nucleus. (C) Viperin redistribution does not affect resident ER proteins. Fibroblasts were costained 4 days after infection for viperin and calnexin. (D) Viperin moves initially to the Golgi apparatus during HCMV infection. Fibroblasts were harvested at 3 days after infection and costained for viperin and the Golgi marker GM130. (E) Viperin redistributes after infection to the Golgi apparatus and undefined cytoplasmic vesicles expressing the HCMV envelope glycoprotein, gB.

Figure 6

Prior expression of viperin inhibits the HCMV-induced redistribution of viperin and gB. (A) Fibroblasts expressing viperin (Lower) or control fibroblasts (Upper) were infected with HCMV, harvested 3 days after infection, fixed, permeabilized, and the subcellular distribution of viperin and gB was examined by immunofluorescence. (B) Viral gene(s) sensitive to ganciclovir are required for viperin redistribution to cytoplasmic vesicles. Fibroblasts were infected with HCMV for 3 days in ganciclovir (50 μM), fixed, permeabilized, and costained with Abs to viperin and either gB or GM130.