Kaposi’s Sarcoma-Associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of Kaposi’s sarcoma, Castleman’s disease, and primary effusion lymphoma. Although the functions of the viral thymidine kinases (vTK) of herpes simplex virus-1/2 are well understood, that of KSHV ORF21 (an ortholog of vTK) is largely unknown. Here, we investigated the role of ORF21 in lytic replication and infection by generating two ORF21-mutated KSHV BAC clones: ORF21-kinase activity deficient KSHV (21KD) and stop codon-induced ORF21-deleted KSHV (21del). The results showed that both ORF21 mutations did not affect viral genome replication, lytic gene transcription, or the production of viral genome-encapsidated particles. The ORF21 molecule-dependent function, other than the kinase function of ORF21, was involved in the infectivity of the progeny virus. ORF21 was expressed 36 h after the induction of lytic replication, and endogenously expressed ORF21 was localized in the whole cytoplasm. Moreover, ORF21 upregulated the MEK phosphorylation and anchorage-independent cell growth. The inhibition of MEK signaling by U0126 in recipient target cells suppressed the number of progeny virus-infected cells. These suggest that ORF21 transmitted as a tegument protein in the progeny virus enhances the new infection through MEK up-regulation in the recipient cell. Our findings indicate that ORF21 plays key roles in the infection of KSHV through the manipulation of the cellular function.

Kaposi’s Sarcoma-Associated Herpesvirus ORF67.5 Functions as a Component of the Terminase Complex

“…Anti-KSHV ORF67.5 rabbit pAb was purified from the immunized rabbit serum using antigen peptide affinity chromatography. The following primary antibodies were used: anti-K-bZIP mouse monoclonal antibody (MAb; F33P1; Santa Cruz Biotechnology, TX, USA), anti-K8.1 A/B mouse MAb (4A4; Santa Cruz Biotechnology), anti-ORF21 rabbit pAb (previously produced in our laboratory) ( 52 ), anti-β-actin mouse MAb (AC-15; Santa Cruz Biotechnology), anti-FLAG mouse MAb (FLA-1; MBL, Nagoya, Japan), anti-S rabbit pAb (sc-802; Santa Cruz Biotechnology), and anti-HA mouse MAb (TANA2; MBL). Anti-mouse IgG-horseradish peroxidase (HRP; NA931; Cytiva, Tokyo, Japan) and anti-rabbit IgG-HRP (7074; Cell Signaling Technology, MA, USA) were used as the secondary antibodies.…”

Sofosbuvir Suppresses the Genome Replication of DENV1 in Human Hepatic Huh7 Cells

“…The cells were then seeded in 96-well plates at 5 × 10 3 cells/well in 100 µL of media with or without the compound at various concentrations. Next, the cells were incubated for 48 or 72 h. The number of viable cells was estimated using the Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan) [40]. The optical density at 450 nm (reference wavelength: 620 nm) was measured using a Tecan M200 microplate spectrophotometer (Tecan, Kanagawa, Japan) and was expressed as a percentage of the value of untreated cells (defined as 100%).…”

“…DENV1 RNA replicon-harboring Huh7 cells (3 × 10 5 cells/well) were seeded in 6-well plates and cultured for 12 h. Test drugs were then added to the media, and the cells were subsequently cultured in the presence (or absence) of the drug for 48 h. mRNA was extracted from the cells using RNAiso Plus (Takara, Osaka, Japan). cDNA was synthesized using a ReverTra Ace qPCR kit (Toyobo, Osaka, Japan) and was subjected to SYBR green real-time PCR [40]. The relative mRNA expression levels were determined by ∆∆Ct methods.…”

Tegument proteins of Epstein-Barr virus: Diverse functions, complex networks, and oncogenesis

“…Disruption of HSV UL23 moderately decreased virus replication [ 212 ]. Knockout of KSHV ORF21 had no effect on viral gene expression and DNA synthesis, but markedly suppressed infectious progeny production, presumably by decreasing infectivity [ 213 ]. Upon infection, ORF21 protein in the tegument is released into the cytoplasm, where it stimulates MEK signaling to enhance infectivity.…”