Identification and characterization of a Penaeus monodon lymphoid cell-expressed receptor for the yellow head virus

Abstract

The yellow head virus is a positive-sense, single-stranded RNA virus that causes significant mortality in farmed penaeid shrimp. This study sought to isolate and characterize the receptor protein used by the virus to gain entry into Penaeus monodon Oka (lymphoid) organ cells, a primary target of yellow head virus infections. Virus overlay protein binding assay on Oka organ membrane preparations identified a 65-kDa protein, and antibodies raised against this protein inhibited virus entry in primary Oka cell cultures by approximately 80%. N-terminal sequence analysis of the 65-kDa protein generated a 17-amino acid peptide fragment which was used to design degenerate primers that amplified a 1.5-kbp product from Oka organ total RNA, which was cloned and sequenced. Northern analysis and PCR were used to confirm a single RNA transcript that was expressed in most tissues. Subsequently, the mature cDNA was recloned and the expressed protein shown to cross-react with the antibody raised against the original virus binding band. Down regulation of the message through double-stranded RNA-mediated RNA interference silencing resulted in the complete inhibition of virus entry. While the identity of the clone remains unknown, it nevertheless represents the first invertebrate Nidovirus receptor isolated to date.

FIG. 1.

(A) Coomassie brilliant blue-stained gel and VOPBA of crude membrane proteins (lanes ii and iv) from the lymphoid organs of P. monodon. Lanes i and iii, purified yellow head virus; M, prestained protein markers. (B) Coomassie brilliant blue-stained gel (lane i) and Western blot analysis (lanes ii and iii) of crude membrane proteins from the lymphoid organs. Western analysis was undertaken with an anti-p65 rat polyclonal antiserum (lane ii) or control serum (lane iii). (C) Inhibition of infection of primary cultures of Oka cells by YHV. Oka cells were incubated with an anti-p65 rat polyclonal antiserum or with control serum prior to infection with YHV. Virus titers were calculated by TCID₅₀ analysis and expressed as a percentage of control. Experiment was undertaken in quadruplicate, and error bars represent standard errors of the means. (D) Coomassie brilliant blue-stained gel and Western blot analysis of the pmYRP65-GST fusion protein. Partially purified pmYRP65-GST fusion protein (lanes i) and GST protein (lanes ii) were separated by electrophoresis and visualized by Coomassie brilliant blue staining before transfer to solid matrix support and probed consecutively with a monoclonal antibody against GST and the rat anti-pmYRP65 serum.

FIG. 2.

Alignment of the deduced amino acid sequence of pmYRP65 YHV and the consensus sequence of the ribosomal L22 domain. Potential glycosylation sites are indicated (closed triangles).

FIG. 3.

(A) Northern blot analysis of pmYRP65. Total RNAs from lymphoid organ (Lym) and gill (G) were separated on a 1.2% denaturing agarose gel, transferred, and hybridized sequentially with a riboprobe derived from a unique region of pmYRP65. A single transcript in both lymphoid and gill tissues was observed. M, RNA marker (Promega). (B) RT-PCR detection of pmYRP65. BN, brain and nerve cord; Hep, hepatopancreas; Ov, ovary; Hea, heart; G, gill; Ad, abdomen; Lym, lymphoid organ; −ve, negative PCR control, +ve, positive control of pmYRP65; −RT, negative control of reverse transcriptase reaction.

FIG. 4.

RT-PCR analysis for actin, YHV, and pmYRP65 expression after dsRNA-mediated gene silencing. Control, primary cultures of Oka cells, not transfected, not infected with YHV; Mock, primary cultures of Oka cells mock transfected (no dsRNA) and infected with YHV; GFP, primary cultures of Oka cells transfected with GFP dsRNA; pmYRP65, primary cultures of Oka cells transfected with pmYRP65 dsRNA.