Decreased expression of the immediate early protein, ICP4, by deletion of the tegument protein VP22 of equine herpesvirus type 1

Abstract

VP22 is a major tegument protein of equine herpesvirus type 1 (EHV-1). In the present study, we examined functions of VP22 in EHV-1 replication by viral protein expression analyses in cells infected with the VP22-deficient virus. The expressions of several viral proteins in the cells infected with the VP22-deficient virus were lower than those in the cells infected with the parent virus. One of the weakly expressed proteins was identified as ICP4, which is a major regulatory protein encoded by an immediate early gene of EHV-1. A real-time PCR analysis showed that the mRNA expression of ICP4 was the same in cells infected with the parent and VP22-deficient viruses. Hence, VP22 appears to promote synthesis of ICP4 post-transcriptionally.

Keywords: EHV-1; ICP4; VP22.

Fig. 1.

A schematic diagram of the genome structure of parent virus and mutant viruses [18].

Fig. 2.

Protein expression comparison by Western blotting using anti-EHV-1 antibody (a). MDBK cells were infected with EHV-1 attB (lane 1), EHV-1∆VP22 (lane 2) and EHV-1∆VP22R (lane 3) at an MOI of 3 PFU/cell and analyzed by Western blotting with antibody to EHV-1 virions at 0, 2, 4 and 8 hrpi. Molecular sizes are shown on the left (a). Expression of ICP4s of EHV-1 attB, EHV-1∆VP22 and EHV-1∆VP22R were analyzed by Western blotting using anti-ICP4 antibody (b).

Fig. 3.

mRNA levels of ICP4 (a) and ICP0 (b) in EHV-1 attB-, EHV-1∆VP22- and EHV-1∆VP22R-infected cells. MDBK cells were inoculated with EHV-1 attB, EHV-1∆VP22 and EHV-1∆VP22R at an MOI of 3. After 0 and 1 hrpi, total RNA and DNA was extracted. Quantification of mRNA was carried out by real-time quantitative RT-PCR. Expression levels of ICP4 and ICP0 mRNA were normalized with the GAPDH mRNA levels and genome DNA, and expressed relatively as the ratio against the mRNA levels in EHV-1 attB-infected cells at 0 hrpi. There are no significant (n.s.) differences between EHV-1 attB and EHV-1∆VP22. * means “below the threshold”.