J. Virol., Apr 1996, 2086-2094, Vol 70, No. 4
Copyright © 1996, American Society for Microbiology

Stably expressed antisense RNA to cytomegalovirus UL83 inhibits viral replication

P Dal Monte, C Bessia, A Ripalti, MP Landini, A Topilko, B Plachter, JL Virelizier and S Michelson
Unite d'Immunologie Virale, Institut Pasteur, Paris, France.

The human cytomegalovirus (HCMV) open reading frame UL83 encodes a phosphoprotein of 64 to 68kDa (pp65) which is a major constituent of this virion and dense bodies. To determine the importance of the HCMV gene in the virus cycle, we studied HCMV replication in astrocytoma cells stably transfected with a retroviral vector carrying an antisense UL83 cDNA. Reverse transcription-PCR detected antisense RNA in the cytoplasm. The steady-state level of a 4-kb RNA containing coding sequences for pp65 was significantly reduced after infection of antisense cells. Concomitant with this, levels of expression of pp65 and pp71 (UL82) were severely reduced. Extracellular HCMV production was almost completely blocked, irrespective of the multiplicity of infection or the time after infection studied. The block occurred at an early phase, since immediate-early protein synthesis occurred normally, while several late proteins (e.g., pp150 [ppUL32] and assembly protein [UL80]) were absent or strongly inhibited. Normal replication of herpes simplex virus and of a pp65 deletion mutant of HCMV (RVAd65), lacking target sequences of antisense RNA, demonstrated the specificity of the block for wild-type HCMV in the antisense-stabilized cells and indicated that the block was not due to indirect interference with cellular genes. Our results appear to contradict those of Schmolke et al (S. Schmolke, H.F. Kern, P. Drescher, G. Jahn, and B. Plachter, J. Virol. 69:5959-5968, 1995), which show that UL83 is a nonessential gene for HCMV replication in vitro. This contradiction is discussed in light of the fact that the 4-kb mRNA, which codes for pp65 and was targeted in UL83-antisense cell lines, may be a bicistronic mRNA which also codes for pp71 (UL82). Thus, interference of expression from the genes encoding pp65 and pp71 by blocking of this putative bicistronic message leads to severe impairment of viral replication.

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