STABLY EXPRESSED ANTISENSE RNA TO CYTOMEGALOVIRUS UL83 INHIBITS VIRALREPLICATION

The human cytomegalovirus (HCMV) open reading frame UL83 encodes a pho sphoprotein of 64 to 68 kDa (pp65) which is a major constituent of the virion and dense bodies. To determine the importance of this HCMV gen e in the virus cycle, we studied HCMV replication in astrocytoma cells stably transfected with a retroviral vector carrying an antisense UL8 3 cDNA. Reverse transcription-PCR detected antisense RNA in the cytopl asm. 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) w ere severely reduced. Extracellular HCMV production was almost complet ely 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 lat e proteins (e.g., pp150 [ppUL32] and assembly protein [UL80]) were abs ent or strongly inhibited. Normal replication of herpes simplex virus and of a pp65 deletion mutant of HCMV (RVAd65), lacking target sequenc es of antisense RNA, demonstrated the specificity of the block for wil d-type HCMV in the antisense-stabilized cells and indicated that the b lock was not due to indirect interference with cellular genes. Our res ults 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 replicatio n 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 p p71 by blocking of this putative bicistronic message leads to severe i mpairment of viral replication.