Abstract： Background:Natural anti-sense transcripts (NATs),which are transcribed from the complementary DNA strand of annotated genes,exert regulatory function of gene expression.Increasing studies recognized anti-sense transcription widespread throughout human cytomegalovirus (HCMV) genome,whereas the anti-sense transcription of RNA1.2 gene locus has never been investigated.In this study,the transcription of the RNA1.2 anti-sense strand was investigated in clinically isolated HCMV strain.Methods:Strand-specific high-through RNA-sequencing (RNA-seq) was performed to find possible anti-sense transcripts (ASTs).For analyzing and visualization of RNA-seq data sets,Integrative Genomics Viewer software was applied.To confirm these possibilities,Northern blotting and rapid amplification of cDNA ends (RACE) were used.Results:Transcription of the opposite strand of RNA1.2 gene locus was detected by RNA-sequencing using RNAs extracted from human embryonic lung fibroblasts infected with HCMV clinical isolate HAN.At least three HCMV NATs,named RNA1.2 AST 1,RNA1.2 AST2,and RNA1.2 AST3,were characterized by Northern blotting and RACE analyses.These RNA1.2 ASTs orientated from the complementary strand of RNA1.2 locus during the late phase of HCMV infection.The 5'-and 3'-termini of these transcripts were located within the opposite sequence of the predicted RNA1.2 gene.Conclusion:A cluster of novel NATs was transcribed from the opposite sequence of the HCMV RNA1.2 gene region.