Fig. 1.

HSV-2 LAT region encodes a miRNA. (A) Schematic diagram of HSV-2 LAT region and HSV-2 miR-I. Restriction endonucleases used to create mutant viruses and plasmids are labeled. Stable RNAs including primary LAT, the LAT intron, ICP0, ICP34.5, ICP4, ORF-O (putative), and ORF-P (putative) are labeled based on their relative transcription-starting sites and transcription direction. The location of a TATA box in the LAT intron, which is mutated in the ΔYAT virus, is also labeled. The HSV-2 mature miRNA (bold) was identified by small-RNA cloning and maps to HSV-2 LAT exon 2 (nucleotides 569–547 and 126681–126703). The predicted anti-sense strand of miR-I is shown in italics. ≈50% of miR-I sequences cloned had one additional “C” at their 3′ end. The arrows indicate Dicer cutting sites. Mutant HSV-2 viruses and HSV-2 LAT plasmids are also shown. TRL, terminal repeat long; IRL, internal repeat long; IRS, internal repeat short; US, unique short; TRS, terminal repeat short; UL, unique long. The open boxes on ICP0 and ICP34.5 represent introns. (B) HSV-2 miR-I detection by Northern blot in 293 and HeLa cells transfected with plasmids containing full-length LAT gene but not with truncated LAT plasmids. Total RNAs from HEK 293 cells and HeLa cells transfected with or without plasmids pSSK and pCMV-SSK, which include the ICP34.5 coding region, and plasmids pSSB and pCMV-SSB, which are truncated and lack the ICP34.5 region of LAT, were blotted with ³²P-labeled oligo probe for miR-I. The same membrane was stripped and reprobed with an oligonucleotide probe for the predicted anti-sense strand of miR-I and a probe for U6 small nuclear RNA (snRNA). (C) Mature miR-I was significantly reduced, but pre-miRNA increased in Dicer exon-5 disrupted cells. Wild-type (WT) and Dicer exon-5 disrupted cells (Dicer^−/−) were studied with or without HSV-2 strain HG52 infection. Total RNAs were hybridized with the HSV-2 miR-I probe and the U6 probe. (D) LAT promoter is not the sole promoter for HSV-2 miR-I production in infected cell cultures. Vero cells were infected with HSV-2 strain HG52, HSV-2 strain 333, and HSV-2 mutant viruses including ΔLAT, CMV-LAT, ΔYAT, and ΔNot-SalI at 0.1 multiplicity of infection or mock-infected. miR-I, the anti-sense strand of miR-I, and U6 snRNA were detected by Northern hybridization after stripping the same membrane. (E) The sequences directly upstream of miR-I contribute to miR-I expression but to a lesser extent than LAT promoter sequences in transfected cells. HEK 293 cells and HeLa cells were transfected with pSSK and pPstI-HincII, which does not contain the LAT promoter region but contains ≈3 kb of sequence upstream of miR-I, or mock-transfected. Total RNA from these transfected cells were hybridized with HSV-2 miR-I and U6 snRNA probes.