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Roizman. of the largest of the HSV type 1 (HSV-1) LAT ORFs (274 amino acids) greatly enhances virus growth in cell types that are normally relatively nonpermissive for HSV replication and also that it complements mutations to the immediate-early (IE) gene ICP0 (S. K. Thomas, G. Gough, D. S. Latchman, and R. S. Coffin, J. Virol. 73:6618-6625, 1999). Here we show that LAT ORF expression overcomes the repression of expression from exogenous promoters introduced into the HSV-1 genome which normally occurs in the absence of IE gene expression. To further explore LAT ORF function, we have generated an epitope-tagged LAT ORF, LATgene into the pR20.5/vhs shuttle vector (35) to yield pR20.5/vhs/LAT, which was recombined into an HSV-1 1764-/27-/4-/P2- (35) genome (Fig. ?(Fig.1b).1b). HSV-1 1764/27-/4-/P1-/P2-/CMVGFP/27 is like 1764/27-/4-/P2- (described in reference 35) with the additional deletion of LAP1 sequences (nucleotides 6151 to 8366 and 113273 to 120470) and the insertion of a CMV GFP cassette so as to replace ICP27 and one LAT region between nucleotides 113273 and Rabbit Polyclonal to POU4F3 120470. The DNA structures of all the viruses were confirmed by Southern blot analysis. Cell culture and viruses. Vero cells were cultured in Dulbecco’s Piperidolate hydrochloride modified Eagle’s medium supplemented with 10% fetal calf serum, and BHK and ND7 cells were cultured as previously described (57). The ND7 and BHK cell lines expressing the LAT ORF and LATmut (a mutant LAT ORF in which a frameshift mutation has been inserted Piperidolate hydrochloride at amino acid 163) have been described previously (57); LAT ORF RNA expression was confirmed by Northern blotting using RNA extracted by standard methods (59). Cell lines expressing VP22tag (Invitrogen). Detection was performed with the enhanced chemiluminescence (ECL) system (Amersham). Indirect immunofluorescence and antibodies. Cells were grown on glass coverslips in 24-well dishes. At Piperidolate hydrochloride the times indicated, the cells were washed twice with phosphate-buffered saline (PBS), then fixed either with ice-cold ethanol (for the anti-ICP0 antibody; 4C for 20 min) or with formaldehyde (for the anti-tubulin, anti-promyelocytic leukemia protein [anti-PML], anti-cyclin D3, and anti-ICP8 antibodies; 1% formaldehyde in PBS, room temperature for 10 min; 1% Triton X-100 in PBS, 4C for 20 min), and again washed twice with PBS. Both fixation methods are suitable for use with the anti-antibody. Cells were then blocked with 10% fetal calf serum at room temperature for 15 min, incubated with the appropriate primary antibodies for 60 min at 37C, washed twice with PBS, incubated with the appropriate secondary antibody for 60 min at 37C, and then washed twice again with PBS. Coverslips were mounted by using the ProLong Antifade kit (Molecular Probes, Eugene, Oreg.) to reduce photobleaching. All images were obtained using a Zeiss Axiophot2 microscope and the software supplied. The anti-monoclonal antibody (MAb) (immunoglobulin G1 [IgG1]) (Invitrogen) was used at a 1:500 dilution. The anti-neuron-specific tubulin (111) MAb TUJI (IgG2a) (Covance) was used at a 1:500 dilution. The anti-ICP0 (exon 2) rabbit polyclonal antibody, a gift from Bernard Roizman (University of Chicago) (32), was used at a 1:200 dilution. The anti-PML rabbit polyclonal antibody (Chemicon) was used at a 1:200 dilution. The anti-cyclin D3 rabbit polyclonal antibody (Santa Cruz) was used at a 1:50 dilution. The anti-ICP8 rabbit polyclonal antibody 383, a gift from David Knipe (Harvard Medical School, Boston, Mass.) (9), was used at a 1:200 dilution. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse and rhodamine (tetramethyl rhodamine isocyanate [TRITC])-conjugated swine anti-rabbit antibodies (DAKO) were used at a 1:200 dilution. TRITC-conjugated goat anti-mouse IgG2a and FITC-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates) were used at a 1:100 dilution. PNGase F and -PPase treatment of cell lysates. Samples were prepared from Vero cells infected at an MOI of 5. For peptide-antibody. Initially the LATMAb. (b) Growth curves using wild-type (17+) virus performed.