Ranscription units (Han et al., 2004; Lewinski et al., 2005). In the present work, we extended this knowledge by a detailed analysis of how TI impacts viral transcription in latent cells. Although TI occurs regardless of the orientation of the two promoters, we studied TI phenomenon in two J-Lat cell lines with the same orientation of the host and viral promoters, which allowed us to detect and an
Effect of Tat on transcription of the provirus. In agreement with the results in Figure 4, expression of Tat alone exerted minimal stimulatory effect on the 5'LTR (Figures 7A-7C, Tat). However, in combination with TNF-, Tat expression activated viral transcription greatly (70-80 of GFP-expressing cells) in all three cell lines (Figures 7A-7C, Tat + TNF-). We further investigated the ability of PM
Ranscription units (Han et al., 2004; Lewinski et al., 2005). In the present work, we extended this knowledge by a detailed analysis of how TI impacts viral transcription in latent cells. Although TI occurs regardless of the orientation of the two promoters, we studied TI phenomenon in two J-Lat cell lines with the same orientation of the host and viral promoters, which allowed us to detect and an
Vels of viral particles, we speculate that average levels of host-viral chimeric transcripts in these cells exceed those in the two J-Lat cell lines. These results thus suggest that TI plays a key role in primary CD4+ T cells. In J-Lat 9.2 and 15.4 cells, the host-viral chimeric transcripts terminated at the pA site in the 5'LTR, but transcripts initiating at the host promoter and terminating at t
Vels of viral particles, we speculate that average levels of host-viral chimeric transcripts in these cells exceed those in the two J-Lat cell lines. These results thus suggest that TI plays a key role in primary CD4+ T cells. In J-Lat 9.2 and 15.4 cells, the host-viral chimeric transcripts terminated at the pA site in the 5'LTR, but transcripts initiating at the host promoter and terminating at t
Ormed using NorthernMax kit (Ambion) according to the manufacturer's instructions. The probes were amplified from cDNA synthesized with MMLV reverse transcriptase (Invitrogen). PCR products were labeled with BioNick Labelling System (Invitrogen). Stimulation of J-Lat cells and detection of activated cells by flow cytometry Tat protein was expressed from pCDNA3 vector using electroporation of 107 J
Tiful in genes and they are recognized by the spliceosome when there is no stronger splice site downstream of them, such aberrant splicing probably takes place in many cases of integration of the viral genome. On one hand, these transcripts reflected active transcription upstream of the provirus in both J-Lat cell lines. On the other hand, LTR sequence in the transcripts enabled us to use them as
Ent (Pharmacia). Isolation of PBLs, their activation with PHA/IL-2 and infection with HIV-1LAI followed by isolation of resting CD4+ T cells by negative selection using magnetic beads (Invitrogen) was performed as previously described (Contreras et al., 2007). Resting CD4+ T cells were treated with 1mM HMBA and 10 ng/ml TNF- for 24 h. Viral release in the supernatant was quantified by p24 ELISA (P