Nificant levels of the host-viral chimeric transcripts. Because they indicate active transcription upstream of the 5'LTR, these findings suggest that viral transcription is subjected to TI in these cells. Transcriptional interference by the actively transcribed host gene reverses viral transcription from the 5' to the 3'LTR Next, we investigated in detail how active transcription of the upstream P
Ost Microbe. Author manuscript; available in PMC 2014 November 03.Lenasi et al.PageWestern blotting 107 J-Lat or J-Lat shER cells were lysed in 0.8 ml of lysis buffer A (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 2 mM EDTA, 1 NP-40, 0.1 protease inhibitor) for 40 min at 4 . Proteins in the lysates were separated on SDS-PAGE electrophoresis. Western blotting was performed according to the standard pro
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
Lation than the 5'LTR, suggesting that the latter LTR is much stronger subjected to TI. Even if there is transcription from host gene `reading through' both LTRs in some latent cells, this does not diminish the impact of TI on the 5'LTR.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCell Host Microbe. Author manuscript; available in PMC 2014 November 03.Lenasi et al.Page
Ns dormant. In contrast, we demonstrated that TI could be counteracted by specific inhibition of the upstream transcription or by cooperative activation of transcription initiation and elongation from the 5'LTR by the viral and host TFs. In the latter scenario, we envision that RNAPII initiating from the viral promoter competes successfully with the RNAPII that is elongating through the 5'LTR. Bec
Pelleted in a conical tube and washed with cold phosphate-buffered saline. Sonication and immunoprecipitation were performed using Chromatin Immunoprecipitation (ChIP) Assay Kit (Upstate) according to the manufacturer's instructions. Antibodies used are presented in Table S2. As negative control, normal rabbit or mouse serum (Sigma-Aldrich) was used. Appropriate primer pairs (Table S1) were used t
K of the latent provirus in J-Lat 9.2 cells is TI of the 5'LTR caused by the transcription of the host PP5* gene. This interfering transcription results in the lack of Sp1 and the presence of the elongating RNAPII at the 5'LTR. Moreover, TI can be partially rescued by TNF- stimulation, as documented by the appearance of Sp1 and initiating RNAPII at the 5'LTR. The slight enrichment of the RNAPII in
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