Ly 20 activation of viral transcription (see Figure 7) already precludes detection of the upstream transcription. This finding is not surprising, as we demonstrated that there is 11-fold or 38-fold more viral mRNA than *mRNA in TNF- treated cells (Figure 2D, bars 3 and 4). Predictably, our control experiments demonstrate that the ratio between the Rev- and Env-containing transcripts was 1 in untr
Tream of the 5'LTR Existence of the host-viral chimeric transcripts that originate from a host promoter and include 5'LTR could be useful for identifying ongoing transcription upstream of the 5'LTR independently of the site of viral integration. Therefore, we next devised an assay, which determines the ratio between transcripts containing LTR sequences and those containing sequences present solely
Stream transcription could be detected independently of the viral integration site. HIV-infected primary CD4+ T cells contain significant levels of transcription upstream of the 5'LTR To identify transcription upstream of the 5'LTR in HIV-infected peripheral blood lymphocytes (PBLs), we used the above method. After activation of HIV-infected PBLs with IL-2 and PHA, we rested them and isolated CD4+
Es obtained with the LTR-specific primers represent a sum of transcripts originating from the host promoter and of viral transcripts, which also contain LTR sequence at their 3' ends. Therefore, the measured ratio corresponds to the equation a+b/b, where `a' is the number of host-viral chimeric transcripts and `b' the number of viral transcripts (see Supplemental text 2). First, we determined this
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
Nce it has been demonstrated that transcription from the PP5 promoter could be reduced through inhibiting estrogen receptor (ER) (Urban et al., 2001). Therefore, we established J-Lat 9.2 and control J-Lat 8.4 cell lines that stably express miRNA-adapted shRNA (shRNAmir) against the ER mRNA (J-Lat 9.2 shER and J-Lat 8.4 shER, respectively). In both J-Lat shER cell lines, the ER levels were decrease
Ipt Author Manuscript Author Manuscript Author ManuscriptExtended Data Figure 2. Development of anti-MAb immune responses in recipients of antiHIV-1 bNAbsNature. Author manuscript; available in PMC 2016 November 29.Gautam et al.Pagea, b, c, d, Longitudinal analysis of anti-VRC01, anti-3BNC117, anti-10-1074 and antiVRC01-LS antibody responses respectively, following a single intravenous infusion of
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