Signal transduction and HIV transcriptional activation after exposure to ultraviolet light and other DNA-damaging agents.

Short wavelength (254 nm) ultraviolet light (UVC) radiation was much more potent in activating transcription of human immunodeficiency virus 1 (HIV) reporter genes stably integrated into the genomes of human and monkey cells than ionizing radiation (IR) from a 137Cs source at similarly cytotoxic doses. A similar differential was also observed when c-jun transcription levels were examined. However, these transcription levels do not correlate with activation of nuclear factor (NF)-kappa B and AP-1 measured by band-shift assays, i.e. both types of radiation produce similar increases in NF-kappa B and AP-1 activity, suggesting existence of additional levels of regulation during these responses. Because of the well-established involvement of cytoplasmic signaling pathways in the cellular response to tumor necrosis factor-alpha (TNF-alpha), UVC, and IR using other types of assays, the role of TNF-alpha in the UVC response of HIV and c-jun was investigated in our cell system. We demonstrate that UVC and TNF-alpha activate HIV gene expression in a synergistic fashion, suggesting that it is unlikely that TNF-alpha is involved in UVC activation of HIV transcription in stably transfected HeLa cells. Moreover, maximum TNF-alpha stimulation resulted in one order of magnitude lower levels of HIV expression than that observed after UVC exposure. We also observed an additive effect of UVC and TNF-alpha on c-jun steady-state mRNA levels, suggestive of a partial overlap in activation mechanism of c-jun by UVC and TNF-alpha; yet these responses are distinct to some extent. Our results indicate that the HIV, and to some extent also the c-jun, transcriptional responses to UVC are not the result of TNF-alpha stimulation and subsequent downstream cytoplasmic signaling events in HeLa cells. Additional levels of regulation that do not directly involve the NF-kappa B and AP-1 transcription factors, such as changes in chromatin structure associated with the UV repair process, may also be important for a full transcriptional response of HIV and c-jun to UVC. In addition to the new data, this report also summarizes our current views regarding UVC-induced activations of HIV gene expression in stably transfected cells.

Activation of human immunodeficiency virus gene expression by ultraviolet light in stably transfected human cells does not require the enhancer element.

Ultraviolet light (UV) exposure of cells infected with human immunodeficiency virus type I (HIV) or transfected with HIV reporter genes increases virus-directed gene expression. Here we report the mapping of the UV response on the long terminal repeat (LTR) by using human cells stably transfected with HIV promoter plasmids harboring different mutations and controlling the expression of the chloramphenicol acetyltransferase (cat) reporter gene. Promoter mutation analysis revealed that no specific upstream region of the LTR was associated with UV activation, although a significant decrease was observed with mutations in the basal promoter elements Spl and TATA. Most importantly, UV activation was not diminished by removal of the - 119 to -69 region encompassing the LTR enhancer region or, more specifically, by point mutations in the NF -kappa B binding elements. Consistent with this result, we found that the phorbol ester (PMA) response, which is known to act through the enhancer, occurred independently and was synergistic with the UV response. Removal of the -119 to -69 region did not affect UV activation; however, it resulted in total abrogation of the PMA response. These results suggest that UV activation is distinct from NF -kappa B activation and does not act through the enhancer in stably transfected cells. This is in dramatic contrast to what is found with transient expression analysis of these responses. Lastly, RNA protection experiments revealed that UV may act on preassembled basal transcription complexes by allowing elongation of nascent short mRNAs generated from the LTR.(ABSTRACT TRUNCATED AT 250 WORDS)

Ionizing radiation activates nuclear factor kappa B but fails to produce an increase in human immunodeficiency virus gene expression in stably transfected human cells.

We have investigated the differential effects of ultraviolet light(UV) and ionizing radiation (IR) on human immunodeficiency virus type 1 (HIV) and c-jun expression in HIVcat/HeLa cells. This cell line harbors integrated copies of the chloramphenicol acetyltransferase (cat) gene under control of the HIV promoter. Both UV and IR increased the binding of nuclear proteins to an oligonucleotide spanning the HIV enhancer region nuclear factor kappa B sites, but only UV increased HIVcat steady-state mRNA and CAT activity. By comparison, transcription of the cellular c-jun gene increased after both types of radiation, but UV was at least 5-fold more effective than IR despite the fact that protein binding to an activator protein 1 oligonucleotide increased similarly after both UV and IR. The lack of HIVcat transcriptional response after IR does not appear to be the result of the repressor binding to upstream promoter elements since cells stably transfected with different HIV promoter deletions showed a lack of response to IR distinguishable from that of the intact promoter. While our findings indicate no correlation between increased binding of transcription factors to upstream promoter elements and increased expression of these genes after radiation, we did observe major differences in how UV and IR affected chromatin structure. UV produced extensive global chromatin decondensation, whereas IR did not, as seen in the microscope and determined by the increased susceptibility of chromatin to micrococcal nuclease digestion.(ABSTRACT TRUNCATED AT 250 WORDS)