The HIV-1 nucleocapsid protein does not function as a transcriptional activator on its own cognate promoter
ABSTRACT The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) is a multifunctional, zinc finger-containing protein known to be involved in almost every step of the viral life cycle. We therefore examined the effects of NC in vivo as a transcription activator on the basal transcriptional activity of the HIV-1 U3 and Rous sarcoma virus (RSV) promoters, as well as HIV-1 long terminal repeats (LTRs) such as the U3R and U3RU5 regions, using promoter-fused reporter gene assays, Western blot analyses, and quantitative real time-polymerase chain reaction. From these studies, we found that the basal transcriptional levels of the HIV-1 U3 and RSV promoters were barely enhanced by the presence of NC. Placing the U3R region upstream of reporter genes greatly increased transcriptional activity compared to that of the U3 promoter alone, and such activity was further increased by Tat expression. However, neither transcription driven by U3R itself nor Tat-mediated transcriptional activation of the U3R was further increased by the addition of NC. Similar results were also observed with U3RU5 of the HIV-1 LTR region in the presence of either NC or Gag protein. Thus, these results indicate that the HIV NC protein is unable to act as a transcriptional activator on its cognate and possibly other retroviral promoters.
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ABSTRACT: Despite inherent limitations, the ease and rapidity of their use make transiently expressed reporter gene assays the most frequently used techniques for analyzing promoters and transcriptional regulators. The results of transient reporter gene assays are generally accepted to reflect transcriptional processes correctly, though these assays study regulatory sequences outside of the chromosomal environment and draw conclusions on transcription based on enzyme activity determination. For transient reporter gene assays, often more than one promoter is introduced into one cell. In addition to the one driving the primary reporter gene expression, a further one might serve to ensure the production of an internal control second reporter or/and a trans-acting factor. We demonstrate here by various examples that interference between physically unlinked promoters can profoundly affect reporter expression. Results of reporter gene assays performed by combinations of the cytomegalovirus promoter and various other promoter constructs (human immunodeficiency virus [HIV], Human T-cell Leukemia Virus Type I (HTLV-I), NF-κB-responsive, and p53-responsive) and trans-activator factors (HIV-Tat and p53) in different host cell lines (U2OS, HeLa, and L929) prove that interference between active transcription units can modify transcription responses dramatically. Since the interference depends on the promoters used, on the amount of transfected DNA, on the host cells, and on other factors, extra caution is required in interpreting results of transient reporter gene assays.DNA and cell biology 09/2012; 31(11):1580-4. DOI:10.1089/dna.2012.1711 · 1.99 Impact Factor