Regulation of HIV-1 provirus transcription

Transcription of the HIV-genome is regulated by the interaction of viral long terminal repeat (LTR) cis-acting sequences with a complex of viral regulatory proteins and cellular transcription factors. The complex interplay between the cellular-, and/or viral proteins and LTR of different subtypes may play a role in the control of HIV-1 latency versus activation. An up/down regulation of transcription could result from a ternary complex formed between a high mobility group (HMG)- like protein and Rel-protein-DNA as in the case of dorsal switch protein (DSP1) which converts Dorsal and nuclear factor kappa-B (NFkB) from transcription activators to repressors as described in Drosophila and human interferon beta (IFN-beta) enhancer. Therefore, the aims of this study were (i) to analyse the polymorphisms in transcription factor binding sites within the LTR in different HIV-1 subtypes, (ii) to study the impact of identified cellular transcription factors such as NFkappaB and upstream stimulatory factor (USF), and/or novel cellular proteins on the proviral transcription (iii) to investigate the role of HMG-1 on HIV-1 transcription in human cells.

The env V3, gag p17 and LTR regions of HIV-] isolates from seven Swedish and 22 African infected individuals were sequenced and phylogenetically analysed. Based on these phylogenetic analysis the sequence variations in the LTRs were linked to the subtype specific sequences (A-G or the intersubtype recombinant strains A/C, A/G, A/D and E/A) in the V3-, and p17 region. Two major subtype C specific sequences were an insertion that created a potential third NFkappaB site and the sequence of the USF binding site (E box) in the negative regulatory element (core-NRE). The LTR regions were then sub- cloned and analysed for promoter/enhancer activity in transient transfection of epithelial (HeLa) cells, and T-cells, in the context of a reporter gene or the complete virus genome. The promoter/enhancer activities of the subtype C LTR were higher than those of the other subtypes suggesting that the potential third NF-kappaB site may confer a higher LTR activity or/and that the subtype C NRE may be less potent. In in vitro binding assays the extra NF-kappaB site was interacted with various recombinant NFkappaB proteins. Furthermore, deletion of this site by PCR directed mutagenesis lead to downregulation of LTR mediated transcription in PMA-stimulated HeLa and T-cells.

The functional importance of the core-NRE was further analysed with regard to USF. USF was found to repress transcription directed from representative HIV-] LTRs of all the subtypes tested in HeLa cells, yet activated transcription in T cells. Mutation of the core-NRE USF site did not affect the cell-specific dual role of USF. In vitro binding assays showed that rUSF 43 interacted with the core-NRE only from subtypes B and C. The dual repressor/activator role of USF may play a role in the control of HIV- I latency versus activation.

HIV- I LTR has emerged as a model system for studying human positive transcription elongation factor b (HP-TEFbn) because it is exquisitely regulated at the level of elongation, via apparent ternary interactions of HIV-1 Tat-TAR-P-TEFb. In addition to previously identified components involved in HP-TEFbN complex, HSP90, CDC37 and a novel protein named MCEF (Major CDK9 Elongation Factor) was identified by immune precipitation from a stable FLAG-tagged-CDK9 expressing cell line. MCEF could be classified as a member of the AF4/FMR2 family of transcription factors and was found to reduce HIV-] replication by more than 50% in HeLa cells. Hence MCEF may contribute to a latent state of HIV- I in cells.

HMG-1 binds to DNA in an independent manner and serves as a DNA-bending, -wrapping, and -looping factor that can be required for transcription, DNA repair, and recombination. Here we showed that transiently overexpressed HMG-1 specifically interacted with LTR and down-regulated HIV-] replication in HeLa cells. Over expression of HMG-1 gave a 2x higher inhibitory activity on a subtype C LTR containing two conserved NFkappaB sites compared to the wt subtype C LTR in HeLa cells. HIV-] subtype C strains may have gained the extra NFkappaB site to overcome an inhibitory effect of HMG-1. As an explanation we suggest that HMG-1 interaction with NFkappaB may brings about a repression state of the chromatin at the LTR by recruiting other proteins necessary for repression. HMG-1 may therefore function as the DSP 1 homologue in human cells as an evolutionary conserved system.

List of scientific papers

I. Naghavi MH, Salminen MO, Sonnerborg A, Vahlne A (1970). "DNA sequence of the long terminal repeat of human immunodeficiency virus type 1 subtype A through G. " AIDS Res Hum Retroviruses 15(5): 485-8
https://pubmed.ncbi.nlm.nih.gov/10195759

II. Naghavi MH, Schwartz S, Sonnerborg A, Vahlne A (1999). "Long terminal repeat promoter/enhancer activity of different subtypes of HIV type 1. " AIDS Res Hum Retroviruses 15(14): 1293-303
https://pubmed.ncbi.nlm.nih.gov/10505678

III. Naghavi MH, Estable MC, Schwartz S, Roeder RG, Vahlne A (2001). "Upstream stimulating factor affects human immunodeficiency virus type 1 (HIV-1) long terminal repeat-directed transcription in a cell-specific manner, independently of the HIV-1 subtype and the core-negative regulatory element. " J Gen Virol 82(Pt 3): 547-59
https://pubmed.ncbi.nlm.nih.gov/11172096

IV. Estable MC, Naghavi MH, Kato H, Xiao H, Qin J, Vahlne A, Roeder RG (2001). "MCEF, the newest member of the AF4 family transcription factors involved in leukemia, is a P-TEFb-associated protein that can repress HIV-1." (Submitted)

V. Naghavi MH, Nowak P, Andersson J, Sonnerberg A, Yang H, Tracey K, Vahlne A (2001). "High-mobility group 1 protein (HMG-1) represses human immunodeficiency virus type 1 (HIV-1) replication." (Manuscript)