The kappaB sites in the human immunodeficiency virus type 1 long terminal repeat enhance virus replication yet are not absolutely required for viral growth

B K Chen, M B Feinberg, D Baltimore
1997 Journal of Virology  
The dependence of human immunodeficiency virus type 1 (HIV-1) on its NF-B binding sites (B sites) for replication in transformed and primary T-cell targets was examined by infecting cells with HIV-1 reporter viruses containing B site enhancer mutations. Viral transcription was measured either with luciferaseexpressing HIV-1 that infects for a single round or by flow cytometric analyses with HIV-1 expressing placental alkaline phosphatase (PLAP) or green-fluorescent protein (GFP). Both PLAP-and
more » ... FP). Both PLAP-and GFP-expressing viruses spread from cell to cell and allowed analysis of viral gene expression patterns in single cells. Infection of a panel of T-cell lines with different basal levels of NF-B demonstrated a direct correlation between the amount of constitutive nuclear NF-B and the degree to which a wild-type virus outperformed B site mutants. One T-cell line with a constitutively high level of nuclear NF-B, PM1, showed a 20-fold decrease in transcription when its B sites were mutated. In contrast, in a T-cell line with a low basal level of NF-B, SupT1, mutation of the B site in the enhancer had no effect on viral transcription or growth rate. Phytohemagglutinin-activated peripheral blood mononuclear cells showed a large dependence on the B sites for optimal virus growth. Viruses without marker genes corroborated the finding that mutations to the B sites impair virus production in cells with a high basal level of NF-B. These data show that in T cells, HIV-1 can use NF-B to enhance its growth but the virus is clearly able to grow in its absence. * Corresponding author. 5495 on May 9, 2020 by guest Downloaded from MATERIALS AND METHODS Cells. SupT1 is a CD4 ϩ CD8 ϩ CD3 Ϫ T-leukemic line isolated from a pleural effusion of a non-Hodgkin's lymphoma patient (27) . Jurkat clone E6 is a CD4 ϩ CD3 ϩ T-leukemic line derived by limit dilution cloning over macrophages (29). PM1, a derivative of HUT78, is a CD4 ϩ T-leukemic line that has been found to support infection of a wide variety of primary and laboratory-adapted isolates of HIV-1 (18). SupT1 from James Hoxie, Jurkat clone E6 from Arthur Weiss, and PM1 from Paolo Lusso and Marvin Reitz were obtained from the AIDS Reagent Repository. PBMC were purified twice by centrifugation over a Ficoll-Hypaque (Pharmacia) gradient and incubated in RPMI medium containing 10% fetal calf serum, either with or without 2 g of phytohemagglutinin (PHA) per ml for 36 to 40 h prior to infection and maintained in medium containing 20 U of recombinant human IL-2 (Genzyme) per ml. Electrophoretic mobility shift assays (EMSA). Nuclear extracts for EMSA were prepared with a modification of the protocol of Dignam et al. (7, 17) . Briefly, 20 ϫ 10 6 cells, treated with 20 ng of recombinant human TNF-␣ (Genzyme) per ml or untreated, were washed once with ice-cold phosphate-buffered saline, resuspended in ice-cold buffer A (10 mM N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid [HEPES, pH 7.9], 1.5 mM MgCl 2 , 10 mM KCl), and then lysed by addition of an equal volume of buffer A containing 0.2% Nonidet P-40. Nuclei were pelleted by low-speed centrifugation and resuspended in buffer C (25% glycerol, 20 mM HEPES [pH 7.9], 1.5 mM MgCl 2 , 0.6 M KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol) with vigorous pipetting. Extracts were cleared by high-speed ultracentrifugation and stored at Ϫ70°C. EMSA was performed as described previously (17) , with a hairpin consensus HIV-immunoglobulin probe (10). Viruses. Viruses were produced from molecular clone HXB-2D in proviral vector R7 (9). PCR-mediated, site-directed mutagenesis was performed by amplifying a 772-bp fragment encompassing the 3Ј LTR region and subcloning the recombinant fragment into the place of the 3Ј LTR via a unique XhoI site and a recombinant AscI site engineered into the 3Ј end of the proviral sequence. The primers used for PCR amplification were 5Ј-GATGGGGTGGGAGCAGCAT CT-3Ј and 5Ј-ATGCTCTAGAGGCGCGCCAGAGTCACACAACAGACGG GCA-3Ј. The resulting provirus contains a deletion of a portion of the sequence flanking the 3Ј end of the provirus. LTR sequences were confirmed by dideoxy sequencing (ABI Sequenator; Applied Biosystems). The HXB-⌬B-alt provirus was made by replacing the XhoI-SacI fragment containing the 3Ј HIV-1 LTR in proviral clone R7 with a mutant LTR from proviral clone BH8 with site-directed mutations to both B sites. Construction of HXB-Luc (luciferase) and HXB-PLAP (placental alkaline phosphatase have been described previously (5, 6). HXB-GFP (greenfluorescent protein) was engineered in an analogous fashion to the luciferaseand PLAP-expressing viruses, by using a modified allele of the Aequorea victoria GFP from Clontech, Enhanced GFP. Enhanced GFP was PCR amplified and cloned into unique NotI and XhoI sites in HXB-Luc. HXB virus that has not been engineered to express marker genes does express a functional repaired nef open reading frame. Virus was produced by calcium phosphate methods and harvested at 48 h following transfection as previously described (5). Virus was quantitated by p24 enzyme-linked immunosorbent assay (19) to normalize all infections to equivalent antigenic input. Luciferase assays and flow cytometry. Luciferase assays were performed as described previously (6) . Flow cytometry was performed on a FACScan (Becton Dickinson) as previously described (5). A modified allele of GFP which has had its coding sequence changed to allow optimal translation in mammalian cells was obtained from Clontech and detected by FACScan on the FL1 channel, 530/30 nm. Reverse transcriptase-mediated PCR of cultured HIV-1. Cell-free supernatants precleared of cellular debris by low-speed centrifugation were ultracentrifuged at 28,000 rpm (Beckman SW60.1) to pellet virions, and viral RNAs were prepared with Tri-Reagent (Molecular Research Center) in accordance with the manufacturer's instructions. Random hexamer (for the U3 region) and oligo(dT) (for the U5 region) primers were used to prime cDNA syntheses. The U3 and U5 regions were amplified with 26 cycles of PCR with native Pfu polymerase (Strategene) from the viral cDNAs with the following
doi:10.1128/jvi.71.7.5495-5504.1997 fatcat:odwq456mmvel7eiqem6yfkjo74