Tag-expressing cells support high levels of transient gene expression from D116A.oriT. (A) CV-1 cells infected with the WT (□) virus or the D116A (⋄) or D116A.oriT (○) mutant virus were analyzed for GFP expression at the indicated times. (B) CV1/EBNA1 cells infected with the WT (□) virus or the D116A (⋄) or D116A.oriP (•) mutant virus were analyzed as described for panel A. (C) COS-1 cells infected as described for panel A. (D) Raji T4/R5 cells infected as described for panel B. Similar gene expression profiles were obtained in three independent sets of experiments.

N/N.oriT replication in 1D Jurkat cells. Supernatants of cells infected with the WT virus (□) or the N/N (×) or N/N.oriT (⋄) mutant virus were analyzed for RT activity at the indicated times. Similar results were observed in three independent experiments.

N/N.Tag-oriT replication in Jurkat cells requires functional Tag protein. (A) Jurkat cells infected with WT (□), N/N.Tag-oriT (▵), N/N.U19-oriT (○), or mock-treated (+) supernatant were analyzed for RT activity at the indicated times. (B and C) Jurkat cells infected with the indicated levels of virus derived from peak days of WT and N/N.Tag-oriT replication in panel A. Arrows in panels A and C indicate cultures lysed by Hirt extraction. Similar replication curves were obtained following a second set of primary and secondary infections of Jurkat cells. pssg, passage.

N/N.oriT replication in the absence of normal provirus formation. (A) 1D Jurkat cells (1.6 × 10⁸) infected with 8.0 × 10⁸ RT cpm of WT (W), N/N (N), N/N.oriT (T), or mock-treated (m) supernatant for 4 h at 37°C were lysed at the indicated times, and unintegrated nuclear DNA was detected by Southern blotting. Whereas the first four lanes were exposed to X-ray film overnight, the remaining lanes were exposed to film for 3 h. Linear DNA was not detected in this analysis, likely because of its loss during alkaline lysis (3). (B) Inverse PCR. Lanes: 1, genomic DNA isolated from mock-infected cells; 2, 1 ng of plasmid pNL4-3 (1) mixed with 10 μg of mock-infected genomic DNA prior to digestion with HindIII; 3 and 4, genomic DNA isolated at 3 dpi with the indicated viruses; 5, mock-infected DNA mixed with Hirt supernatant from approximately 10⁴ N/N.oriT-infected 1D Jurkat cells prior to HindIII digestion; 6, mock-infected DNA plus 1 ng of pN/N.oriT. Whereas the gel in lane 3 was exposed to film overnight, the gel in lanes 1, 2, and 4 to 6 was exposed to film for 5 h. The migration positions of the 1,102-, 358-, and 261-bp internal PCR products are indicated on the right; migration positions of molecular mass standards (sizes in base pairs are shown) are indicated on the left. (C) Inverse PCR strategy. Solid lines, HIV-1 DNA; open boxes, HIV-1 LTRs; vertical arrows, relevant HindIII sites; dashed lines, cellular DNA; black box, oriT; P, location of PCR primers (not drawn to scale); X, variably sized products indicative of provirus distribution. Whereas the HindIII sites at positions 8,131 and 9,606 in WT HIV-1_NL4-3 yielded a 1,102-bp internal PCR product (38), oriT introduced an additional HindIII site, thereby reducing the size of the WT product to358 bp. The WT and N/N.oriT viruses each yielded a 2-LTR-specific 261-bp product (38).

WT and N/N.Tag-oriT viruses support similar kinetics of gene expression in single-round infections. (A) RT activity (▪) was plotted against the WT Alu RQ-PCR (⋄) signal from Fig. 8A. (B) Comparison of WT RT (▪) and LRT (○) profiles. (C) RT activity (▪) plotted against the N/N.Tag-oriT RQ-PCR Alu signal (⋄) from Fig. 8C. (D) N/N.Tag-oriT RT (▪) and LRT (○) profiles. Although N/N.Tag-oriT and the WT supported similar kinetic profiles of gene expression, we noted that the level of N/N.Tag-oriT expression was approximately 50% of that of the WT. We infer that this reduced level of mutant gene expression was in large part responsible for the delay in N/N.Tag-oriT replication observed in spreading infection assays (Fig. 5). AU, arbitrary units.

SV40 Tag and oriT yield high levels of transient IN mutant expression in nonpermissive human T cells. (A) Jurkat cells infected with the WT (□) virus or the D116A (⋄) or D116A.oriT (○) mutant virus were analyzed for GFP activity at the indicated times. (B) Jurkat TagC15 cells infected as described for panel A. Nearly WT levels of D116A.oriT expression in Jurkat TagC15 cells were observed in two independent experiments.

Spreading N/N.oriT replication in 1D Jurkat cells. (A) 1D Jurkat cells (2 × 10⁶) infected with 10⁶ RT cpm of WT (▵), N/N.oriT (▴), or mock-treated supernatant (+) were assayed for RT activity at the indicated times. (B) RT assays of cell supernatants following coculture of 2 × 10⁵ cells from panel A with 1.8 × 10⁶ Jurkat TagC15 cells. (C and D) RT assays following coculture of 2 × 10⁵ cells from the experiment whose results are shown in panel A with 1.8 × 10⁶ Jurkat and 1D Jurkat cells, respectively.

Functional analysis of N/N.Tag-oriT replication. (A) Agarose gel stained with ethidium bromide. Lanes: 1, IN-containing pol fragment amplified from plasmid pN/N.Tag-oriT; 2, PCR of Hirt supernatant from approximately 5 × 10⁴ second-round N/N.Tag-oriT-infected cells (Fig. 5C, arrow); 3, same as lane 2 except that lysate was treated with DpnI to degrade residual plasmid DNA prior to PCR; 4, Tag amplified from pN/N.Tag-oriT; 5 and 6, minus and plus DpnI treatment of first-round Hirt supernatant (Fig. 5A, arrow), respectively; 7 and 8, minus and plus DpnI treatment of second-round lysates (Fig. 5C, arrow), respectively. (B) Ethidium bromide-stained polyacrylamide gel. Lanes: 1, oriT amplified from pN/N.Tag-oriT; 2 and 3, minus and plus DpnI treatment of first-round lysates, respectively; 4 and 5, minus and plus DpnI treatment of second-round lysates. Whereas migration positions of PCR products are marked to the right of each gel, sizes of molecular mass standards in kilobases are marked to the left.

Real-time profiles of HIV-1 LRT, 2-LTR, and Alu RQ-PCR DNAs in WT-, N/N-, and N/N.Tag-oriT-infected cells. (A) Total cellular DNA was isolated from WT-infected Jurkat cells at the indicated times. Levels of LRT (♦), 2-LTR (□), and Alu (▴) RQ-PCR products were normalized to ERV-3. (B and C) RQ-PCR values for N/N- and N/N.Tag-oriT-infected cells, respectively, were determined as described for panel A. Error bars indicate variation between duplicate RQ-PCR assays. AU, arbitrary units.

Efficient N/N.Tag-oriT replication in MDM requires Tag DNA replication and transforming activities. (A) MDM infected with macrophage-tropic WT (□), N/N (⋄), N/N.Tag-oriT (○), N/N.GKH-oriT (▵), N/N.D402H-oriT (▴), N/N.U19-oriT (▪), or mock-treated supernatant (×) were assayed for RT activity at the indicated times. Similar to N/N.GKH-oriT, neither N/N.E107K-oriT nor N/N.C105G/E107K-oriT supported detectable levels of HV-1 replication in MDM (not shown). (B) Growth curves in panel A replotted to highlight the various IN mutant viruses.