Since previous studies of HIVD116N demonstrated persistence and expression in resting T cells, we chose to focus on macrophages, another non-dividing target of HIV/SIV. PCR was used to investigate whether SIVsmD116N was capable of entering and replicating in monocyte derived macrophages (MDM) in vitro. Total cellular DNA from WT and SIVsmD116N infected macrophages was isolated at different times post infection and PCR amplified with primers specific for SIV late DNA, 1-LTR-circles and 2-LTR-circles DNA. Production of SIV full length DNA was indicated by the detection of a LTR product using SIV/DNA-5 and SIV/DNA-3 primers (see Table 1). Circular viral DNA forms were also detected using SIV/Circle-5′ primer and SIV/Circle-3′ primer. Based on detection of full length viral DNA, the SIVsmD116N mutant was able to synthesize viral DNA in MDM with equal efficiency to the WT virus. Viral DNA synthesized from SIVsmD116N persisted for 20 days (the termination of culture) at a level comparable with the WT virus (Fig. 3A). In addition, 1-LTR-circles and 2-LTR circles were found to be present and persisted in both WT and SIVsmD116N infections, and 1-LTR circles were much more abundant than 2-LTR circles. Although this assay was not quantitative, the SIVsmD116N infected cells appeared to have significantly higher levels of 2-LTR-circles than the WT infected cells (Fig. 3B). This result is consistent with previous research in HIV-1 non-integrating mutant (Wiskerchen and Muesing, 1995). To confirm that the PCR products came from SIV viral DNA rather than residual plasmid DNA, we used cells infected with heat-inactivated (56°C for 60 min) as a control. As shown in Fig. 3C, no products were detected in PCR reactions for SIV DNA or LTR circles from cells infected with heat-inactivated virus at 2 days post-infection, whereas these products were readily detected in the untreated virus infection. These results indicated that the viral DNA detected by PCR came from replicating virus rather than residual plasmid DNA from the transfection.

To determine whether SIVsmD116N was capable of integration into the host genome, we used an Alu sequence-based PCR strategy (Zhuge et al., 2001). The Alu sequence is a ubiquitous repeat element found in the human/monkey genome but absent in the HIV/SIV genomes which can be used to specifically demonstrate whether viral DNA is integrated into the host chromosome. As expected, DNA was amplified from as few as 10³ CEM×174 cells infected with SIVsmE543-3, consistent with the presence of proviral DNA in these cells (185 bp product). In contrast, no product was detected in up to 2.5×10⁴ CEM×174 T cells infected with SIVsmD116N virus (Fig. 4A). Similarly, no integrated viral DNA was observed in SIVsmD116N infected macrophages, while in WT infected macrophages, integrated viral DNA persisted until the termination of the cell culture at 20 days (Fig. 4B). These results suggested that SIVsmD116N is not capable of integration into genomic DNA in either T cells or macrophages.

To determine transcriptional activity of non-integrated viral DNA in macrophages, total cellular RNA was isolated and amplified by semi-quantitative RT-PCR at different times post infection (Wu and Marsh, 2001). Briefly, reverse transcription of cellular RNA was accomplished with random decamers as the first-strand primers. Following cDNA synthesis, PCR was carried out using primers to detect the specific messages of SIV using the primers detailed in Table 1. Primer combinations used the same forward primer, SIV/5′ and four different reverse primers, SIV/nef-tat-rev, SIV/3′env, SIV/3′vif and SIV/3′gag-pol to detect multiply, singly and unspliced messages. For relative quantification of RT-PCR, cellular β-actin transcripts (294 bp) were co-amplified using QuantumRNA β-actin Internal Standards (Ambion, Austin, TX), with a ratio of 3/7 for actin primer/competitor. The PCR results were compared between WT and SIVsmD116N based upon the reference of co-amplified cellular β-actin transcripts. Consistent with the persistence of non-integrated DNA, the viral transcription in SIVsmD116N infected macrophages was also persistent. In contrast to the transient presence of viral DNA and its transcripts in T cells (Wu and Marsh, 2003b), the non-integrated viral DNA in macrophages remained transcriptionally active until the termination of the cultures at 20 days. However, RNA expression levels of the SIVsmD116N mutant were much lower than that of WT virus. As shown in Figure 5A, PCR amplification with primers specific for different viral transcripts demonstrated that Nef, Tat and Env were the predominant transcripts in SIVsmD116N infection. Expression level of the Rev transcript was very low and Vif message also declined over time in SIVsmD116N infected macrophages. To make sure the RT-PCR products came from SIV viral gene expression, we used uninfected cells and heat-inactivated virus infected cells as controls. As shown in Fig. 5B, these controls were negative in contrast to amplification from cells infected with untreated virus. These results indicated that the viral gene expression detected by RT-PCR came from viruses instead of any residual DNA plasmid.

The SIVsmD116N and WT SIVsmE543-3 clones were transfected into 293T cells to generate cell free viruses using FuGENE transfection reagent (Roche, Indianapolis, IN) according to the manufacturer’s instructions. Cell free virus supernatants used for subsequent infections were treated with DNase I (Invitrogen, Carlsbad, CA) at 25°C for 10 min. to remove contaminating plasmid DNA. CEM×174 T cells, PBMC, and MDM were infected with cell free supernatant normalized for p27 antigen content from 293T cells transfected with SIVsmD116N or SIVsmE543 viruses (a multiplicity of infection 0.2). Cells were incubated with virus for 2–3 hours, then pelleted and resuspended in fresh culture medium. Virus replication was detected by ³²P reverse transcriptase (RT) assay as previously reported (Sears, Repaske, and Khan, 1999). For experiment control, SIVsmD116N or SIVsmE543 viruses were heated on 56°C for 60 min to be inactivated and then were used to infect cells.

To obtain MDMs, PBMCs were separated from whole blood by Percoll density centrifugation and were cultured in 48-well plates in RPMI 1640 medium (Life Technologies, Rockville, MD) containing 15% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, Utah), 10% human type AB serum (Sigma, St.Louis, MO) and 500 units/ml macrophage colony-stimulating factor (M-CSF, R&D systems, Minneapolis, MN) for five days. Cells were washed 3–5 times with pre-warmed RPMI 1640 medium to remove non-adherent cells. Adherent cells were maintained in fresh RPMI 1640 medium containing 15% heat-inactivated FBS, 5% human type AB serum and 200 units/ml M-CSF for two days, after which they were infected with viruses.

Total cellular DNA and RNA were purified using SV total RNA isolation kit (Promega, Madison, WI, USA), as recommended by the manufacturer. The primer sequences used to detect viral RNA and DNA are listed in Table 1. For the detection of full-length viral DNA by PCR, forward SIV/DNA-5 primer and anti-sense SIV/DNA-3 primer were used. PCR was carried out in 1 x Ambion PCR buffer, 125μM dNTP, 50 pmol each primer, 1U SuperTaq Plus (Ambion, Austin, TX) with 30 cycles of 94°C for 20s, 60°C for 30s, 68°C for 40s. Both the 1-long terminal repeat (LTR) circles and 2-LTR-circles, SIV/Circle-5′ primer and SIV/Circle-3′ primer were used for detection of circular viral DNA forms. For detection of viral RNA species, reverse transcription of cellular RNA was accomplished with random decamers as the first-strand primers. Following cDNA synthesis, PCR was performed using SIV/5′ primer and SIV/nef-tat-rev primer to amplify doubly spliced viral transcripts Nef, Tat and Rev. For relative quantification of RT-PCR, cellular β-actin transcripts were co-amplified using QuantumRNA β-actin Internal Standards (Ambion, Austin, TX), with a ratio of 3/7 for actin primer/competitor. For analysis of singly spliced viral transcripts such as the Env transcript, SIV/5′ primer and SIV/3′ env primer were used, whereas for the Vif transcript, SIV/5′ primer and SIV/3′ vif primer were used. For analysis of Gag-Pol transcript, SIV/5′ primer and SIV/3′ gag-pol primer were used.

To detect SIV integration into the host genome, we used an Alu sequence-based PCR strategy to amplify the junction between the nearest Alu sequence and the SIV LTR. Nested PCR was used for Alu-PCR, with Alu 1-1 primer corresponding to human Alu sequence position 172–196, and Alu 1–2 primer located in the U3 region (bp230-207) of SIV LTR (Zhuge et al., 2001). Following the first round of Alu-PCR, a second round of PCR was carried out by using a 1:500 dilution of the first round PCR product as a template and the LTR specific primers Alu 2-1 and Alu 2-2 (Table 1). The amplification conditions for Alu-PCR were 35 cycles of 94°C for 20s, 65 ° C for 30s, 68° C for 40s. To ensure that the second round PCR products were derived from first round PCR product (integrated DNA) rather than non-integrated DNA carry-over, a 1:500 dilution of the original DNA samples were used as templates for second round PCR as a negative control.