Persistent transcription of a nonintegrating mutant of simian immunodeficiency virus in rhesus macrophages

Abstract

A nonintegrating mutant, SIVsmD116N, was derived from the infectious pathogenic SIVsmE543-3 clone by introducing an Asp (D) to Asn (N) mutation into the catalytic domain of integrase. Although SIVsmD116N generated all viral proteins following transfection, cell-free virus did not productively infect CEMx174 cells, macaque peripheral blood mononuclear cells (PBMCs) or monocyte-derived macrophages (MDM). Viral DNA and transcripts were observed transiently in SIVsmD116N-infected CEMx174 cells and macaque PBMC but persisted in MDM for as long as 20 days. Circular forms of viral DNA were detected but there was no evidence of integration detected by Alu PCR. We found that SIV D116N mutant remained transcriptionally active and expressed low levels of viral proteins persistently in MDM. These data are consistent with a role for macrophages as a persistent latent reservoir for AIDS viruses. The capacity of nonintegrating SIV to persistently generate viral products in macrophages suggests that nonintegrating lentiviral vectors could be engineered to efficiently and safely express proteins for vaccine purposes.

Figure 1

SIVsmD116N virus can generate all the viral proteins at the same level as WT SIVsmE543. The SIVsmD116N and WT SIVsmE543 clones were transfected into 293T cells to generate cell free viruses. Viruses were concentrated and western blotting was performed to analyze the viral proteins. The proteins shown here are gp120, p66, p55, gp41, gp31, p27 (from top to bottom).

Figure 2

Non-integrated mutant virus SIVsmD116N does not replicate in host cells. Reverse transcriptase assay was carried out to evaluate infectivity of the WT and SIVsmD116N mutant in CEM×174 cells (2A), macaque PBMC (2B) and macaque MDM (2C). The WT virus replicated well in CEM×174 cells, macaque PBMCs as well as in MDM. In contrast, no RT activity was observed in cell-free media following infection with the integrase mutant SIVsmD116N in any of these cell types.

Figure 3

Persistence of unintegrated viral DNA in macrophages. A. Total cellular DNA from WT and D116N infected cells was extracted at different times post infection and PCR amplified. The non-integrated DNA persisted for 20 days and the viral DNA synthesized from SIVsmD116N was at a comparable level with the WT virus. B. Two of the non-integrated viral DNA forms, both the 1-LTR-circle and 2-LTR-circle were found to be present and persist with viral infection. 1-LTR circles were much more abundant than 2-LTR circles. The D116N infected cells had higher levels of 2-LTR-circles than the wild type infection. Cellular actin DNA was co-amplified from each sample to ensure equal amount of cellular DNA was used. C. Uninfected cells and heat-inactivated virus infected cells were used as control to compare with WT or D116N infected cells.

Figure 4

The SIVsmD116N mutant does not integrate into cell chromosome. CEM×174 cells and macaque macrophages were infected with SIVsmE543 and SIVsmD116N viruses. Total cellular DNA harvested was subjected to Alu-PCR amplification. A. No integrated viral DNA was amplified in up to 25×10⁴ CEM×174 T cells infected with integrase mutant SIVsmD116N, while integrated DNA was amplified well in just 1×10³ CEM×174 T cells infected with SIVsmE543. B. In D116N infected macrophages, Alu-PCR demonstrated that no integrated viral DNA persisted for 20 days until the termination of the cell culture. While in WT infected macrophages, integrated DNA amplified well from day 1 to day 20. For the “Alu-” controls without first round PCR, no product was amplified.

Figure 5

Persistence of transcriptional activity from non-integrated viral DNA in macrophages. A. Total cellular mRNA from WT or D116N infected macrophages was purified and amplified with RT-PCR. Multiple viral transcripts including all classes of viral splicing isoforms were detected. The non-integrated viral DNA in macrophages remained transcriptionally active for 20 days. RNA expression level of non-integrated D116N mutant was much lower than that of wild type. PCR products from RT-PCR were shown in the figure using primers specific for Tat/Rev/Nef, Env, Gag-pol, and Vif (from top to bottom). Nef, Tat and Env were the predominant transcripts in D116N infection. Expression level of Rev transcript was very low and Vif diminished in D116N infected macrophages over time. The PCR results were compared between WT and SIVsmD116N based upon the reference of co-amplified cellular β-actin transcripts. B. To confirm that the RT-PCR products were the result of SIV viral gene expression, we add uninfected cells and heat-inactivated virus infected cells as controls. No SIV transcripts were amplified from uninfected cells or heat-inactivated virus infected cells. The appropriate transcripts were detected in WT and D116N infected cells, but not in uninfected cells or cells infected with heat-inactivated virus.

Figure 6

Proteins synthesis from SIVsmE543 and SIVsmD116N viruses in macrophages detected by western blotting. A. Macrophages were cultured and divided into 3 groups, infected with SIVsmE543 or SIVsmD116N, and uninfected cells. Cells were harvested at different times and western blot was performed using plasma from a SIV-infected rhesus macaque E544 as primary antibody. In WT infected cells, viral proteins including gp160, gp120, p66, p55, gp41, p31 and p27 were strongly and persistently expressed from day 1 to day 18. In D116N infected cells, expression of viral p66, p55, p31 proteins were also observed, although the expression levels were much lower than those in wild type infection. The proteins also expressed persistently for 18 days in D116N infected cells. B. Macrophages were infected with WT SIVsmE543, SIVsmD116N, heat-inactivated WT, or heat-inactivated D116N viruses and harvested at day 3 and 5 for western blotting with a SIV anti-p27 antibody. P27 was detected in both D116N and WT infected cells at day 3 and 5 post infection, with much higher levels in the WT infection.