Clonal expansion of SIV-infected cells in macaques on antiretroviral therapy is similar to that of HIV-infected cells in humans

Abstract

Clonal expansion of HIV infected cells plays an important role in the formation and persistence of the reservoir that allows the virus to persist, in DNA form, despite effective antiretroviral therapy. We used integration site analysis to ask if there is a similar clonal expansion of SIV infected cells in macaques. We show that the distribution of HIV and SIV integration sites in vitro is similar and that both viruses preferentially integrate in many of the same genes. We obtained approximately 8000 integration sites from blood samples taken from SIV-infected macaques prior to the initiation of ART, and from blood, spleen, and lymph node samples taken at necropsy. Seven clones were identified in the pre-ART samples; one persisted for a year on ART. An additional 100 clones were found only in on-ART samples; a number of these clones were found in more than one tissue. The timing and extent of clonal expansion of SIV-infected cells in macaques and HIV-infected cells in humans is quite similar. This suggests that SIV-infected macaques represent a useful model of the clonal expansion of HIV infected cells in humans that can be used to evaluate strategies intended to control or eradicate the viral reservoir.

Fig 1. SIV integration sites in human and macaque PBMCs and HIV integration sites in human PBMCs.

We prepared large integration site libraries from SIV infected human and macaque PBMCs, and we previously prepared a large library from HIV infected human PBMCs. To permit a direct comparison of the integration site data, the SIV/macaque integration sites were remapped onto the human genome (hg19, see text). Numbered chromosomes are indicated and the frequency of integrations in different loci is indicated the heights of the peaks, shown in green for HIV integrations in human PBMC, orange for SIV integrations in human PBMC and red for SIV integrations into macaque PBMC, mapped onto the corresponding loci in the human genome.

Fig 2. Both HIV and SIV preferentially integrate their DNA into highly expressed genes.

Genes were placed in bins of 100, based on the levels of RNA in stimulated human PBMCs. Panel A shows the relative levels of RNA in each bin. Panel B shows the distribution of HIV integration sites in human PBMCs, placing the genes in the same bins as in panel A. Panel C shows the distribution of SIV integration sites in human PBMCs, using the same bins for the genes. Panel D shows the distribution of SIV integration sites into macaque PBMCs (the integration sites used in the analysis were mapped to the human genome).

Fig 3. Preferred sites for SIV integration in macaque PBMCs and HIV integration in human PBMCs can be used to link syntenic regions of the human and macaque genomes.

There are large syntenic blocks that can be used to connect homologous regions of the human genome (23 pairs of chromosomes) and the macaque genome (21 pairs of chromosomes). Regions of the macaque genome into which SIV preferentially integrates in PBMCs can be linked to the corresponding syntenic regions of the human genome by connecting the regions of the two genomes into which HIV and SIV preferentially integrate.

Fig 4. SIV and HIV preferentially integrate into regions that contain highly expressed genes.

The figure shows a region of human chromosome 11 approximately 6 megabases in length that is centered around the long noncoding gene NEAT1. The top panel shows the distribution of 100,000 HIV integration sites in human PBMC, chosen at random from the 385,000 member library we prepared (reducing the number of HIV/human PBMC integration sites makes it easier to compare the data). The proviruses that are inserted in the same orientation as the numbering of the chromosome are shown in red, those in blue are in the opposite orientation. The second panel shows the human PBMC RNA-Seq data, mapped to the human genome, on a log scale. The third panel shows the distribution of SIV integration sites in human PBMC. The fourth panel shows RNA-Seq data from the macaque PMBCs, mapped to the human genome, and plotted on a log scale. The bottom panel shows the distribution of SIV integration sites in macaque PBMCs, mapped onto the human genome.

Fig 5. The overall distribution of SIV integration sites in macaque PBMC infected in vitro is similar to the distribution of integration sites in samples taken from infected macaques pre-ART (both 2-week and 4-week samples) and on-ART.

For the in vivo pre-ART and on-ART analysis, the data from all the infected macaques was combined. Genes were placed in bins of 100 based on the frequency of integration sites in the genes in SIV infected macaque PBMC. Each point on the Y axis represents the cumulative fraction of integration sites in all of the bins to the left of that point of the X axis. The in vitro integration site data are shown in blue, the in vivo pre-ART data are shown in yellow, the in vivo on-ART data are shown in green, and a computer-generated random control data are shown in gray.

Fig 6. The overall distribution of SIV integration sites in macaque PBMC infected in vitro, and mapped to the macaque genome, is similar to the distribution of the SIV integration sites in samples taken from all of the SIV infected macaques both pre-ART (both 2-week and 4-week data) and on-ART.

The integration site data from the macaque PBMC infected in vitro are shown in green. The combined pretherapy integration site data are shown in orange, and the combined on-ART integration site data are shown in red.