Genetic variation within the HIV-1 genome is a naturally occurring phenomenon because of the low fidelity of reverse transcriptase as well as selective pressures present within the host [17, 18]. These viral quasispecies are potentially able to establish niches and reservoirs within HPCs, cells of the monocyte-macrophage lineage, as well as T cells that can reseed infection into the periphery and CNS of infected patients and possibly other end organs as well [20–28]. Nucleotide changes within the NF-κB-proximal C/EBP and Sp transcription factor binding sites within the viral promoter have been observed in both the pre-HAART and HAART eras and have been shown to guide differential regulation of viral expression by the LTR [20–22, 55–57]. Interestingly, the prevalence of the 3 T, 5 T, and 3T5T LTR variants in the HAART era, at least in the DrexelMed HIV/AIDS Genetic Analysis Cohort is lower when compared to the levels observed from published studies in the pre-HARRT era [23–27, 37]. However, the DrexelMed HIV/AIDS Genetic Analysis Cohort is overall much healthier because of the prolonged use of combination antiretroviral therapy. However, we hypothesize that the prevalence of the 3 T, 5 T, and 3T5T LTR variants will become more prevalent with the development of greater disease severity across this HIV-1-infected patient population. In fact, one of the patients that has shown severe CD4 decline and neurologic impairment carries both of these variations as we have previously published .
The TF-1, U-937, and Jurkat T-cell lines were used as models of HPCs, monocytes, and CD4+ T cells, respectively, in order to study the effects of the previously described SNPs on LTR-driven gene expression within a chromatin-based microenvironment. Each cell type was stably transfected with a specific LTR variant of interest and then stored at low temperature for prolonged periods prior to experimentation. Flow cytometric analysis showed that subsequent recovery of the stored cell populations containing the HIV-1 variant LTR resulted in two different LTR-driven GFP expression phenotypes with each model cell line (TF-1, U-937, and Jurkat) and parental or variant LTR (3 T, 5 T, and 3T5T) (Figure 2). We theorized that once multiple cell samples were stored at low temperature for prolonged periods, the same expression phenotype would be maintained for each sample that was recovered from storage and passaged to approximately the same level. However, as shown in Figure 2, this was not the case; several phenotypes were associated with the specific genotypic changes within the LTRs stably transfected across all model cell lines. Alterations in gene expression from cells recovered from low-temperature storage could be caused by differences in the stably transfected cells that survive the thaw and reculturing process. Cell death is a major problem when the cell line populations are thawed and recultured, as cells with different phenotypes may be the ones surviving each time this cycle occurs. The cell clones that have very distinct expression profiles, and maintain their expression profiles during subsequent thaws and reculture from low-temperature storage, provide further evidence of this phenomenon. In another sense, a recent study showed that when rheumatoid arthritis synovial fibroblast (RASF) cells were in culture, as the culture passages increased from 2 to 8, up to 10% of the genes within this cell line are differentially expressed . In contrast to our observations, however, after long-term storage, gene expression within the RASF after passage 2 was comparable to gene expression prior to freezing . This is the first study to show that long-term storage of stably transfected cells of the HPC, monocytic, and T-cell lineages can result in alterations in gene expression patterns.
TNF-α treatment of each of the stably transfected cell line populations was performed to determine how each SNP of interest might impact the way the LTR drives GFP expression when observed from cell populations recovered from long-term low- temperature storage. Stably transfected U-937 cells containing the 3 T and 3T5T LTRs exhibited 2.34- and 2.55-fold increases in MFI, respectively, with TNF-α stimulation. The difference in LTR-driven gene transcription with LTRs containing the WT, 3 T, 5 T, and 3T5T SNPs between stably transfected TF-1 and U-937 cells could be attributed to the difference in cell types and the availability of specific transcription factors and inherent viral gene activities within those cells. Within the stably transfected TF-1 cells treated with TNF-α, the 5 T LTR resulted in a higher level of change in LTR-driven GFP expression than the WT LTR (Figure 3). This observation indicates that although this genotype (which occurs frequently in the DrexelMed HIV/AIDS Genetic Analysis Cohort) could be involved in evading immune activation within the host under basal conditions, under stimulatory conditions it might be activated to produce a higher level of gene expression and virus production. Additionally, TNF-α stimulation of the stably transfected TF-1 and U-937 cells (Figure 3) suggested that a 3T5T LTR-containing proviral DNA could remain quiescent under certain metabolic conditions or in a basal viral gene expression mode. Only minimal intracellular stimulatory conditions would be required to drive low levels of viral gene expression and virus production within these cell populations, allowing evasion of HAART elimination of the infecting cell. The viral genome containing the 3T5T LTR could promote the evasion of effective HAART elimination, potentially contributing to the development of a niched viral reservoir for virus with specific genetic signatures that favor the quiescent or low-level gene expression mode. Subsequently, when an infected patient has a proinflammatory response to either the HIV-1 infection or an opportunistic infection, the 3T5T double-variant LTR-containing viral genotype could again result in an activation of the viral genome from a latent viral reservoir. This conclusion has been based on the increase in NF-κB binding to the 3T5T double variant and enhanced operation of these low-affinity C/EBP and Sp binding sites in the presence of elevated levels of the corresponding activation factors and a repopulation of circulating virus within the patient’s peripheral blood and lymphoid tissues and within specialized reservoirs. These changes would thereby make HAART less effective in removing and reducing virus from within the immune and nervous systems as well as other end organs.
The cell clones from each stably transfected cell line containing the WT, 3 T, 5 T, or 3T5T LTRs were developed to facilitate studies to explain the observable differences between each of the LTRs within each cell line. The low-GFP-expressing 3T5T LTR-containing TF-1 cell clone exhibited a larger difference in MFI between unstimulated and stimulated conditions compared with the intermediate-GFP-expressing cell clone. This observation may suggest that virus containing the 3T5T LTR genotype with a low enough expression profile in the body to facilitate evasion of HAART and the immune response, could, under stimulated conditions, produce a large amount of virus, reseeding infection into the body, possibly with viral strains that may be more resistant to treatment. These observations suggest that across LTRs containing each of the SNPs of interest (3 T, 5 T, and 3T5T), there is a differential expression within individual GFP-expressing clones. These results also show that within an LTR, in this case the 3T5T-containing LAI LTR, stimulation by cytokines naturally occurring during inflammation and infection can lead to differential regulation of the LTR between different cell lines. In addition, non-GFP-expressing cell clones, regardless of the LTR or cell type, could not be induced into expression with TNF-α stimulation, whereas the intermediate- and high-GFP-expressing cell clones containing each SNP of interest could be induced. This important result shows that differences in regulation of LTR-driven expression may also be attributable to the location of integration within the human genome, proviral DNA copy number, or possibly differences in epigenetic controls operative on the LTRs.