In a study conducted by Zimmerman et al. (2004) [24], they explored on the mechanism which eukaryotes have adopted in order to naturally combat DNA damage during replication. This pathway leaves the cells with two options: either to prevent mitosis to allow DNA repair or to commit to apoptosis in instances of irreparable conditions. In situations of replication complications, the ataxia-telangiectasia-mutated and Rad3-related kinase ATR would initiate damage signal.
In a related study, the researchers have found that the human immunodeficiency virus type I (HIV-1) gene product viral protein R (Vpr) causes infected cells to arrest at the G2 phase through ATR activation. In this present study, it was demonstrated that ATR activation by Vpr is analogous to activation caused by various genotoxic agents in both the mechanistic aspect and downstream consequences (Fig. 6).
It was further shown that Rad17 and Hus1 are both needed in order to stop the cell cycle at the G2 phase as well as Vpr-induced phosphorylation of histone 2A variant X (H2AX) and nuclear foci with H2AX formation and breast cancer susceptibility protein 1. The experiment’s results indicate the mediation of HIV-1 gene product Vpr in arrest at G2 phase, which uses the cellular signaling pathways especially designed to detect replication stress.
These are significant discoveries that can lead to a more through understanding of how HIV-1 manipulates the CD4+-lymphocyte cycle and apoptosis induction in the progressive CD4+-lymphocyte depletion characteristic of HIV-1 pathogenesis [24]. Figure 6. Schematic representation of ATR pathway [24] In another research conducted by Klase et al. (2007) [23], they investigated the dicer processing of HIV-1 TAR element in order to form a viral micro RNA that is utilized in viral LTR chromatin remodeling.
It has been mentioned that there is a conserved regulatory mechanism n eukaryotic cells called RNA interference. This pathway results in the formation of small interfering RNAs (siRNAS) or micro RNA (miRNA). Respectively, these two are synthesized by long dsRNA or RNA hairpins. An effector complex is guided by the siRNA or miRNA to a sequence homolgous of mRNA and it regulates genetic expression. The silencing of endogenous genes is utilized by the cells to protect themselves from foreign nucleic acid sequqnces. In the study, the researchers wanted to create a viral miRNA.
According to previous studies, dicer enzymes cleave miRNA and siRNA from longer RNA sequences and it is expressed in CD4+ T-cells. Observations suggest that RNAi may have a role in maintaining T-cell latency, as dicer levels in monocytes are found to be suboptimal. It is then demonstrated that dicer binds to this structure, as shown by results of biotin labeled TAR element. The new dicer actually ahs the ability of cleave TAR elements in vitro and that TAR derived miRNA is present in HIV-1 infected cell lines and primary T-cell blasts.
TAR elements from miRNA are also found to be capable of regulating viral gene expression, suppressing it through transcriptional silencing. Successful results indicated the possibility of creating a viral miRNA though the processing dicer enzymes of HIV-TAR element. This viral miRNA is detectable in infected cells and have apparent effect in viral latency [23]. Figure 7. Generation and Action of HIV-1 TAR derived miRNA [23] A similar study was done exploring the possibility of trans-inhibition of HIV-1 by a long hairpin RNA expressed within the viral genome. Konstantinova et al.
(2007) [22]recognized that HIV-1 gene expression can be regulated and silenced through RNAi using short synthetic interfering RNAs (siRNAS) or gene constructs that code for short hairpin RNAS (shRNAs) or long hairpin RNAs (lhRNAs). But due to the emergence of viral escape mutants, there is a limited antiviral therapeutic use of shRNAs and siRNAs. This is a setback that is theoretically countered by the prevention of intracellular expression of lhRNAs generating multiple siRNAs that target the virus simultaneously, which reduced the probability for viral escape.
In reality, there are complications with gene constructs encoding lhRNA molecules. These molecules are not properly delivered to the targeted cells of the infected individual [10]. The research synthesized an HIV-1 variant of 300 bp long hairpin structure in the 3’ end of the genome homologous to the Nef gene. Results of the study yielded that HIV-lhNef potently stopped the expression of wild-type HIV-1 products in trans. But this demonstrated a severe production and replication defect, causing the researchers to devise a solution by selecting spontaneous variants of virus with truncated hairpin structures.
These escape variants effectively outgrew the wild-type virus when competed against each other, despite the former’s loss of ability to trans-inhibit HIV-1. Therefore, lhNef hairpin gene expression in the HIV-1 genome results in potent transinhibition of wild-type HIV-1 [22]. Figure 8. Trans Inhibition of HIV-1 by HIV- lhNef [22] There are two chemokine receptors significant in the antiviral studies on RNA interference. CXCR4 and CCR5 are both chemokine receptors that are important in the entry of HIV-1 into cellular systems.
Also, as the infection progresses from HIV-1 to AIDS, a switch in the co-receptro usage of the virus from CCR5 to CSCR4 is required. Qureshi et al. (2006) [21]therefore took interest in the silencing of genes through the use of ribozymes and single stranded antisense RNAs. The scientists of this research demonstrated that using ribozymes, CXCR4 and CCR5 mRNAs can simultaneously deplete in human peripheral blood mononuclear cells (PBMCs).
The activity of the ribozyme leads to the inhibition of cell-surface expression of both CR5 and CXCR4. HIV-1 replication is significantly inhibited when PBMCs are infected by the virus. The small single stranded antisense RNAs can aso be used to silence CCR5 and CXCR4 genes upon delivery to PBMCs, caused by selective degradation of receptor mRNAs [21]. Figure 9. Schematic Illustration of CXCR4 and CCR5 ribozymes and antisense molecules binding to their respective targets [21].