Applied Immunohistochemistry & Molecular Morphology:
Role of Micro-RNAs in Regulation of Lentiviral Latency and Persistence
Bagasra, Omar MD, PhD*; Stir, Ariana E. MD*; Pirisi-Creek, Lucia MD†; Creek, Kim E. PhD‡; Bagasra, Alexander U. MD§; Glenn, Nancy PhD∥; Lee, Jeremy S. PhD¶
*Department of Biology, South Carolina Center for Biotechnology, Orangeburg
Departments of †Pathology
‡Microbiology and Immunology, University of South Carolina, School of Medicine
∥Department of Statistics, University of South Carolina, Columbia, SC
§Medical University of South Carolina, Charleston, SC
¶Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada
Reprints: Omar Bagasra, South Carolina Center for Biotechnology, nb Claflin University, 400 Magnolia Street, Orangeburg, SC 29115 (e-mail: firstname.lastname@example.org).
Received for publication July 21, 2005; accepted November 21, 2005
Small interfering RNAs have been demonstrated to serve as a molecular defence against numerous retroviruses in plants and insects and, more recently, in primates. With the recent findings of micro-RNAs (miRNAs) that seem to play a pivotal role in the survival of the host, we have explored the role of miRNAs in lentiviral (LV) replication. We have previously hypothesized that, at least in the case of lentivirus infection, small interfering RNAs are involved in the inhibition of these types of viruses by the formation of intramolecular triplex formation (triplexes) between the polypurine tracks sequences of LV provirus and miRNAs and blocking the viral replication at the preintegration complex levels, placing these viruses into a suspended latency. Using several latently and chronically infected LV cell lines and human PBMCs from HIV–1-infected individuals, we show that perinuclear triplexes are formed in LV-infected cells. The number of triplexes decreased in cells with productive replication of LVs. Therefore, the degree of replication of HIV-1 and other LVs, both in the HIV-1 or other LV-infected cell lines and the HIV-1 infected PBMCs, inversely correlate with the number of cytoplasmic triplexes present in a particular cell. This correlation was further confirmed by the stimulation of PBMCs and LV-infected cell lines with appropriate mitogens. Treatment with Tagetin, a RNA polymerase III inhibitor, resulted in a significant decrease in triplexes and a dramatic increase in the LV replication. Our data suggest that triplex formation may be an important mechanism of LV latency mediated by endogenous miRNAs.
Development of a suitable vaccine against HIV-1 has been an unsuccessful task. The initial hope of identifying the specific anti–HIV-1 antigenic epitopes, which can protect HIV-1–infected individuals and serve as a potential vaccine, has been replaced by the realization that we have yet to identify a clear correlate of protective immunity against HIV-1 infection.1,2 Understanding the anti–HIV-1 protective factors and their potential role in the development of a vaccine or inexpensive therapy remains one of the major obstacles in HIV-1 research. In the last quarter century, since the realization of AIDS, all of the studies performed to establish the role of humoral or cellular immune responses in protecting human hosts against HIV-1 have been inconclusive.1–5
One of the most interesting and still unexplained aspects of HIV-1 infection is the presence of a large number of latently infected cells in the host body.1,2,5–7 After infection of the resting CD4+ T cells, the HIV-1 RNA is reverse transcribed into dsDNA and forms a preintegration complex (PIC). In the majority of HIV-1–infected cells, HIV-1 is placed in a suspended latency state.8,9 HIV-1 latency is a natural consequence of the fact that the virus replicates in activated CD4+ T cells, and until these cells are activated the virus remains in a suspended latency. Upon activation CD4+ T cells undergo a profound but reversible change in state. After responding to an antigenic challenge, the majority of the activated CD4+ T cells return to a resting state and persist as memory cells. The lifespan of these cells is, by necessity, long because they provide the cellular basis for immunologic memory.10–14
HIV-1 latency is a complex multifactorial phenomenon that ultimately results from the profound differences between resting and activated CD4+ T cells.14 HIV-1 PIC can avoid host immune responses and antiretroviral drugs through the latent infection of resting memory CD4+ T cells.14 However, the molecular mechanisms that keep the lentiviral (LV) PICs in the latent state are poorly understood and may hold the key in the development of vaccine against HIV-1. In this study, we have explored the molecular basis of differences in the HIV-1 versus other LV-latencies in the resting, nondividing cells. We have explored the role of micro-RNAs (miRNAs) in LV-latency and reactivation.
miRNAs are small RNAs that regulate the expression of complementary messenger RNAs. Hundreds of miRNA genes have been found in diverse animals, and many of these are phylogenetically conserved. miRNAs represent a class of noncoding RNAs that seem to regulate gene expression via translational repression. miRNAs are initially transcribed as several hundred-nucleotide pre-miRNAs, and are then processed to approximately 60-nucleotide (nt) hairpin pre-miRNAs in the nucleus by the double-stranded RNA (dsRNA) specific ribonuclease (Drosha).5,15,16 The ribonuclease Drosha requires a dedicated dsRNA binding protein to convert long, nuclear primary pri-miRNA transcripts into shorter pre-miRNA stem-loops. The pre-miRNAs are exported to cytoplasm where they are further excised by a RNA-induced silencing complex (RISC)-like enzyme. These miRNAs are further processed into numerous specific 19 to 23 nt miRNAs, with the ability to target various endogenous and exogenous genes. The difference between small interfering RNAs (siRNAs) and mature small (19 to 23 nt) miRNAs is the mechanism of posttranscription silencing.5 siRNAs lead to total degradation of the target mRNA, whereas miRNAs inhibit gene expression by preventing the translation process. The exact mechanism is still unknown and is the subject of our report.5,15–20 However, it is very difficult to decipher the origin of mature 19 to 23 nt miRNAs and siRNAs, therefore, occasionally we would use this term interchangeably.5 Several hundred miRNA genes have been identified in Caenorhabditis elegans, Drosophila, and also in certain plants and mammals.15–18 The most striking role of miRNAs is the discovery that organisms have developed miRNAs that are designed to serve as an intracellular molecular defense against viral infection.5,19–21 Both plants and insects have been known to carry these endogenous noncoding genes that are processed into miRNAs by Drosha/Dicer and then cut down viral transcripts into precise 19 to 23 nt fragments upon infection.5,18–21 Recently, a miRNA has been described in humans that protects against primate foamy virus, a complex retrovirus similar to HIV-1.22 The abundance of siRNAs throughout the animal kingdom and amongst all fauna and flora suggests the existence of an extensive molecular immune network (reviewed in Refs. 5, 19).
We have hypothesized that the miRNA-based molecular immunity has evolved to disable the LV infection by forming an intramolecular triplex-forming complex (TF) between the polypurine tracks (PPTs) of LV-PICs and miRNAs.19,20 Here, we show that in the resting LV-infected cell lines, PICs are kept in a suspended latency by the formation of TF that can be directly observed by the specific TF antibodies (Abs). Upon activation, LV-infected cells lose their TFs and LV enters a productive state. Surprisingly, inhibition of RNA polymerases, specifically Pol III results in abrogation of TF and LV latency, suggesting that TF siRNAs are generated by miRNAs. The possibilities for diagnostic value of triplexes in the measurement of the latency state and their therapeutic application in treating HIV-1 infection are also discussed.
METHODS AND MATERIALS
The monocytoid cell line U937, and over 3 dozen LV-infected cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, penicillin, and streptomycin or other media described by the donor. A subclone of HIV-1–infected U937 cells, U1,23 and a subclone of the CEM T-lymphocytic line, ACH-2, exhibit minimal amounts of constitutive expression of HIV-1.24 All the cells infected with HIV-1, HIV-2, various SIVs and FIV, and uninfected cell lines were a gift from the NIH AIDS Repository (McKesson BioServices Corp, 621 Lofsrtand Lane, Rockville, MD 20850) and included the following LV-infected cell lines: ACH-2 (HIV-1), U1/HIV-1, Hela/LAV, Hela/CD4-HIV-1, J1.1 (HIV-1), H9/HTLV III, MT-4/HTLVIII), H9/HIV-2, U937/HIV-2, HUT 78/SIVmac251, HUT 78/SIV BK28, SIV B670, H9/HIV-2 MVP11971, LuSIV, H9/SIV 186, SIVhu, SIVagm, SIVsm, SHIV KU-1, STLV-1, 3201/61E (FeLV), CRFK/FIV AZR-1 and many others. Uninfected cell lines included Sup-T1, Molt-4, H9, A3.01, CEM, U937, and 3T3.CD4.
PBMCs and HIV-1 Assays
Cell lines were plated at 5×105 cells/mL in 24-well plates (Nunc) in 10 mL of complete media. In some experiments, U1 cells were pretreated with GM-CSF (Sigma) at 500 units/mL for 48 hours before harvest. In other cultures, cells were treated with granulocyte–monocyte colony stimulating factor (GM-CSF) for 24 hours before addition of LPS from Escherichia coli 0127:B8 (Difco) (10 μg/mL), as described previously.25,26 After an additional 24 hours, cells were harvested for analysis by indirect immunofluorescence. PBMCs from 4 HIV-1–infected LTNPs, 3 typical progressors, 1 rapid progressor, and 8 HIV-1 seronegative individuals were tested by indirect immunofluorescence for the presence of triplexes before and 48 hours after stimulation with 1% PHA to activate HIV-1 transcription. Nonlymphoid LV-infected cell lines were stimulated with a cocktail containing sodium butyrate (1 mM; Sigma), ionomycin (500 nM; Sigma), and interleukin 1β (3.5 ng/mL; Sigma). All the reagents were dissolved in complete RPMI-1640.
As described previously,25 cells were harvested for single and dual staining immunofluorescence. For triplex formation (TF) staining, 2 murine monoclonal antibodies, Jel 318 and Jel 466,26,27 were mixed in equal volume, at 20 μg/mL. These monoclonal anti-triplex antibodies can recognize triplexes between any combinations of DNAs and RNAs, but more strongly between dsDNA and ssRNA.26,27 We used the pooled sera from multiple HIV-1–infected individuals containing polyclonal antiserum at 1:40 dilution for HIV-1 staining. In some cases polyclonal anti–HIV-1 Abs increased in goats, containing HIV-1-gag, gp120, gp41, gp160, p24, and p17 were also used. These Abs were used at 20 μg/mL concentrations.25 Anti–HIV-1, HIV-2, and Anti-SIV Abs were a gift from the NIAID AIDS Research and REFERENCE Reagent Program (operated by McKesson BioServices Corporation, Losftrand, MD). Detection of FIV infection was carried out by using a serum sample from a PCR confirmed FIV infected cat and was a kind gift from a Vet Clinic (Lincoln, PA). A fluorescent conjugated secondary antibody for mouse antibody and a rhodamine-conjugated appropriate secondary antibody were used for staining HIV-1 antigens and other LV antigens were used for staining triplexes and LV antigens, respectively. Cocktails of Abs were used to detect replication of various lentiviruse. LV replication markers were visualized by the appropriate rhodamine-conjugated secondary Abs (Fc-specific, anti-goat or sheep) and triplexes by FITC-conjugated anti-mouse Fab-specific secondary Abs.25
Small RNA Inhibition
To inhibit the RNA inhibition, we used Tagetin, a RNA Polymerase Inhibitor. It is the only compound known to potently inhibit RNA polymerase III from a variety of eukaryotic organisms including mammalian cells.28 It also seems that it is involved in the transcription of polypurine track of small RNAs.28 We used 100 pμ of Tagetin inhibitor which resulted in >90% inhibition of RNA polymerase in the mammalian cell lines and PBMCs were used for our analyses (data not shown).
Restriction Digestion and Immunohistochemistry for Triplexes
To confirm that the observed structures are triplexes, cells were digested with ds DNA cutters: BglII, EcoRI, and BamHI as well with single-strand-specific (ss) DNA/RNA nuclease S1, for 1 hour or 12 hours as described by Pearson et al.29 Cells were treated with 30 U of S1 nuclease (Promega) at room temperature for 45 minutes or 12 hours in S1 buffer (pH 5.0). In certain experiments, cells were treated with both types of enzymes. All the cells were analyzed by Abs to triplexes as described above.26,27
Statistical analyses were performed to determine whether the number of triplexes correlated with the relative degrees of LV replications in the cell lines UI, ACH-2, H9/IIIB, Hela-LAV, HIV-2, FIV, SIVagm, and SIVsm. We carefully counted the numbers of triplexes per cell in each of the respective cell lines and then performed analysis of variance (ANOVA) to infer whether mean triplex numbers differ among cell lines. Once it has been determined that differences exist among cell lines, we focus on all possible cell line pairs to specify which pairs have differing mean triplex numbers. We independently selected random samples from 39 different subjects, and determined the number of triplexes in a single cell for 8 different cell line types. The data are presented in Tables 1A and B.
Detection of Triplexes in LV-infected Cell Lines
Multiple studies with lentiviruses, including HIV-1, have shown that less than 1% of the retroviruses make it to the integration step—even with completely naive cells (reviewed in Refs. 14,19). Previously, we have hypothesized that the main cause of low integration rate of LV is the formation of triplexes between the endogenously expressed siRNAs and LV-preintegration complexes (LV-PICs), which prevent nuclear entry of the invading LV.19,20 The stability of the TF complex depends on the degree of perfect match between the invading PPT of LV and endogenously expressed siRNAs.19,30 Furthermore, the anti-LV miRNAs and siRNAs have coevolved with the LV prevalent in the African primates, and humans have a limited evolutionary history to complex LV and lack perfectly matching siRNAs to form stable TF.19,20,31 However, upon stimulation cells enter mitosis and initially the nuclear membranes become leaky and the cells lose their nuclear membrane barrier and LV-PICs enter the area that was occupied by the nucleoplasm. Owing to the change in cellular pH, triplex complexes dissociate and LV-PICs become available for integration into the host genome.19,20 Therefore, regardless of the presence of protective siRNAs all primates infected with SIVs exhibit certain viral load.32 This hypothesis is consistent with our current understanding of LV latency in HIV-1–infected individuals and in experimental models of AIDS (reviewed in Refs. 14, 19). Therefore, at any given time, most CD4+ T lymphocytes are in a resting G0 state. Resting lymphocytes are profoundly quiescent cells with a low metabolic rate and a small cytoplasmic volume. In adults, approximately 50% of the resting cells are virgin cells and have yet to encounter an appropriate antigen (Ag). The remaining cells are memory cells that have previously responded to numerous Ags (reviewed in Refs. 12–14). Antigenic or mitogenic stimulation results in a burst of cellular proliferation and differentiation, giving rise to effector cells. Most effector cells die quickly, but a subset survives and reverts to the resting G0 state as memory cells. These cells persist with the ability to rapidly respond to a future Ag. If CD4+ T lymphocytes become infected they still remain in a latent state unless stimulated. Thus, to detect HIV-1 from PBMCs from an HIV-1 seropositive individual, the PBMCs must be cultured in media that mitogenically stimulate these cells [ie, medium containing IL-2 and T-cell mitogen, phytohemagglutinin-P (PHA-P)]. The virus replicates in activated CD4+ T cells and kills them, typically within a few days after infection.33
It has long been recognized that, under the proper conditions, certain nucleic acid (NA) sequences preferentially adopt a structure composed of triplexes.30,34–36 Triplexes are thermodynamically favored structures characterized by a third pyrimidine-rich (Py triplex) or purine-rich (Pu triplex) NA strand located within the major groove of a homopurine/homopyrimidine stretch of duplex NA (reviewed in Refs. 34–36). In intermolecular and intramolecular triplexes, stable interaction of the third strand is achieved through either specific Hoogsteen (Py triplex) or reverse Hoogsteen (Pu triplex) hydrogen bonding to the homopurine strand of the duplex, with the third strand adopting either a parallel (Py triplex) or an antiparallel (Pu triplex) orientation relative to the homopurine acceptor.19,34,35 Base triplets in the pyrimidine motif include T*AT and C*GC, whereas those in the purine motif include G*GC, A*AT, and T*AT. Because cytosine protonation requires acidic pH35 and the G*GC base triplet is the most stable in the purine motif, T-rich Py motif or G-rich Pu motif triplexes would be expected to predominate under physiologic conditions.35 We analyzed over 21,136 HIV-1 sequences submitted to GenBank and found that all of the HIV-1 genes contain multiple polypurine tracks, ranging in length from 6 to 35 nt and ranging in number from 11 to 35 in the full-length HIV-1 sequences (Table 2).
We examined the presence of NA triplexes and the relative degree of LV replication, by double indirect immunocytochemistry, in a variety of lentivirus-infected cell lines. For this purpose, we used 2 murine monoclonal Abs (Jel 318 and Jel 466: 26-27) for the detection of triplexes, and a cocktail of polyclonal Abs to various proteins of HIV-1, HIV-2, SIVsm, SIVagm, FIV and other LV Ags, for detection of HIV-1, HIV-2, and SIV (SIVsm and SIVagm), and FIV replications.
As shown in Figure 1, the majority (>99%) of unstimulated, latently HIV-1–infected cell lines U1 and ACH-2 (Figs. 1A, B) exhibited numerous cytoplasmic triplexes. U1 and ACH-2 both exhibit minimal amounts of constitutive expression of HIV-1.23–25 In these lines, the majority of cells exhibited the green triplexes that seemed to be so abundant in the perinuclear areas that the area looked grainy under the microscope and especially in the photographic images. In addition, the majority of chronically HIV-1–infected cells (ie, H9/IIIB, Hela/LAV) and cells infected by other lentiviruses (ie, HIV-2, FIV, SIVsm, SIVsm, and SIVagm) also exhibited triplexes similar to those observed in the latently HIV-1–infected cell lines. Figures 1C, D show the representative pictures from Hela/LAV and CrFK/FIV-ARR-1 (FIV) cell lines. The average number of triplexes per cell varied significantly among different cell lines, but seemed to have remained relatively constant within a specific latently infected cell line at the single cell level, under similar culture conditions. Significantly, the cells that were positive for the specific LV Ags for viral replication (ie, anti-gag, pol, and env) have lost green cytoplasmic triplexes and exhibited prevalently red staining for those LV Ags. Of note, the degree of relative LV production in the infected cell lines was inversely related to the frequency of triplexes in a particular cell line. Therefore, latently infected cell lines like U1 and ACH-2 that produce a very low amount of HIV-1 proteins under unstimulated conditions showed very low percentages of cells that were producing HIV-1 (∼%). On the contrary, the cell lines that are highly productive for a specific LV (ie, Hela/LAV or CrFK FIV) exhibited on average significantly lower numbers of triplexes percell and markedly higher percentages of cells that were productively infected (Tables 1A and B). For example, the estimations of TF in the U1 and ACH-2 cells showed an average of 302 and 270 TF/cell, respectively, and less than 1% of cells were productively infected. Whereas, Hela/LAV and CrFK FIV cell lines showed an average of 27 and 40 TF/cell and significantly higher percentages of cells that were productively infected (ranging from 14% to 17% and 28%to 35%, respectively).
Triplexes were also observed in HIV-1–uninfected cells, but the numbers of triplexes per cell were significantly less and of lower intensity than what was observed in the LV-infected cells (data not shown). Less than 5±2 triplexes were observed in SUPT1, CEM, U937, and several other uninfected cell lines. These observations indicate that TF may be an evolutionary mechanism of miRNA/siRNAs against certain retroviruses in particular or transposons in general, and triplexes may be a normal physiologic phenomenon to silence endogenous and exogenous genes. Their numbers increase as the cells are infected with a particular LV.5,19 In addition, the presence of a large number of TFs suggests that resting cells are continuously being infected with LVs being released from the productively infected cells but they are unable to cross the nuclear membrane in the resting permissive cells.6,7
Stimulation and Activation Result in Decreased Triplexes and Increased LV Replication
In a second series of experiments, we evaluated the second part of our hypothesis: the maintenance of LV-latency is based on intramolecular TF between LV-proviruses, miRNAs and intact nuclear membranes.30 Therefore, HIV-1 or other LVs latency is broken only after dissolution of the nuclear barrier and dissociation of intramolecular triplex complexes. All the LV-infected cells can be stimulated by a variety of agents to greatly augment LV replication.25,33 We reasoned that if the latently infected cells lines like U1 and ACH-2, or chronically infected cells H9/IIIB, Hela-LAV, HIV-2, FIV, and SIVagm and SIVsm were stimulated with an appropriate agent (GMCSF/LPS for U1 cells25 and a cocktail of stimulants containing: PHA-P, ionomycin, sodium butyrate, phorbol myristate acetate and IL-2 for the other cell lines) then significant percentages of cells will enter mitosis, lose their nuclear membrane and allow LV replication to proceed.6–10,33 Therefore, U1 cells stimulated with GM-CSF/LPS showed a gradual reduction in triplex complexes, and cells positive for a high number of triplexes began to lose their cytoplasmic triplexes and started to produce HIV-1 as they entered mitosis. We observed a direct association at the single-cell levels in the cells that lost cytoplasmic triplexes and began to produce HIV-1 as determined by double labeling for triplexes (FITC labeled: green) and HIV-1 Ags (rhodamine labeled: red). Therefore, there seems to be a direct correlation between the dissociation of triplexes and cell stimulation leading to HIV-1 production. It takes several days for the majority of the U1 or ACH-2 cells to enter mitosis (shown in Figs. 2A, B). Thus, we were able to observe a progressive disassociation of triplexes and subsequent production of HIV-1. Similar results were found for a variety of cell lines chronically infected with HIV-1, and with all the other LVs. Figures 2C and D show the representative pictures from H9/IIIB, and FIV, respectively. Of note, as compared with chronically infected cell lines, the well-characterized latently infected cell lines, U1 and ACH-2, maintained significant percentages of cells that did not enter the HIV-1 productive stage (Figs. 2A, B). It seems that these cell lines are able to maintain true latency even after stimulation/activation.11,14
PBMCs From Latently Infected Cells Exhibit Increased Numbers of Triplexes per Cell and Cells With Least Number of Triplexes Show Increased HIV-1 Production
HIV-1 infection in man results in a highly variable disease course ranging from rapid progression to long-term nonprogression.1–4 The rate of disease progression is tightly linked with the extent of virus replication. Therefore, after seroconversion, viral RNA load (number of copies/mL) is predictive of disease outcome. However, which factors control viral replication in the PBMCs is still elusive.14,19 HIV-1–infected quiescent CD4+ T cells are refractory to viral replication in vitro. It has been reported that HIV-1 provirus exists in a silent or latent state in these cells.5–11 Lymphocytes from infected individuals that carry viral DNA (but do not seem to express it) and viral expression seem to be suspended at the PIC. Therefore, after observing an inverse correlation between the numbers of triplexes noted per cell and the relative degree of viral replication in the LV-infected cell lines, we evaluated the presence of triplexes in the PBMCs from 3 HIV-1–infected groups: LTNPs, typical progressor (TP), and rapid progressors (RP). LTNP was defined as HIV-1 seropositive individuals with a normal CD4+ T-lymphocyte count and manifesting no AIDS-related symptoms for over 10 years, and no history of antiretroviral treatment. TP was defined as HIV-1–infected patients with development of AIDS in less than 10 years but more than 2 years. RP was defined as individuals with a rapid development of AIDS-related symptoms and decline in CD4+ cell count in less than 2 years. We used double-labeling immunocytochemistry to visualize triplexes (green) and HIV-1 replicating cells (red). Figure 3 shows the representative pictures from the PBMCs from each of the 3 groups of HIV-1–infected individuals. Therefore, LTNPs exhibited a large percentage of PBMCs (∼100%) with triplexes and close to 0% of cells showed HIV-1 replication (Fig. 3A). This particular patient was HIV-1 seropositive in 1985 that was later confirmed by the Western blot and has shown no evidence of AIDS. His CD4+ cell counts have fluctuated but have remained within the normal limits. However, the other 3 LTNP's PBMCs have shown an average of 99.6% cells with triplexes and 0.4% of cells with HIV-1 replication. The triplexes and HIV-1 replicative cells in the TPs showed a relatively higher percentage of HIV-1–producing cells (between 5% and 7% and over 85% of cells with triplexes) (Fig. 3B). We also estimate that the number of triplexes per cell was significantly less in this group as compared with LTNPs. On the other hand, RPs showed a significant percentage of cells in HIV-1–replicative state (>45%) and a much lower percentage of cells exhibiting triplexes (60% to 70%). The number of triplexes per cell was also markedly lower than what was observed in LTNPs and TPs (Fig. 3C, and Tables 1A and B). As it was noted with the cell lines, PBMCs from uninfected individuals also exhibited triplexes, but the number of such triplexes was significantly lower compared with HIV-1–infected PBMCs. These observations suggest the presence of the genetically protective miRNAs disabling HIV-1 in LTNPs or relative lack of these types of miRNAs in RPs.19–22 A relatively large number of TPs in the LTNPs further confirms our thesis.19 In many cells the green dots (triplexes) appeared in the nuclear spaces. However, this was due to fine focusing. We are certain that these triplexes are at the perinuclear areas. Upon stimulation/activation of the PBMCs (with IL-2 and PHA) each group showed a different pattern with regard to triplexes. As shown in Figures 3D to F (bottom panel), the PBMCs from all 3 HIV-1–infected groups exhibited a significant reduction in triplexes and a simultaneous, cell-associated, and interdependent increase in HIV-1 replication. However, in LNTPs (Fig. 3D) there was a significantly lower percentage of cells that showed productive infection. Therefore, when the LTNPs were stimulated with PHA, a large percentage of PBMCs remained nonproductive for HIV-1. We have shown previously that this was not due to the absence of HIV-1, but was due to the resistance to stimulation-associated HIV-1 replication, as less than 0.4% of the cells showed HIV-1 replication (Figs. 3A vs D), whereas, PBMCs from TPs exhibited 30% of productive cell population (Fig. 3B vs E) and from a RP (Fig. 3C vs F) showed HIV-1 replication in 80% of the cells. The yellow-appearing cells represent a combination of red and green, where triplexes are rapidly dissociating and HIV-1 proteins are being synthesized. Of note, one can also observe the intranuclear shifting of LV-PICs and intense yellow staining due to staining with HIV-1 proteins, that is, tat, tar, and rev.45
Endogenous Nature of Triplex siRNAs
The nature and origin of siRNAs forming triplexes was evaluated by using a potent small RNA polymerase III inhibitor—Tagetin. This inhibitor seems to be necessary for the transcription of PPT in vivo.28 We have hypothesized that PPT-specific siRNAs are endogenous in nature, their molecular origin is the noncoding endogenous transposons and retroelements, and they are synthesized mainly by RNA polymerase III.28,37,38 Furthermore, we theorize that the PICs observed in LV-infected cells are the endogenously expressed siRNAs evolved to control reintegration of endogenous retroelements and to inhibit nuclear entry and integration of exogenous viruses.19 To explore this hypothesis, we treated HIV-1 and other LV-infected cell lines (ie, U1, ACH-2 Hela-LAV, FIV, and HIV-2) with Tagetin, a selective DNA-dependent RNA polymerase III inhibitor.28 We reasoned that if small endogenous RNAs are responsible for the siRNA-based molecular defences and TF, then an inhibition of RNA polymerase III activities should produce a breakdown in LV latency. Figure 4 shows a representative chronically infected FIV cell line that was treated with Tagetin, in which a significant decrease in the cytoplasmic triplexes was observed. Most interestingly, the observed decrease in siRNA synthesis was accompanied by a sudden burst of LV replication, indicating that siRNA-mediated triplexes were most likely responsible for containing the LV replication observed in various LV-infected cell lines. Figure 1D shows untreated FIV cells. One can observe multiple green dots representing clusters of LV-PIC triplex complexes, surrounding the perinuclear areas. Of note, one may suspect that these triplexes may be intranuclear owing to bright staining of some of the nuclei, but fine focusing has allowed us to determine that these are indeed perinuclear in the unstimulated cells. A time course analysis of the Tagetin effect suggested that as early as 6 hours post-RNP–inhibition, the cytoplasmic triplex numbers began to decrease (Fig. 4A). Around 24 to 48 hours post-Tagetin exposure, the triplexes in these cells began to disappear from the perinuclear areas and were being replaced by FIV-specific proteins (arrow, Fig. 4B). In 48 to 96 hours following post-Tagetin treatments, an increasing number of cells exhibited a marked decrease in nuclear triplexes and subsequent cytopathic effects of FIV infection (Fig. 4C). A similar pattern of triplex dilution and subsequent break in latency were observed in latently infected cell lines ACH-2 and U1 after 96 hours. Remarkably, both latently infected ACH-2 and U1 cell lines also exhibited a total breakdown in latency after Tagetin treatment. In contrast to the activated/stimulated ACH-2 and U1 cells where a significant percentage of cells were able to maintain latency, Tagetin-treated cells were unable to maintain latency (data not shown).
Restriction Digestion (RE) of ds and ss NAs
To confirm that the observed structures described in the preceding sections were triplexes, we used enzymes that would digest dsDNA and ssNA. For this purpose, we used a strategy that can destroy all ssNA and dsDNAs. Therefore, we incubated various LV-infected cell lines described above with a mixture of 3 RE: BglII, EcoRI, and BamHI, that will destroy dsDNAs, and S1 nuclease that will destroy ssRNAs or ssDNA. We used 1 hour and overnight incubations to assure the complete digestion of the ds and ss NA structures. After digestion, we performed indirect immunohistochemistry to detect triplexes. As shown in Figures 5A and B, both Hela LAV and FIV cell lines treated with the cocktails of RE+SI nuclease, maintained triplex structures. As compared with untreated cells, the triplex dots were significantly distinct and well defined. Similar results were observed when each of the LV-infected cell lines were treated with RE and SI nuclease separately (data not shown). From these figures one can clearly observe that triplex surrounds the perinuclear area, where one would expect the accumulation of LV-PICs. The clarity of the triplex dots after enzymatic digestion may be owing to digestion of the ss and ds fragments of the LV-PICs, leaving only the triplex complexes intact, thus making them more clearly visible.
Molecular Mechanisms for the Regulation of LV Replication, Latency, and Persistence: Role of Endogenous SiRNAs
Previous studies have focused on HIV-1 latency as a barrier in stymieing HIV-1 infection whereas, in this study, we have attempted to decipher the molecular mechanism of LV latency and how this process can be used to silence HIV-1. As it is well documented, SIVs in the African primates do not cause AIDS even though significant populations of the African primates are infected with various strains of SIVs in the wild (reviewed in Refs. 19, 32). Most significant among these primates are chimpanzees that carry SIVcpz that is very similar to HIV-1 and is considered to be the origin of HIV-1.19,20,32 The major goal of our study was to evaluate the differences in the degree of latency between PBMCs infected with HIV-1 and chronically infected cell lines that carry SIVs, HIV-2, and FIV. The major premise of our studies was that the African primates are able to contain SIVs owing to the presence of miRNAs that have coevolved with the species and can block their respective SIVs by forming triplexes with the PICs.19,20 Generally, in all primates the mechanism that appears to be the most significant in LV latency is that in resting CD4+ T cells, LVs can avoid host immune responses and antiretroviral drugs through the latent infection of resting memory CD4+ T cells.6–14 This kind of latency is a natural consequence of the fact that the virus replicates in activated CD4+ T cells.6–14 After Ag recognition, clonal expansion, and differentiation, a significant percentage of the effecter immune cells (ie, mature CD4+ and CD8+ T cells) revert back to the resting state as memory cells.6–14 The memory cells are not permissive for viral replication.6–14 However, these cells can be reactivated by the specific Ag or mitogens.6–14 In these cells, LV-PICs seem to accumulate in the cytoplasm and preferentially around the perinuclear spaces, allowing provirus to integrate into the host genome only after the nuclear membrane dissolves during cell division (ie, mitosis). This concept of LV latency explains why in the infected African primates SIVs continue to replicate. The reactivation of memory cells guarantees the viral persistence for the lifespan of the infected host.1,2,14,19,20
In our current study we have attempted to answer one of the most important questions regarding HIV-1 latency: What factors keep the LVs in the infected cells in a latent state? And what is the difference between SIV-infected cells versus HIV-1 infected cells? One of the most interesting findings regarding LV latency relates to the presence of a large number of cytoplasmic and perinuclear triplexes that seem to keep the LVs in a suspended latency, therefore, effectively inhibiting their replication by blocking their nuclear entry and hence preventing integration in the resting cells.35 In our studies, we observed 2 correlations that seem to determine the LV latency: (i) a direct correlation between the percentage of triplex-containing cells in a given population of LV-infected cells and degree of latency in the LV-infected cells (Tables 1A and B), and (ii) a direct correlation between numbers of triplexes per cell and latency. As it is evident from Tables 1A and B, the cell populations that exhibited the highest percent of triplex-containing cells or higher number of triplexes per cell were the most latent with regards to their respective LV infection. Therefore, SIVagm, SIVsm, ACH-2, and U1 cell lines were the least productive cell lines with regards to their respective LVs. Several cell lines exhibited a very low number of triplexes per cell and a low percentage of triplex-containing cells (ie, Hela/LAV, and Cre.FIV). These cells lines were also the most productive with regards to their respective LV infection (17% and 35%, respectively). We used 24 different cell lines and found these observations to be consistent. Therefore, it seems that the higher the number of triplexes per cell, the higher the degree of latency. We applied these criteria to HIV-1 infected PBMCs and found a significant difference in the HIV-1–productive PBMCs versus HIV-1 latently PBMCs. Therefore, as compared to LNTPs, the percentage of triplex-containing cells and triplexes per cell were significantly lower in the PBMCs of TPs and RPs (P>0.005 and P>0.001, respectively). Similarly, in other LV-infected cells the degree of latency appeared to be directly related to the numbers of triplexes present per cell (ie, HIV-2, SIVagm, and SIVsm). Most striking observations were made in the cell lines that are well-established HIV-1 latently infected cell lines: ACH-2 and U1. Both of these cell lines exhibited a large number of triplexes per cell and a significant percentage of cells with triplexes. On the other hand, HIV-1–producing cell lines like Hela/LAV and H9/IIIB, as well as FIV-producing cell lines exhibited a reverse pattern with regards to triplexes as determined by a double immunocytochemical method.
We have hypothesized that the mechanisms through which these factors block LV replication in various primates are owing to the presence of specific miRNAs, products of which form triplexes with LV-PICs, potentially masking or distorting the binding sites of nuclear translocation proteins.19,20,31,39 In our previous studies, we hypothesized that a pan-eukaryote, gene silencing, small RNA-based immune system must have developed from evolutionary exposure of organisms to a myriad of transposons, retroviruses and other retro-elements beginning long before the appearance of classical immunity.19,20 We further postulated that small RNA-based immunity against retroviruses is mediated by miRNA repertoires. Some of the miRNAs precisely match the sequences of the invading retroviruses at the PPTs and form triplexes by binding to the specific gene sequences within the provirus and block its integration. It is hypothesized that as PICs are relatively resistant to the classical immune responses, the host must have evolved the means to stymie the LV replication by using the noncoding genetic sequences it has accumulated throughout the evolution from various retroelements.5,19,20,37–44 The presence of a large number of integrated retroviruses (HERVs), introns and retrotransposons, is well documented in human genomes as well as other life forms.19,20 Noncoding small endogenous miRNAs derived from intergenic regions of the genome, have recently emerged as important regulators of gene expression in plants, animals, and primates.15,16,18 We suggest that one of the main reasons that SIVs are unable to replicate in many of the nonhuman primate cells is owing to the presence of specific miRNAs in these cells that carry a near-perfect homology to certain gene sequences of these SIVs. Recently, Lecellier et al22 have shown that a cellular miRNA effectively restricts the accumulation of the retrovirus primate foamy virus type 1 (PFV-1) in human cells. Therefore, through miRNAs the recognition of foreign NA, cellular miRNAs have direct antiviral effects in addition to their regulatory functions.22 We postulate that the detection of the relatively increased numbers of triplexes in LV-infected cells as compared to HIV-1–infected PBMCs may reflect the increased numbers of miRNAs that can form triplexes with the increasing number of invading LVs.19,20 Multiplicity of infection (moi: virion particle to cell ratio) has not been explored extensively and its relationship to latency is still a relatively unexplored area (reviewed in Ref. 25). In all LV-infected cells, the number of virions/target cells can vary from moi of 1 to >1000 (reviewed in Ref. 45). Because, in an acutely infected person the virus particle/mL can reach over 100,000particles/mL and generally in a healthy human the number of CD4+ T cells would be no more than 1500/mL, therefore, the moi would be more than 1:60. Usually, the viral RNA load decreases to <50 copies/mL within 2 to 3 weeks postinfection in both humans and African primates well before the development of classical immune responses, presumably due to the molecular immunity by miRNAs.1,2,5 However, in the African primates, the viral load remains low without the development of AIDS.32 Whereas, in humans, the course is highly variable with AIDS generally developing in 5 years postexposure.1,2 In a typical HIV-1–infected man, the viral load increases gradually after a few years and remains so if no antiviral therapy is administered.1,2,5 Most interestingly, all HIV-1–infected humans exhibit “blips,” where viral loads go up for a short time and then become undetectable. It would be easy to imagine that all resting CD4+ T cells are being exposed to HIV-1 and are being infected by the new waves of “blips” increasing the number of PICs per cell.6–14 We suggest that in a HIV-1–infected person the number of repertoires that can almost perfectly match and form stable triplexes are relatively much less than what would be found in the African primates. Therefore, after saturation of these miRNA repertoires the intracellular block would reach a viral threshold and even in the resting cells the HIV-1 latency would break. Then, the provirus would integrate and become productively infected (reviewed in Refs. 19, 45).
On the basis of our data we hypothesize that the latency state is based on 2 types of factors: the endogenous and the exogenous.19,20 The endogenous factors are the miRNA repertoires that can block the invading LVs through miRNAs and stymie it by forming triplexes.19,20 It seems that there may be several exogenous factors. One seems to be the moi of the virus. An initial high dose of virus that can saturate the complementary miRNAs and can overcome the limited number of repertoires of complementary miRNAs can play an important role.19,31,45 The number of miRNAs that can perfectly bind the LV provirus is the function of the endogenous miRNA repertoires.15,16,19,20 It is expected that the African primates where SIVs have coevolved would contain many protective complementary miRNAs which would recognize various SIVs and block their replication at postreverse transcription, PIC levels.19,20 There is another important exogenous factor that may play an important role at the initial stage of a LV infection, especially in HIV-1 infection. This is the activation state of the target cells (CD4+ T cells and other Ag presenting cells) at the initial stage of infection.14,19 If an individual contains a relatively large percent of activated target cells at the time of the initial exposure to a LV, then the number of cells that could be recruited to produce HIV-1 would be relatively large. This, firmly establishes a large reservoir of integrated HIV-1–infected cells at the early phase of infection, which can be reactivated upon antigenic or mitogenic stimulation.14,19 Of note, both alcohol and cocaine consumption have been shown to nonspecifically activate lymphocytes and are known to increase the number of cells infected at the initial exposure (reviewed in Refs. 46–48). A third factor that has been a matter of great concern in HIV-1 latency is superinfection. Superinfection is defined as an infection by a second virus (from a different clad or the same clad as the primary virus) during a steady-state viral load, after infection by a primary virus. It is now well established that superinfection with HIV-1 commonly occurs in humans.49 Detection of an increasing number of circulating recombinant forms, which result from infection of a cell by 2 or more clads, suggests that superinfection occurs more frequently than previously thought. The second virus can superinfect some time after the first and this is associated with rapid viral rebound and decline in CD4+ T cell count.49 The primary infection with a specific clad seems not to provide cross-protection against superinfection with a different clad or the same clad.49 It can be easily summarized from our data that if an individual was able to contain a specific HIV-1 clad by miRNAs, then a superinfection with a viral clad would allow the 2 different viruses to form an array of new recombinant viruses in the activated cells that would eventually form a HIV-1 quasi-strain that would overcome the endogenous miRNAs.19
The differences between LTNPs, TPs or RPs seem to be the percentages of cells that can enter into a HIV-1–productive state. From the numerous studies conducted on HIV-1–infected individuals it seems that LTNPs have a lower HIV-1 RNA viral load as compared with TPs or RPs. Similarly, in animals naturally infected with LVs (ie, chimpanzees with SIVcpz or African Green Monkeys with SIVagm), the viral loads of the respective LVs are generally relatively low.32 Most striking are observations made in a small percentage of HIV-1–infected individuals termed LTNPs, who remain free of symptoms for a decade or longer.6–11 Our observation confirms, and provides an explanation for these observations. As it is evident from our data, latency is determined at the single cell level and each of the LV-infected cells seems to mount an anti-LV–specific defense in the cytoplasm. If the cytoplasm of a specific cell contains miRNAs that can form triplexes with the invading genes at the PPTs then, most likely, these viruses would be placed in a suspended latency state. These latent PICs may accumulate at the perinuclear spaces. However, superinfection or infection with a very large dose of virus would saturate the complementary miRNAs and (as well activation) could abrogate the latency state, as has been described previously.5,19,31,45,50 The presence of a low-to-moderate load of SIVs even in the primates naturally infected with these viruses in the wild is the result of a small percentage of activated cells that would naturally allow these LVs to replicate via antigenic stimulation. This concept is depicted in Figure 6.
Our current findings suggest the endogenous nature of miRNAs and their important role in maintenance of viral latency. Inhibition of miRNAs by a selective RNA polymerase III inhibitor-Tagatin resulted in marked reduction of triplexes suggesting that endogenous miRNAs form triplexes and a sudden burst of LV replication in the Tagetin-treated cells strongly confirms our thesis that endogenous miRNAs may be the major resistance factors described by various investigators (reviewed in Refs. 5, 22). Interestingly, Tagetin has been shown to interfere in the transcription of PPT-specific small RNA.28
We also confirmed that the triplexes we observed in the infected cells and the PBMCs were actually triplexes and not an artifact. Therefore, treatment of cells with restriction enzymes that can destroy ss and dsNA still retained the observed triplexes.
The next step would to determine whether a long-term HIV-1 latency in the susceptible cell lines and in the PBMCs can be induced by expression of vectors that can produce miRNAs with perfect or near complementary to HIV-1 PPTs. This study is in progress in our laboratory.
In summary, we have forwarded a new model of HIV-1 persistence, and maintenance of latency based on endogenous miRNA. Our investigation reconciles many of the unexplained observations and explains how miRNAs may be responsible for LV latency, its persistence and reactivation. We have provided evidence that triplexes seem to play a pivotal role in RNA-based molecular immunity in LV infections, including HIV-1. We believe that further studies may provide the importance of triplexes as a means to stabilize the HIV-1 latency, making HIV-1 infection a nonpathogenic chronic infection similar to the SIV infections in chimpanzees and other African primates.32 Once the correct PPT sequences that form the crucial triplexes with the majority of the clades of HIV-1 are determined, they may serve as molecular therapy against this viral infection. Most interestingly, this form of therapy can potentially be administered to already infected individuals. These studies are currently underway in our laboratories.
The authors thank the excellent editorial assistance of Ms Twaina Harris and Mr Sajid Saleem and Dr Sibrina Collins. They are also very grateful to M. Iqbal Hussein for his excellent artwork and to the National Institutes of Health, NIAID division, AIDS Research and REFERENCE Reagent Program (operated by McKesson BioServices Corp, 621 Lofsrtand Lane, Rockville, MD 20850) for the gift of most of the cell lines and many antibodies.
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This article has been cited 1 time(s).
Libyan Journal of MedicineComputational analysis to predict functional role of hsa-miR-3065-3p as an antiviral therapeutic agent for treatment of triple infections: HCV, HIV-1, and HBVLibyan Journal of Medicine
Lentivirus; HIV-1; HIV-2; regulation; HIV-1 persistence; HIV-1 latency; HIV-1 persistence; polymerase III; replication; long-term non-progressors; rapid progressors; miRNAs; RNAi; siRNA; triplex formation; and typical progressor
© 2006 Lippincott Williams & Wilkins, Inc.
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