Identification of Host Micro RNAs That Differentiate HIV-1 and HIV-2 Infection Using Genome Expression Profiling Techniques

While human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2) share many similar traits, major differences in pathogenesis and clinical outcomes exist between the two viruses. The differential expression of host factors like microRNAs (miRNAs) in response to HIV-1 and HIV-2 infections are thought to influence the clinical outcomes presented by the two viruses. MicroRNAs are small non-coding RNA molecules which function in transcriptional and post-transcriptional regulation of gene expression. MiRNAs play a critical role in many key biological processes and could serve as putative biomarker(s) for infection. Identification of miRNAs that modulate viral life cycle, disease progression, and cellular responses to infection with HIV-1 and HIV-2 could reveal important insights into viral pathogenesis and provide new tools that could serve as prognostic markers and targets for therapeutic intervention. The aim of this study was to elucidate the differential expression profiles of host miRNAs in cells infected with HIV-1 and HIV-2 in order to identify potential differences in virus-host interactions between HIV-1 and HIV-2. Differential expression of host miRNA expression profiles was analyzed using the miRNA profiling polymerase chain reaction (PCR) arrays. Differentially expressed miRNAs were identified and their putative functional targets identified. The results indicate that hsa-miR 541-3p, hsa-miR 518f-3p, and hsa-miR 195-3p were consistently up-regulated only in HIV-1 infected cells. The expression of hsa-miR 1225-5p, hsa-miR 18a* and hsa-miR 335 were down modulated in HIV-1 and HIV-2 infected cells. Putative functional targets of these miRNAs include genes involved in signal transduction, metabolism, development and cell death.


Introduction
Human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2) are closely related retroviruses that share 30%-60% overall genetic homology and similar structural and genomic organization [1]. In addition to structural similarities, these two related viruses exhibit functional similarities like cluster of differentiation 4 (CD4) binding, T-cell and mononuclear cell tropism, viral transmission, replication and pathogenesis [2][3][4]. However, in spite of the similarities between HIV-1 and HIV-2, several studies have shown that the disease outcomes presented by these two viruses are distinct [5]. HIV-2 is less transmissible than HIV-1 and the disease free survival time in HIV-2 infected patients is

Peripheral Blood Mononuclear Blood Cells Isolation and Cell Culture
Buffy coats from individual seronegative donors were provided by the National Institute of Health (NIH) Blood bank. Informed consent was obtained from all of the healthy, normal blood donors according to the ethical principles of international ethical guidelines for biomedical research involving human subjects. A categorical exemption is in place for Center for Biologics Evaluation and Research (CBER)/Food and Drug Administration (FDA) for experimental studies by CBER/ FDA researchers using existing, deidentified samples of blood and/or blood products originally obtained under the NIH institutional review board (IRB) approved protocol and consent form 99-CC-0168. This study was approved by the FDA IRB. Written informed consent to participate in a clinical research study involving the collection and distribution of blood components from healthy donors for in vitro research use and for publication of this Case Report and any accompanying images was obtained from healthy, normal blood donors according to the ethical principles of international ethical guidelines for biomedical research involving human subjects. This study was approved by the NIH ethics committee (study number: 99-CC-0168, principal investigator: Susan F. Leitman, MD). A single step Ficoll-Hypaque density gradient centrifugation method was used for isolating PBMC from buffy coats. The PBMC were stimulated with 10 µg/mL of -Phytohemagglutinin (PHA) for 72 h by culturing in complete Roswell Park Memorial Institute (RPMI)-1640 media (RPMI medium containing 10% heat inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin, 10 U/mL interleukine-2 (IL-2)), and were cultured for an additional 24 h in complete RPMI media prior to infection. Jurkat cells were cultured in complete RPMI-1640 media.

Viruses and Infection
Jurkat cells were seeded at 1ˆ10 6 cells/mL for 24 h, and infected with known amounts (10 ng/mL p24/p27 units per 10 6 cells) of HIV-1 (MN) [36,37] and HIV-2 (ROD) and cultured for different days as indicated. PBMC cells were infected with known amounts (5 ng/mL p24/p27 units per 10 6 cells) of HIV-1 (MN) and HIV-2 (ROD). Two hours post-infection, tubes were washed with PBS to remove un-adsorbed virus and cells were fed with complete RPMI (RPMI 1640, 10% FBS, 1% penicillin-streptomycin for Jurkat cells and supplemented with IL-2 for PBMC) and cultured in T75 flask. The cells were harvested at different time points as indicated. HIV-1 and HIV-2 replication was quantitated by Alliance HIV-1 p24 Antigen ELISA Kit (Perkin Elmer, Waltham, MA, USA) and RETRO-TEK SIV p27 Antigen ELISA Kit (ZeptoMetrix, Buffalo, NY, USA).

RNA Extraction
Total RNA was extracted from infected and uninfected cells using miRNeasy total RNA isolation kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's protocol. Infected and uninfected cells were lysed with 700 µL of QIAzol lysis reagent. DNA was sheared using the QIAshredder columns (QIAGEN) and total RNA was extracted from this lysate using the RNeasy ® Mini column (QIAGEN). Total RNA was estimated by spectrophotometry using the Nanodrop 1000 (Thermo Fisher, Waltham, MA, USA).

miRNA Profiling Using RT 2 MicroRNA PCR Array
Qiagen RT 2 microRNA PCR Array system (Cat#MAH200, based on miRBase version 14), an optimized real-time PCR assay was used for miRNA profiling studies, which allows the simultaneous detection of 352 miRNAs, representing most functional miRNAs, as well as appropriate control miRNAs and RNA quality controls. Extracted total RNA including miRNAs (20 ng/µL concentration) was first reverse transcribed into cDNA using the miScript II RT kit following manufacturer's recommendations (QIAGEN). One ng cDNA per well was then mixed with SYBR Green PCR Master Mix and placed into a 96-well PCR-array plate containing a panel of 88 mature miRNAs sequences for Real-time PCR analysis on an Applied Biosystems 7500 Real Time PCR system. Relative amounts were calculated by the ∆∆CT method.

PCR Array Data Analysis
The expression of individual miRNA was analyzed using Ct values obtained with a threshold of 0.2. All array plates were tested with endogenous controls, RT negative controls, and genomic DNA contamination controls. Ct values of 35 or greater for any particular miRNA in either the control or the experimental samples was excluded from the analysis and marked as undetectable or undetermined. The Ct values that passed through these stringent criteria were uploaded into the Qiagen/SABiosciences software (RT 2 Profiler PCR Array Data Analysis) and the fold change calculated for each miRNA.

Validation of Differentially Expressed miRNA
Validation of miRNA expression was performed by real-time PCR (RT-PCR) experiments. Based on the data analyses, a subset of miRNAs was selected for validation by RT-PCR using specific primers and probes (Applied Biosystems, Waltham, MA, USA). We used total RNA samples from five independent donors to validate the high throughput PCR array results. Briefly, miRNA reverse transcription was done with the Taqman miRNA reverse transcription kit (Applied Biosystems); using 15 mM dNTPs, MultiScribe™ Reverse Transcriptase 50 U, 1.5 µL reverse transcription buffer, RNase inhibitor 3.8 U and 3 µL of 5ˆRT primers for miRNA specific. Reverse transcription was carried out using the following conditions: 30 min at 16˝C, 42˝C for 30 min and 85˝C for 5 min. Real-time PCR was carried out using the following conditions: 10 min at 95˝C, followed by 40 cycles at 95˝C for 15 s and 60˝C for 1 min. The results were expressed as n-fold difference in expression of miRNA of interest relative to endogenous controls (RNU44) for infected and uninfected samples; n-fold=2ˆ´( ∆Ct infected´∆Ct uninfected) .

miRNA Targeted Gene Prediction
We utilized miRNA target gene prediction with the MirTarget2 prediction tool in miRDB database version 4.0. We selected top ten genes with a highest target score to validate the interaction between host genes and differentially expressed miRNA.

Quantification of Predicted Target Genes by RT-PCR
To validate the predicted miRNA target genes, we tested the expression of selected genes by RT-PCR using total RNA samples isolated from five independent donors. Quantitative RT-PCR was performed using the two-step RT-PCR with SYBR Green and ROX (Invitrogen, Waltham, MA, USA) according to manufacturer's protocols. Total RNA was reverse transcribed with Superscript ® III First-Strand Synthesis SuperMix (Invitrogen). SYBR Green PCR mix (Invitrogen) and gene specific primers for real-time PCR were used for amplification. The program for thermal cycling was 20 s at 95˝C followed by 40 cycles of 3 s at 95˝C, 30 s at 57˝C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control for each donor. Each sample was run in triplicate to ensure accurate fold change estimation. Relative gene expression from each donor RNA was calculated from Ct values for the gene of interest and GAPDH. The Ct value of the gene-of interest was normalized to the Ct value of GAPDH and the final results expressed as n-fold difference in expression of gene-of-interest relative to GAPDH gene for infected and uninfected samples.

PCR-Array and Differential miRNA Expression Analysis
In our studies, the expression profile of 352 host cellular miRNAs was assessed in PBMC isolated from three independent donors productively infected with HIV-1 (MN) or HIV-2 (ROD) ( Table 1) to identify putative biomarkers of infection that could differentiate between the two viruses. Differential expression of host miRNA expression profiles were analyzed using the miRNA profiling PCR arrays (QIAGEN/SA Biosciences). The miRNA PCR arrays represent a large panel of the most relevant miRNAs in the human genome (as annotated by the Sanger miRBase Release 14) and is designed to analyze miRNA expression using real-time PCR. The results of the expression profile of 352 host cellular miRNAs indicate that infection with HIV-1 and HIV-2 differentially regulated the expression of several miRNAs. The miRNA expression levels in HIV-1 and HIV-2 infected cells were compared with miRNA expression in uninfected PBMC cells, and internal controls were used for normalization and calculation of fold differences. The scatter plot of the differentially expressed miRNAs ( Figure 1) indicates that the fold differences ranged between 2-and 7-fold for a number of miRNAs. regulated the expression of several miRNAs. The miRNA expression levels in HIV-1 and HIV-2 infected cells were compared with miRNA expression in uninfected PBMC cells, and internal controls were used for normalization and calculation of fold differences. The scatter plot of the differentially expressed miRNAs (Figure 1) indicates that the fold differences ranged between 2-and 7-fold for a number of miRNAs.

Validation of miRNA Expression by RT-PCR
To validate the differentially regulated miRNAs identified using PCR arrays, a subset of 20 differentially expressed miRNAs in HIV-1 and HIV-2 infected PBMC were selected ( Figure 3). We tested total RNA isolated from the infected and uninfected PBMC derived from five independent donors for specific miRNAs along with endogenous controls using miRNA specific primers and probes by reverse transcription (RT)-PCR ( Figure 4).
Results indicate that among the twenty differentially expressed miRNAs tested, only nine miRNAs (hsa-miR 193a-3p, hsa-miR 195-3p, hsa-miR 499-3p, hsa-miR 518f-3p, hsa-miR 541-3p, hsa-miR 654-3p, hsa-miR 125b-1*, hsa-miR 24-1* and let-7e*) showed consistent differential expression by independent RT-PCR assay ( Figure 4). This could be due to the donor to donor variation. The results obtained from five independent PBMCs infected with HIV-1 and HIV-2 indicates that these miRNAs are differentially regulated across multiple donors in response to infection by HIV-1 or HIV-2. Using RNA isolated from two independent sets of Jurkat cells infected with HIV-1 and HIV-2 ( Figure 5) as the template we identified three miRNAs (hsa-miR 195-3p, hsa-miR 518f-3p, and hsa-miR-541-3p) that were consistently upregulated in HIV-1 infected cells. This result is in agreement with our previous RT-PCR results obtained from PBMC, further validating the reproducibility of our PCR array analyses.

Validation of miRNA Expression by RT-PCR
To validate the differentially regulated miRNAs identified using PCR arrays, a subset of 20 differentially expressed miRNAs in HIV-1 and HIV-2 infected PBMC were selected ( Figure 3). We tested total RNA isolated from the infected and uninfected PBMC derived from five independent donors for specific miRNAs along with endogenous controls using miRNA specific primers and probes by reverse transcription (RT)-PCR ( Figure 5).
Results indicate that among the twenty differentially expressed miRNAs tested, only nine miRNAs (hsa-miR 193a-3p, hsa-miR 195-3p, hsa-miR 499-3p, hsa-miR 518f-3p, hsa-miR 541-3p, hsa-miR 654-3p, hsa-miR 125b-1*, hsa-miR 24-1* and let-7e*) showed consistent differential expression by independent RT-PCR assay ( Figure 5). This could be due to the donor to donor variation. The results obtained from five independent PBMCs infected with HIV-1 and HIV-2 indicates that these miRNAs are differentially regulated across multiple donors in response to infection by HIV-1 or HIV-2. Using RNA isolated from two independent sets of Jurkat cells infected with HIV-1 and HIV-2 ( Figure 5) as the template we identified three miRNAs (hsa-miR 195-3p, hsa-miR 518f-3p, and hsa-miR-541-3p) that were consistently upregulated in HIV-1 infected cells. This result is in agreement with our previous RT-PCR results obtained from PBMC, further validating the reproducibility of our PCR array analyses.

Validation of miRNA Targets Genes
Validation of targeted mRNA is essential because miRNA target prediction algorithms are characterized by high false discovery. To further establish the potential for functional relationship between miRNA and target genes, we validated the targeted mRNA expression with the same sample sets where the miRNAs were differentially expressed. The predicted host target genes were validated in Jurkat cells as well as in PBMC samples. Among the twelve putative mRNA targets that were tested for miR 195-3p, we found four genes, CUL-2, poly(A) binding protein interacting protein 2 (PAIP2), methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2-like (MTHFD12L) and UBE2I genes were downregulated in HIV-1 infected Jurkat cells. However, in PBMC only CUL-2 and UBE2I genes were found to be down modulated ( Figure 6A). In the same way we validated ten putative mRNA targets each for miRNAs miR-518f-3p and miR-541-3p. Out of the ten predicted target genes tested for miR-518f-3p we found that only one gene, SLCO4C1 gene was downregulated in HIV-1 infected Jurkat and PBMC cells ( Figure 6B). Likewise, the predicted miR 541-3p target genes were validated in both Jurkat and PBMC cells. The results showed that in infected Jurkat cells, out of the ten target genes tested, HOOK3, RAB6A, LSM12, and PCDH11X were down regulated. However, when these genes were tested in HIV-1 infected PBMC cells only HOOK3 and PCDH11X genes showed consistent downregulation ( Figure 6C). 1 infected Jurkat and PBMC cells ( Figure 6B). Likewise, the predicted miR 541-3p target genes were validated in both Jurkat and PBMC cells. The results showed that in infected Jurkat cells, out of the ten target genes tested, HOOK3,RAB6A, LSM12, and PCDH11X were down regulated. However, when these genes were tested in HIV-1 infected PBMC cells only HOOK3 and PCDH11X genes showed consistent downregulation ( Figure 6C). The expression of target genes at the protein level was validated to further determine the functional relationship between the miRNAs and target genes. Expression levels of selected miRNA target genes were assessed by Western Blotting. The expression of CUL-2 and UBE21 the predicted targets of miR 195-3p, SLCO4C1 the predicted target of miR 518f-3p, and HOOK3 the predicted target of miR 541-3p were determined in cell lysates of PBMCs infected with HIV-1 or HIV-2. The results indicate (Figure 7) that protein expression of CUL-2, SLCO4C1 and HOOK3 were all down modulated, which is in concordance with the real-time PCR data. The expression of target genes at the protein level was validated to further determine the functional relationship between the miRNAs and target genes. Expression levels of selected miRNA target genes were assessed by Western Blotting. The expression of CUL-2 and UBE21 the predicted targets of miR 195-3p, SLCO4C1 the predicted target of miR 518f-3p, and HOOK3 the predicted target of miR 541-3p were determined in cell lysates of PBMCs infected with HIV-1 or HIV-2. The results indicate (Figure 7) that protein expression of CUL-2, SLCO4C1 and HOOK3 were all down modulated, which is in concordance with the real-time PCR data. The expression of target genes at the protein level was validated to further determine the functional relationship between the miRNAs and target genes. Expression levels of selected miRNA target genes were assessed by Western Blotting. The expression of CUL-2 and UBE21 the predicted targets of miR 195-3p, SLCO4C1 the predicted target of miR 518f-3p, and HOOK3 the predicted target of miR 541-3p were determined in cell lysates of PBMCs infected with HIV-1 or HIV-2. The results indicate (Figure 7) that protein expression of CUL-2, SLCO4C1 and HOOK3 were all down modulated, which is in concordance with the real-time PCR data.

Discussion
HIV-1 and HIV-2 are closely related retroviruses that share considerable similarities in their genome architecture, pathogenesis and clinical outcomes. Both HIV-1 and HIV-2 infections lead to acquired immune deficiency syndrome (AIDS), the main difference observed in HIV-2 infected patients being the relative slower disease progression and greater immunological control of HIV-2 replication [13,14,39]. The exact cause of this phenomenon is not known. In order to understand the molecular basis of the differences in disease progression between HIV-1 and HIV-2, previously we examined differences in apoptotic signaling pathways during infection with either HIV-1 or HIV-2 in Jurkat cells and PBMC. Our studies demonstrated that HIV-1 infection induced greater cell death, activation of apoptosis and autophagy, generation of reactive oxygen species (ROS) and increased the expression of diverse molecules involved in cell signaling [16,40].
HIV infection induces multiple changes in the expression profile of host genes including noncoding RNA species, all of which contribute to host response. In order to delineate the differences in virus-host responses to infection with HIV-1 or HIV-2, we wanted to characterize the expression profiles of host miRNAs in cells infected with HIV-1 and HIV-2. Thus, the differential expression of host miRNA expression profiles was analyzed using the miRNA profiling PCR arrays.
Previous studies comparing miRNA expression profiles in in vitro HIV-1 infected T cell lines, PBMCs and cells from infected patients identified several miRNAs that were differentially regulated after HIV-1 infection or exposure to the virus [11,16,19,20,27,[41][42][43][44][45][46]. Studies have shown that cellular miRNA expression upon viral infection contributes to HIV-1 latency in resting T-lymphocytes [47], interferes with Nef protein expression and tat protein function and inhibits gag protein assembly

Discussion
HIV-1 and HIV-2 are closely related retroviruses that share considerable similarities in their genome architecture, pathogenesis and clinical outcomes. Both HIV-1 and HIV-2 infections lead to acquired immune deficiency syndrome (AIDS), the main difference observed in HIV-2 infected patients being the relative slower disease progression and greater immunological control of HIV-2 replication [13,14,39]. The exact cause of this phenomenon is not known. In order to understand the molecular basis of the differences in disease progression between HIV-1 and HIV-2, previously we examined differences in apoptotic signaling pathways during infection with either HIV-1 or HIV-2 in Jurkat cells and PBMC. Our studies demonstrated that HIV-1 infection induced greater cell death, activation of apoptosis and autophagy, generation of reactive oxygen species (ROS) and increased the expression of diverse molecules involved in cell signaling [16,40].
HIV infection induces multiple changes in the expression profile of host genes including non-coding RNA species, all of which contribute to host response. In order to delineate the differences in virus-host responses to infection with HIV-1 or HIV-2, we wanted to characterize the expression profiles of host miRNAs in cells infected with HIV-1 and HIV-2. Thus, the differential expression of host miRNA expression profiles was analyzed using the miRNA profiling PCR arrays.
Previous studies comparing miRNA expression profiles in in vitro HIV-1 infected T cell lines, PBMCs and cells from infected patients identified several miRNAs that were differentially regulated after HIV-1 infection or exposure to the virus [11,16,19,20,27,[41][42][43][44][45][46]. Studies have shown that cellular miRNA expression upon viral infection contributes to HIV-1 latency in resting T-lymphocytes [47], interferes with Nef protein expression and tat protein function and inhibits gag protein assembly leading to decreased HIV-1 infectivity [16,27,[48][49][50][51][52]. In contrast, other studies have reported that upregulation of specific host miRNAs [29,46,53,54] enhances HIV-1 replication. While several studies, including the present study identified many miRNAs that were commonly regulated, a greater variation was noted between these studies [20,27,41,42,55,56]. The differences observed could be due to differences in cell type, patient population and the methodology used to identify miRNAs. Our comparative analysis of miRNA expression profiles using PCR arrays in HIV-1 and HIV-2 infected PBMC indicate that miRNA expression profiles were considerably altered in HIV-1 and HIV-2 infected cells compared to uninfected controls. Analysis of the differentially expressed miRNA between the two groups (HIV-1 and HIV-2 infected PBMC) indicates significant overlap as well as group specific expression of miRNAs ( Figure 2). The PCR array results demonstrate that five miRNAs were commonly upregulated and four miRNAs were commonly downregulated in both HIV-1 and HIV-2 infected cells. Similarly, 16 miRNAs were upregulated and 11 miRNAs downregulated in HIV-1 infected PBMC. Eight (8) miRNAs were upregulated and 19 miRNAs were downregulated in HIV-2 infected PBMC cells. Consistent with reports in literature, our study also identified several miRNAs (hsa-miR 195-3p, hsa-miR 335, hsa-miR 424, hsa-miR 518c, hsa-miR 541-3p, and hsa-miR 518f-3p) that were modulated by HIV-1 infection [20]. In addition, our PCR array results identified several miRNA species that were uniquely regulated by HIV-1 and HIV-2 infection. Micro-RNA species hsa-miR-195-3p, hsa-miR-513c and hsa-miR-518f-3p were found to be upregulated in HIV-1 infected cells only. Micro-RNA species hsa-miR-939-5p, hsa-miR-125b-1, and hsa-miR 654-3p were found to be upregulated only in HIV-2 infected cells. MicroRNA species hsa-miR-548m, hsa-193a-3p, and hsa-miR-148b* were found to be upregulated in both HIV-1 and HIV-2 infected cells. Similarly, several miRNA species were found to be dowregulated only in HIV-1 or in HIV-2 infected cells. Independent validation of the PCR array results identified three miRNAs, hsa-miR-195-3p, hsa-miR-518f-3p, and hsa-miR-541-3p that were consistently upregulated in HIV-1 infected cells.
Some of the differentially expressed miRNAs are associated with a wide variety of cancers and are implicated to play an important role in tumor progression. Studies have demonstrated the involvement of hsa-miR 195 in cancer. MiRNA hsa-miR-195 was found to be downregulated in several cancers [16,27,57] and is believed to function as a tumor suppressor by targeting BCL2 [16,57,58]. In addition, studies have demonstrated that the overexpression of hsa-miR 195 in tumor cells leads to the inhibition of cell proliferation, invasion and migration [46,53,59]. Similar studies have identified that hsa-miR-541-3p is associated with the inhibition of tumor cell proliferation by inhibiting telomerase activity and transcription factors involved in Wnt signaling pathway [60]. MiRNA hsa-miR 541 is also implicated in the growth of HER-2 positive breast cancer cells and acts as a negative regulator in the HER-2 pathway [61]. Similarly, studies have demonstrated that hsa-miR 193a-3p downmodulated intestinal inflammation and suppress metastasis of non-small cell lung cancer [62]. While the exact mechanism of how these miRNAs modulate HIV infection is not known, high miRNA expression following HIV-1 infection may target cellular factors that act as host restriction factors and facilitate the infection process and promote viral replication. Among the predicted host factors targeted by the upregulated miRNAs in HIV-1 infected cells, we found CUL 2, UBE2I, SLCO4C1, HOOK3, and PCDH11X genes were consistently down modulated only in HIV-1 infected cells [17].
The host enzyme CUL 2 belongs to a family of structurally related proteins that are involved in the ubiquitin-dependent protein degradation pathway [63]. Protein ubiquitination plays an important role in a wide variety of cellular processes and in HIV-1 infection. HIV-1 integrase enzyme can be actively degraded by the ubiquitin-proteasome pathway [46]. Recent studies have identified that von Hippel-Lindau binding protein 1 (VBP1) and the cullin 2-based von Hippel-Lindau (VHL) ubiquitin ligase are required for HIV-1 expression post integration. The integrase binding protein VBP1 and CUL2-VHL cooperate in the polyubiquitination and degradation of HIV-1 integrase by the ubiquitin-proteasome pathway, an essential step between viral integration and transcription of viral genes [64]. The host gene, UBE2I is an E2 Small Ubiquitin-like Modifier (SUMO) conjugase that adds SUMO residues post-translationally to target proteins [65]. Several studies have reported that UBE2I interacts with HIV-1 viral proteins and plays an important role in viral replication [66]. The knockdown of endogenous UBE2I expression in HIV-1 infected cells caused defects in the trafficking of HIV-1 gag and env proteins resulting in the production of defective virus particles that were not infectious [67].
Our studies identified three miRNAs that were differentially expressed in HIV-1 infected cells. We also observed a direct correlation between the upregulation of the three miRNAs and downmodulation of the cellular genes targeted by these miRNAs. The differentially expressed hsa-miR-195-3p targets host genes that modulate cellular proteins which are essential for viral replication and disease progression. Thus modulation of host miRNAs in HIV-1 infected cells can potentially enhance the antiviral responses triggered by HIV-1 entry and infection, resulting in increased resistance to productive infection and decreased production of replication competent virus particles.