FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1843766852> ?p ?o ?g. }

• W1843766852 endingPage "e259" @default.
• W1843766852 startingPage "e259" @default.
• W1843766852 abstract "Cells from women who are epidemiologically deemed resistant to HIV infection exhibit a 40–60% reduction in endogenous IRF-1 (interferon regulatory factor-1), an essential regulator of host antiviral immunity and the early HIV replication. This study examined the functional consequences of reducing endogenous IRF-1 on HIV-1 replication and immune response to HIV in natural HIV target cells. IRF-1 knockdown was achieved in ex vivo CD4+ T cells and monocytes with siRNA. IRF-1 level was assessed using flow cytometry, prior to infection with HIV-Bal, HIV-IIIB, or HIV-VSV-G. Transactivation of HIV long terminal repeats was assessed by p24 secretion (ELISA) and Gag expression (reverse transcription–polymerase chain reaction (RT–PCR)). The expression of IRF-1–regulated antiviral genes was quantitated with RT–PCR. A modest 20–40% reduction in endogenous IRF-1 was achieved in >87% of ex vivo–derived peripheral CD4+ T cells and monocytes, resulted in >90% reduction in the transactivation of the HIV-1 genes (Gag, p24) and, hence, HIV replication. Curiously, these HIV-resistant women demonstrated normal immune responses, nor an increased susceptibility to other infection. Similarly, modest IRF-1 knockdown had limited impact on the magnitude of HIV-1–elicited activation of IRF-1–regulated host immunologic genes but resulted in lessened duration of these responses. These data suggest that early expression of HIV-1 genes requires a higher IRF-1 level, compared to the host antiviral genes. Together, these provide one key mechanism underlying the natural resistance against HIV infection and further suggest that modest IRF-1 reduction could effectively limit productive HIV infection yet remain sufficient to activate a robust but transient immune response. Cells from women who are epidemiologically deemed resistant to HIV infection exhibit a 40–60% reduction in endogenous IRF-1 (interferon regulatory factor-1), an essential regulator of host antiviral immunity and the early HIV replication. This study examined the functional consequences of reducing endogenous IRF-1 on HIV-1 replication and immune response to HIV in natural HIV target cells. IRF-1 knockdown was achieved in ex vivo CD4+ T cells and monocytes with siRNA. IRF-1 level was assessed using flow cytometry, prior to infection with HIV-Bal, HIV-IIIB, or HIV-VSV-G. Transactivation of HIV long terminal repeats was assessed by p24 secretion (ELISA) and Gag expression (reverse transcription–polymerase chain reaction (RT–PCR)). The expression of IRF-1–regulated antiviral genes was quantitated with RT–PCR. A modest 20–40% reduction in endogenous IRF-1 was achieved in >87% of ex vivo–derived peripheral CD4+ T cells and monocytes, resulted in >90% reduction in the transactivation of the HIV-1 genes (Gag, p24) and, hence, HIV replication. Curiously, these HIV-resistant women demonstrated normal immune responses, nor an increased susceptibility to other infection. Similarly, modest IRF-1 knockdown had limited impact on the magnitude of HIV-1–elicited activation of IRF-1–regulated host immunologic genes but resulted in lessened duration of these responses. These data suggest that early expression of HIV-1 genes requires a higher IRF-1 level, compared to the host antiviral genes. Together, these provide one key mechanism underlying the natural resistance against HIV infection and further suggest that modest IRF-1 reduction could effectively limit productive HIV infection yet remain sufficient to activate a robust but transient immune response. HIV-1 transmission via genital mucosal surfaces is an inefficient process with an estimated risk of 3–50 events per 10,000 sexual exposures or <1% per unprotected sexual exposure. The local and systemic establishment of HIV-1 infection is contingent upon the viral fitness,1Joseph SB Swanstrom R HIV/AIDS. A fitness bottleneck in HIV-1 transmission.Science. 2014; 345: 136-137Crossref Scopus (9) Google Scholar the availability of shared host factor, and the efficacy of antiviral immunity.2Sivro A Su RC Plummer FA Ball TB Interferon responses in HIV infection: from protection to disease.AIDS Rev. 2014; 16: 43-51PubMed Google Scholar,3Pitha PM Innate antiviral response: role in HIV-1 infection.Viruses. 2011; 3: 1179-1203Crossref PubMed Scopus (27) Google Scholar Delineation of molecular events occurring at early stages of infection and determination of the critical events for successful HIV-1 transmission may help develop better prevention measures. However, examining the early events of successful, natural HIV infection in human subjects is technically challenging. Fortunately, not everyone exposed to HIV-1 become infected. In all investigated HIV-exposed cohorts, there are ∼10–15% of these HIV-exposed individuals who remain seronegative (HIV-exposed seronegative (HESN)).4Shearer G Clerici M Historical perspective on HIV-exposed seronegative individuals: has nature done the experiment for us?.J Infect Dis. 2010; 202: S329-S332Crossref Scopus (21) Google Scholar Studies of molecular events that may be involved in hindering the establishment of HIV infection can be performed in HESN,5McLaren PJ Ball TB Wachihi C Jaoko W Kelvin DJ Danesh A et al.HIV-exposed seronegative commercial sex workers show a quiescent phenotype in the CD4+ T cell compartment and reduced expression of HIV-dependent host factors.J Infect Dis. 2010; 202: S339-S344Crossref PubMed Scopus (95) Google Scholar,6Songok EM Luo M Liang B Mclaren P Kaefer N Apidi W et al.Microarray analysis of HIV resistant female sex workers reveal a gene expression signature pattern reminiscent of a lowered immune activation state.PLoS One. 2012; 7: e30048Crossref PubMed Scopus (46) Google Scholar,7Songok EM Osero B McKinnon L Rono MK Apidi W Matey EJ et al.CD26/dipeptidyl peptidase IV (CD26/DPPIV) is highly expressed in peripheral blood of HIV-1 exposed uninfected female sex workers.Virol J. 2010; 7: 343Crossref Scopus (19) Google Scholar and the findings will help identifying genetic and immune correlates of protection.8Guerini FR Lo Caputo S Gori A Bandera A Mazzotta F Uglietti A et al.Under representation of the inhibitory KIR3DL1 molecule and the KIR3DL1+/BW4+ complex in HIV exposed seronegative individuals.J Infect Dis. 2011; 203: 1235-1239Crossref Scopus (37) Google Scholar,9Tomescu C Abdulhaqq S Montaner LJ Evidence for the innate immune response as a correlate of protection in human immunodeficiency virus (HIV)-1 highly exposed seronegative subjects (HESN).Clin Exp Immunol. 2011; 164: 158-169Crossref PubMed Scopus (76) Google Scholar,10Malhotra R Hu L Song W Brill I Mulenga J Allen S et al.Association of chemokine receptor gene (CCR2-CCR5) haplotypes with acquisition and control of HIV-1 infection in Zambians.Retrovirology. 2011; 8: 22Crossref Scopus (23) Google Scholar,11Schellenberg JJ Links MG Hill JE Dumonceaux TJ Kimani J Jaoko W et al.Molecular definition of vaginal microbiota in East African commercial sex workers.Appl Environ Microbiol. 2011; 77: 4066-4074Crossref PubMed Scopus (61) Google Scholar,12Ghadially H Keynan Y Kimani J Kimani M Ball TB Plummer FA et al.Altered dendritic cell-natural killer interaction in Kenyan sex workers resistant to HIV-1 infection.AIDS. 2012; 26: 429-436Crossref Scopus (9) Google Scholar,13Sironi M Biasin M Cagliani R Forni D De Luca M Saulle I et al.A common polymorphism in TLR3 confers natural resistance to HIV-1 infection.J Immunol. 2012; 188: 818-823Crossref PubMed Scopus (91) Google Scholar,14Lajoie J Juno J Burgener A Rahman S Mogk K Wachihi C et al.A distinct cytokine and chemokine profile at the genital mucosa is associated with HIV-1 protection among HIV-exposed seronegative commercial sex workers.Mucosal Immunol. 2012; 5: 277-287Crossref PubMed Scopus (103) Google Scholar,15Habegger de Sorrentino A Sinchi JL Marinic K Lopez R Iliovich E KIR-HLA-A and B alleles of the Bw4 epitope against HIV infection in discordant heterosexual couples in Chaco Argentina.Immunology. 2013; 140: 273-279Crossref Scopus (25) Google Scholar,16Pattacini L Murnane PM Kahle EM Bolton MJ Delrow JJ Lingappa JR et al.Differential regulatory T cell activity in HIV type 1-exposed seronegative individuals.AIDS Res Hum Retroviruses. 2013; 29: 1321-1329Crossref Scopus (14) Google Scholar,17Zapata W Aguilar-Jiménez W Pineda-Trujillo N Rojas W Estrada H Rugeles MT Influence of CCR5 and CCR2 genetic variants in the resistance/susceptibility to HIV in serodiscordant couples from Colombia.AIDS Res Hum Retroviruses. 2013; 29: 1594-1603Crossref Scopus (30) Google Scholar,18Card CM Ball TB Fowke KR Immune quiescence: a model of protection against HIV infection.Retrovirology. 2013; 10: 141Crossref PubMed Scopus (49) Google Scholar,19Aguilar-Jiménez W Zapata W Caruz A Rugeles MT High transcript levels of vitamin D receptor are correlated with higher mRNA expression of human beta defensins and IL-10 in mucosa of HIV-1-exposed seronegative individuals.PLoS One. 2013; 8: e82717Crossref PubMed Scopus (25) Google Scholar,20Shen R Smith PD Mucosal correlates of protection in HIV-1-exposed sero-negative persons.Am J Reprod Immunol. 2014; 72: 219-227Crossref Scopus (16) Google Scholar,21Li H Liu TJ Hong ZH Gene polymorphisms in CCR5, CCR2, SDF1 and RANTES among Chinese Han population with HIV-1 infection.Infect Genet Evol. 2014; 24: 99-104Crossref Scopus (18) Google Scholar,22Van Raemdonck G Zegels G Coen E Vuylsteke B Jennes W Van Ostade X Increased Serpin A5 levels in the cervicovaginal fluid of HIV-1 exposed seronegatives suggest that a subtle balance between serine proteases and their inhibitors may determine susceptibility to HIV-1 infection.Virology. 2014; 458-459: 11-21Crossref PubMed Scopus (19) Google Scholar,23Sironi M Biasin M Gnudi F Cagliani R Saulle I Forni D et al.A regulatory polymorphism in HAVCR2 modulates susceptibility to HIV-1 infection.PLoS One. 2014; 9: e106442Crossref Scopus (12) Google Scholar,24Lima JF Oliveira LM Pereira NZ Mitsunari GE Duarte AJ Sato MN Distinct natural killer cells in HIV-exposed seronegative subjects with effector cytotoxic CD56(dim) and CD56(bright) cells and memory-like CD57+NKG2C+CD56(dim) cells.J Acquir Immune Defic Syndr. 2014; 67: 463-471Crossref Scopus (17) Google Scholar,25Ruiz-Riol M Llano A Ibarrondo J Zamarreño J Yusim K Bach V et al.Alternative effector-function profiling identifies broad HIV-specific T-cell responses in highly HIV-exposed individuals who remain uninfected.J Infect Dis. 2015; 211: 936-946Crossref PubMed Scopus (14) Google Scholar Our earlier work identified interferon (IFN) regulatory factor-1 (IRF-1) to be a genetic and functional correlate of protection against HIV-1 acquisition in a highly HIV-exposed commercial sex worker cohort in Nairobi, Kenya26Su RC Sivro A Kimani J Jaoko W Plummer FA Ball TB Epigenetic control of IRF1 responses in HIV-exposed seronegative versus HIV-susceptible individuals.Blood. 2011; 117: 2649-2657Crossref PubMed Scopus (32) Google Scholar,27Ji H Ball TB Ao Z Kimani J Yao X Plummer FA Reduced HIV-1 long terminal repeat transcription in subjects with protective interferon regulatory factor-1 genotype: a potential mechanism mediating resistance to infection by HIV-1.Scand J Infect Dis. 2010; 42: 389-394Crossref PubMed Scopus (14) Google Scholar,28Ball TB Ji H Kimani J McLaren P Marlin C Hill AV et al.Polymorphisms in IRF-1 associated with resistance to HIV-1 infection in highly exposed uninfected Kenyan sex workers.AIDS. 2007; 21: 1091-1101Crossref PubMed Scopus (73) Google Scholar; these HESN women can be defined as epidemiologically “resistant” to HIV infection.29Horton RE McLaren PJ Fowke K Kimani J Ball TB Cohorts for the study of HIV‐1-exposed but uninfected individuals: benefits and limitations.J Infect Dis. 2010; 202: S377-S381Crossref PubMed Scopus (60) Google Scholar,30Cohen J AIDS vaccine research. HIV natural resistance field finally overcomes resistance.Science. 2009; 326: 1476-1477Crossref Scopus (13) Google Scholar IRFs are a family of transcriptional regulators found in all principle metazoan groups including simple organisms such as sea sponges.31Schneider WM Chevillotte MD Rice CM Interferon-stimulated genes: a complex web of host defenses.Annu Rev Immunol. 2014; 32: 513-545Crossref PubMed Scopus (1805) Google Scholar IRF genes are thought to have coevolved with Rel/NF-κB genes, which together play important roles in regulating host immunity.32Nehyba J Hrdlicková R Bose HR Dynamic evolution of immune system regulators: the history of the interferon regulatory factor family.Mol Biol Evol. 2009; 26: 2539-2550Crossref PubMed Scopus (114) Google Scholar IRF-1, the first IRF identified, functions as a transcription regulator33Ozato K Tailor P Kubota T The interferon regulatory factor family in host defense: mechanism of action.J Biol Chem. 2007; 282: 20065-20069Crossref PubMed Scopus (156) Google Scholar by binding to an IFN-stimulated response element (ISRE), found in numerous genes controlling immune responses and cellular apoptosis.2Sivro A Su RC Plummer FA Ball TB Interferon responses in HIV infection: from protection to disease.AIDS Rev. 2014; 16: 43-51PubMed Google Scholar,31Schneider WM Chevillotte MD Rice CM Interferon-stimulated genes: a complex web of host defenses.Annu Rev Immunol. 2014; 32: 513-545Crossref PubMed Scopus (1805) Google Scholar,34Frontini M Vijayakumar M Garvin A Clarke N A ChIP-chip approach reveals a novel role for transcription factor IRF1 in the DNA damage response.Nucleic Acids Res. 2009; 37: 1073-1085Crossref PubMed Scopus (44) Google Scholar IRF-1 expression is expressed at low basal level in most cell types and can be induced by specific cytokine/chemokines and by viral infection. It was recently shown to be upregulated in CD4+ T cells,35Marsili G Remoli AL Sgarbanti M Perrotti E Fragale A Battistini A HIV-1, interferon and the interferon regulatory factor system: an interplay between induction, antiviral responses and viral evasion.Cytokine Growth Factor Rev. 2012; 23: 255-270Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar,36Sgarbanti M Remoli AL Marsili G Ridolfi B Borsetti A Perrotti E et al.IRF-1 is required for full NF-kappaB transcriptional activity at the human immunodeficiency virus type 1 long terminal repeat enhancer.J Virol. 2008; 82: 3632-3641Crossref PubMed Scopus (74) Google Scholar monocyte-derived dendritic cells, and monocyte-derived macrophages37Harman AN Lai J Turville S Samarajiwa S Gray L Marsden V et al.HIV infection of dendritic cells subverts the IFN induction pathway via IRF-1 and inhibits type 1 IFN production.Blood. 2011; 118: 298-308Crossref PubMed Scopus (94) Google Scholar,38Nasr N Maddocks S Turville SG Harman AN Woolger N Helbig KJ et al.HIV-1 infection of human macrophages directly induces viperin which inhibits viral production.Blood. 2012; 120: 778-788Crossref PubMed Scopus (161) Google Scholar by in vitro HIV infection. In addition to the antiviral role, IRF-1/NF-κB are essential facilitators of the early transactivation of HIV-1 genome.3Pitha PM Innate antiviral response: role in HIV-1 infection.Viruses. 2011; 3: 1179-1203Crossref PubMed Scopus (27) Google Scholar,27Ji H Ball TB Ao Z Kimani J Yao X Plummer FA Reduced HIV-1 long terminal repeat transcription in subjects with protective interferon regulatory factor-1 genotype: a potential mechanism mediating resistance to infection by HIV-1.Scand J Infect Dis. 2010; 42: 389-394Crossref PubMed Scopus (14) Google Scholar Deleting the ISRE39Liang C Li X Quan Y Laughrea M Kleiman L Hiscott J et al.Sequence elements downstream of the human immunodeficiency virus type 1 long terminal repeat are required for efficient viral gene transcription.J Mol Biol. 1997; 272: 167-177Crossref PubMed Scopus (28) Google Scholar or NF-κB site36Sgarbanti M Remoli AL Marsili G Ridolfi B Borsetti A Perrotti E et al.IRF-1 is required for full NF-kappaB transcriptional activity at the human immunodeficiency virus type 1 long terminal repeat enhancer.J Virol. 2008; 82: 3632-3641Crossref PubMed Scopus (74) Google Scholar in the HIV LTR (long terminal repeats) results in a virus with reduced replicative capacity, directly pointing to a role for IRF-1 in regulating HIV replication. Polymorphisms in the IRF-1 gene are associated with disease progression in hepatitis C infection40Wietzke-Braun P Maouzi AB Mänhardt LB Bickeböller H Ramadori G Mihm S Interferon regulatory factor-1 promoter polymorphism and the outcome of hepatitis C virus infection.Eur J Gastroenterol Hepatol. 2006; 18: 991-997Crossref PubMed Scopus (26) Google Scholar and with altered susceptibility to HIV infection.28Ball TB Ji H Kimani J McLaren P Marlin C Hill AV et al.Polymorphisms in IRF-1 associated with resistance to HIV-1 infection in highly exposed uninfected Kenyan sex workers.AIDS. 2007; 21: 1091-1101Crossref PubMed Scopus (73) Google Scholar Several linked IRF-1 polymorphisms were found to associate with reduced susceptibility to HIV-infection,27Ji H Ball TB Ao Z Kimani J Yao X Plummer FA Reduced HIV-1 long terminal repeat transcription in subjects with protective interferon regulatory factor-1 genotype: a potential mechanism mediating resistance to infection by HIV-1.Scand J Infect Dis. 2010; 42: 389-394Crossref PubMed Scopus (14) Google Scholar,28Ball TB Ji H Kimani J McLaren P Marlin C Hill AV et al.Polymorphisms in IRF-1 associated with resistance to HIV-1 infection in highly exposed uninfected Kenyan sex workers.AIDS. 2007; 21: 1091-1101Crossref PubMed Scopus (73) Google Scholar but not disease progression.41Sivro A McKinnon LR Ji H Kimani J Jaoko W Plummer FA et al.Interferon regulatory factor 1 polymorphisms previously associated with reduced HIV susceptibility have no effect on HIV disease progression.PLoS One. 2013; 8: e66253Crossref Scopus (6) Google Scholar These polymorphisms were also functionally linked with reduced endogenous IRF-1 expression and a reduced responsiveness to exogenous IFN-γ signaling.28Ball TB Ji H Kimani J McLaren P Marlin C Hill AV et al.Polymorphisms in IRF-1 associated with resistance to HIV-1 infection in highly exposed uninfected Kenyan sex workers.AIDS. 2007; 21: 1091-1101Crossref PubMed Scopus (73) Google Scholar Importantly, they also correlated with the decreased, transient transactivation of the HIV-1 LTR.27Ji H Ball TB Ao Z Kimani J Yao X Plummer FA Reduced HIV-1 long terminal repeat transcription in subjects with protective interferon regulatory factor-1 genotype: a potential mechanism mediating resistance to infection by HIV-1.Scand J Infect Dis. 2010; 42: 389-394Crossref PubMed Scopus (14) Google Scholar However, not all HESN subjects have these protective IRF-1 polymorphisms; yet, the majority of HESN women who can be epidemiologically defined as relatively resistant to HIV infection in studies from Nairobi, Kenya have reduced endogenous IRF-1 expression (Figure 1a, P < 0.001) that may be regulated through epigenetic mechanisms.26Su RC Sivro A Kimani J Jaoko W Plummer FA Ball TB Epigenetic control of IRF1 responses in HIV-exposed seronegative versus HIV-susceptible individuals.Blood. 2011; 117: 2649-2657Crossref PubMed Scopus (32) Google Scholar In vitro studies have shown that the complete knockdown of IRF-1 in Jurkat T-cell lines reduced HIV-1 transactivation, emphasizing the absolute requirement for IRF-1 in HIV replication.36Sgarbanti M Remoli AL Marsili G Ridolfi B Borsetti A Perrotti E et al.IRF-1 is required for full NF-kappaB transcriptional activity at the human immunodeficiency virus type 1 long terminal repeat enhancer.J Virol. 2008; 82: 3632-3641Crossref PubMed Scopus (74) Google Scholar However, it is unknown if a modest reduction of IRF-1 expression, as observed in vivo in HESN women, could limit HIV replication and, importantly, how this reduction would affect IRF-1–regulated IFN-stimulated genes (ISGs), the antiviral immune responses. Endogenous IRF-1 protein level was assessed in the HESN subjects, women who exhibit natural resistance to HIV-1 acquisition (Figure 1a). Although not all HESN female sex workers (FSWs) have the IRF-1 protective polymorphisms (at least one), which is strongly associated with reduced IRF-1 expression,28Ball TB Ji H Kimani J McLaren P Marlin C Hill AV et al.Polymorphisms in IRF-1 associated with resistance to HIV-1 infection in highly exposed uninfected Kenyan sex workers.AIDS. 2007; 21: 1091-1101Crossref PubMed Scopus (73) Google Scholar most peripheral blood mononuclear cell (PBMC) samples from the HESN FSWs analyzed showed reduced IRF-1 expression, in comparison to the HIV-seronegative, non-HESN FSW controls (P values <0.01), suggesting that mechanisms other than IRF-1 genetic polymorphism are responsible for the reduced IRF-1 expression,26Su RC Sivro A Kimani J Jaoko W Plummer FA Ball TB Epigenetic control of IRF1 responses in HIV-exposed seronegative versus HIV-susceptible individuals.Blood. 2011; 117: 2649-2657Crossref PubMed Scopus (32) Google Scholar and that reduced IRF-1 expression may have a critical role in the resistance phenotype against HIV-1 acquisition in these HESN FSWs.27Ji H Ball TB Ao Z Kimani J Yao X Plummer FA Reduced HIV-1 long terminal repeat transcription in subjects with protective interferon regulatory factor-1 genotype: a potential mechanism mediating resistance to infection by HIV-1.Scand J Infect Dis. 2010; 42: 389-394Crossref PubMed Scopus (14) Google Scholar,28Ball TB Ji H Kimani J McLaren P Marlin C Hill AV et al.Polymorphisms in IRF-1 associated with resistance to HIV-1 infection in highly exposed uninfected Kenyan sex workers.AIDS. 2007; 21: 1091-1101Crossref PubMed Scopus (73) Google Scholar We then determined if transiently reducing endogenous IRF-1 expression in ex vivo PBMCs would limit HIV replication. A complete knockdown of gene expression in primary cells remains a technical challenge and a complete IRF-1 knockout may not be desirable, as IRF-1 regulates cell viability.46Chen FF Jiang G Xu K Zheng JN Function and mechanism by which interferon regulatory factor-1 inhibits oncogenesis.Oncol Lett. 2013; 5: 417-423Google Scholar,47Tamura T Yanai H Savitsky D Taniguchi T The IRF family transcription factors in immunity and oncogenesis.Annu Rev Immunol. 2008; 26: 535-584Crossref PubMed Scopus (915) Google Scholar However, transiently altering IRF-1 expression in primary cells is technically feasible48Mantei A Rutz S Janke M Kirchhoff D Jung U Patzel V et al.siRNA stabilization prolongs gene knockdown in primary T lymphocytes.Eur J Immunol. 2008; 38: 2616-2625Crossref PubMed Scopus (62) Google Scholar,49Nold MF Nold-Petry CA Pott GB Zepp JA Saavedra MT Kim SH et al.Endogenous IL-32 controls cytokine and HIV-1 production.J Immunol. 2008; 181: 557-565Crossref PubMed Scopus (106) Google Scholar and may be more biologically relevant, reflecting the level of IRF-1 expression observed in most HESN women. Here, partial IRF-1 reduction was achieved in ex vivo CD4+ T cells and monocytes using IRF-1–specific siRNA. A significant reduction of endogenous IRF-1 protein could be detected by flow cytometry at 8 hours posttransfection (Figure 1b). The efficiency of siRNA uptake was monitored with fluorescence (Alexa 647)-tagged siRNA spiked into the nontagged siRNA. In unstimulated ex vivo PBMCs, IRF-1 protein expression was reduced in ∼25–40% of total PBMCs (Figure 1b), and similar frequency of PBMCs demonstrated the uptake of siRNA (positive for Alexa 647, Figure 1c), perhaps due to the preferential transfection of T cells with the T-cell–specific Nucleofector solution. To determine the half-time of IRF-1 knockdown, CD4+ T cells and CD14+ monocytes transfected with siRNA specific for IRF-1 were stained for IRF-1 expression at 18, 42, and 66 hours posttransfection (Figure 2). No further reduction in IRF-1 expression level was observed past 18 hours in transfected cells and the half-time of transient IRF-1 knockdown was ∼42 hours posttransfection in both cell types (Figure 2) and was accounted for in later experimental design. Moreover, greater than 90% of the enriched CD4+ T cells and CD14+ monocytes could be successfully transfected with siRNA using human T-cell Nucelofector™ solution and human monocyte Nucleofection solution, respectively (Figures 2 and 3). The effectiveness of IRF-1 knockdown was, hence, measured at 18 hours posttransfection (Figure 3). The efficiency of knocking down IRF-1 protein expression ranged from 25 to 55% in CD4+ T cells (Figure 3a: mean value: 38%, n = 9) and 30–60% in unstimulated monocytes (Figure 3b: mean value: 44%, n = 6). To examine whether cellular stimuli would affect the efficiency of IRF-1 knockdown in monocytes, at 6 hours posttransfection, monocytes were treated with low-dose mitogen (phorbol 12-myristate 13-acetate: 1 ng/ml and ionomycin: 50 ng/ml). Cell viability dropped to <50%, if transfected cells (with IRF-1 siRNA or control) were stimulated prior to 6 hours posttransfection, suggesting a minimum of 6 hours recovery from transfection was required. Low-dose stimulation of CD14+ monocytes consistently increased the endogenous IRF-1 expression level but maintained the efficiency of IRF-1 knockdown (Figure 3c: 60–80%, mean value: 75%, n = 8). The reduction of endogenous IRF-1 expression in CD4+ T cells and monocytes were within the range of that observed in HESN women (Figure 1a, 35–60%). Transfection of CD4+ T and CD14+ monocytes from healthy local blood donors with the scrambled siRNA control showed no effect on IRF-1, HDAC2, GAPDH, IL-12p35, IFN-γ, tumor necrosis factor-α (TNF-α), STAT1α, IL-4 mRNA, or 18s rRNA levels in quantitative reverse transcription–polymerase chain reaction (RT–qPCR) assays. To examine the effects of IRF-1 knockdown after viral entry, transfected cells and controls were infected with HIVVSV-G (vesicular stomatitis virus G glycoprotein pseudotyped, multiplicity of infection (MOI): 0.1) at 18 hours posttransfection (Figure 4). HIVVSV-G consists of an LTR-dependent luciferase gene, replacing the HIV-1 Nef (43Yao XJ Kobinger G Dandache S Rougeau N Cohen E HIV-1 Vpr-chloramphenicol acetyltransferase fusion proteins: sequence requirement for virion incorporation and analysis of antiviral effect.Gene Ther. 1999; 6: 1590-1599Crossref PubMed Scopus (29) Google Scholar) and is capable of entering cells independent of normal CD4/gp120 attachment. At 76 hours following HIVVSV-G infection, a threefold increase in LTR-driven luciferase activity was observed in CD4+ T cells transfected with IRF-1–specific siRNA, compared to a 84-fold increase with control (Figure 4a). A similar reduction of HIV LTR–driven luciferase activity (≥90%) after IRF-1 knockdown was observed in ex vivo unstimulated (Figure 4b) and stimulated monocytes (Figure 4c). These data clearly demonstrate that transactivation of LTR in HIVVSV-G depends on the IRF-1 expression, and that knocking down IRF-1 by as little as 38% markedly inhibits HIV-1 LTR–driven transcription. We further examined the dependence of high IRF-1 for transactivation of the HIV-1 LTR in replication-competent HIV-1 virus, unstimulated CD4+ T cells treated with IRF-1–specific siRNA were infecting with X4- and R5-tropic laboratory isolates (HIV-IIIB and HIV-BaL (MOI: 1.0)).50Philpott SM HIV-1 coreceptor usage, transmission, and disease progression.Curr HIV Res. 2003; 1: 217-227Crossref PubMed Scopus (99) Google Scholar At 96 hours postinfection, a significant reduction in transactivation of the HIV-1 LTR, measured by Gag RNA transcripts (Figure 4d) and p24 secretion (by ≥85%, Figure 4e), was observed in IRF-1 siRNA–treated cells, compared to control siRNA. Furthermore, flow cytometric analyses confirmed that less of the IRF-1 siRNA–transfected cells (with reduced endogenous IRF-1 level) were also positive for HIV-1 p24 (Figure 4f: 4%), compared to the control siRNA–transfected cells (Figure 4f: 20%). To insure that these results were specific to HIV-1, we again infected the transfected CD4+ T cells with a type 5 adenovirus (Ad5), containing a luciferase reporter gene. IRF-1 knockdown did not affect the transactivation of the Ad5 promoter, which contains no ISRE51Yao X Yoshioka Y Morishige T Eto Y Narimatsu S Mizuguchi H et al.Adenovirus vector covalently conjugated to polyethylene glycol with a cancer-specific promoter suppresses the tumor growth through systemic administration.Biol Pharm Bull. 2010; 33: 1073-1076Crossref Scopus (10) Google Scholar (Figure 4g). Together, these data indicate that a mere 38% decrease in endogenous IRF-1 could drastically impair HIV-1 LTR transactivation in unstimulated primary CD4+ T cells and thus, HIV replication. It further supports the hypothesis that naturally reduced IRF-1 expression observed in vivo may, at least partly, be accountable for reduced susceptibility to HIV infection observed in these HESN women.26Su RC Sivro A Kimani J Jaoko W Plummer FA Ball TB Epigenetic control of IRF1 responses in HIV-exposed seronegative versus HIV-susceptible individuals.Blood. 2011; 117: 2649-2657Crossref PubMed Scopus (32) Google Scholar,28Ball TB Ji H Kimani J McLaren P Marlin C Hill AV et al.Polymorphisms in IRF-1 associated with resistance to HIV-1 infection in highly exposed uninfected Kenyan sex workers.AIDS. 2007; 21: 1091-1101Crossref PubMed Scopus (73) Google Scholar IRF-1 binds to the ISRE at the promoter of numerous antiviral ISGs31Schneider WM Chevillotte MD Rice CM Interferon-stimulated genes: a complex web of host defenses.Annu Rev Immunol. 2014; 32: 513-545Crossref PubMed Scopus (1805) Google Scholar,33Ozato K Tailor P Kubota T The interferon regulatory factor family in host defense: mechanism of action.J Biol Chem. 2007; 282: 20065-20069Crossref PubMed Scopus (156) Google Scholar,46Chen FF Jiang G Xu K Zheng JN Function and mechanism by which interferon regulatory factor-1 inhibits oncogenesis.Oncol Lett. 2013; 5: 417-423Google Scholar and also regulates many other ISGs indirectly, not through direct binding to the promoter. We previously showed that HESN women who exhib" @default.
• W1843766852 created "2016-06-24" @default.
• W1843766852 creator A5007276113 @default.
• W1843766852 creator A5020486211 @default.
• W1843766852 creator A5029810760 @default.
• W1843766852 creator A5030906400 @default.
• W1843766852 creator A5034471348 @default.
• W1843766852 creator A5035611558 @default.
• W1843766852 creator A5042629291 @default.
• W1843766852 creator A5044279427 @default.
• W1843766852 creator A5076276906 @default.
• W1843766852 date "2015-01-01" @default.
• W1843766852 modified "2023-09-27" @default.
• W1843766852 title "Reducing IRF-1 to Levels Observed in HESN Subjects Limits HIV Replication, But Not the Extent of Host Immune Activation" @default.
• W1843766852 cites W132604487 @default.
• W1843766852 cites W1539748875 @default.
• W1843766852 cites W1854904866 @default.
• W1843766852 cites W1935842090 @default.
• W1843766852 cites W1954697740 @default.
• W1843766852 cites W1964441398 @default.
• W1843766852 cites W1969818067 @default.
• W1843766852 cites W1970197481 @default.
• W1843766852 cites W1973826424 @default.
• W1843766852 cites W1975766627 @default.
• W1843766852 cites W1977250127 @default.
• W1843766852 cites W1980606152 @default.
• W1843766852 cites W1984399257 @default.
• W1843766852 cites W2001448454 @default.
• W1843766852 cites W2008653158 @default.
• W1843766852 cites W2010609657 @default.
• W1843766852 cites W2015076056 @default.
• W1843766852 cites W2015647512 @default.
• W1843766852 cites W2015829024 @default.
• W1843766852 cites W2028674492 @default.
• W1843766852 cites W2030391162 @default.
• W1843766852 cites W2032478431 @default.
• W1843766852 cites W2032523448 @default.
• W1843766852 cites W2046810464 @default.
• W1843766852 cites W2048273698 @default.
• W1843766852 cites W2048535421 @default.
• W1843766852 cites W2049498011 @default.
• W1843766852 cites W2054682292 @default.
• W1843766852 cites W2059311140 @default.
• W1843766852 cites W2061169429 @default.
• W1843766852 cites W2068366465 @default.
• W1843766852 cites W2071086156 @default.
• W1843766852 cites W2071692035 @default.
• W1843766852 cites W2072963070 @default.
• W1843766852 cites W2078646756 @default.
• W1843766852 cites W2080340741 @default.
• W1843766852 cites W2085417709 @default.
• W1843766852 cites W2086509049 @default.
• W1843766852 cites W2087183134 @default.
• W1843766852 cites W2092894591 @default.
• W1843766852 cites W2096614211 @default.
• W1843766852 cites W2099470418 @default.
• W1843766852 cites W2103367520 @default.
• W1843766852 cites W2104586170 @default.
• W1843766852 cites W2114146889 @default.
• W1843766852 cites W2116412678 @default.
• W1843766852 cites W2123797251 @default.
• W1843766852 cites W2123943200 @default.
• W1843766852 cites W2124277087 @default.
• W1843766852 cites W2130968538 @default.
• W1843766852 cites W2131152496 @default.
• W1843766852 cites W2133362843 @default.
• W1843766852 cites W2133402546 @default.
• W1843766852 cites W2136311436 @default.
• W1843766852 cites W2140941354 @default.
• W1843766852 cites W2147640266 @default.
• W1843766852 cites W2150240056 @default.
• W1843766852 cites W2153317942 @default.
• W1843766852 cites W2153962603 @default.
• W1843766852 cites W2164107473 @default.
• W1843766852 cites W2167017590 @default.
• W1843766852 cites W2168510057 @default.
• W1843766852 cites W2170359168 @default.
• W1843766852 cites W2330029919 @default.
• W1843766852 doi "https://doi.org/10.1038/mtna.2015.29" @default.
• W1843766852 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4881757" @default.
• W1843766852 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/26506037" @default.
• W1843766852 hasPublicationYear "2015" @default.
• W1843766852 type Work @default.
• W1843766852 sameAs 1843766852 @default.
• W1843766852 citedByCount "5" @default.
• W1843766852 countsByYear W18437668522017 @default.
• W1843766852 countsByYear W18437668522019 @default.
• W1843766852 countsByYear W18437668522020 @default.
• W1843766852 countsByYear W18437668522022 @default.
• W1843766852 crossrefType "journal-article" @default.
• W1843766852 hasAuthorship W1843766852A5007276113 @default.
• W1843766852 hasAuthorship W1843766852A5020486211 @default.
• W1843766852 hasAuthorship W1843766852A5029810760 @default.
• W1843766852 hasAuthorship W1843766852A5030906400 @default.
• W1843766852 hasAuthorship W1843766852A5034471348 @default.
• W1843766852 hasAuthorship W1843766852A5035611558 @default.
• W1843766852 hasAuthorship W1843766852A5042629291 @default.
• W1843766852 hasAuthorship W1843766852A5044279427 @default.

• next