Clinical application of bronchoalveolar lavage fluid metagenomics next-generation sequencing in cancer patients with severe pneumonia.

OBJECTIVE: Metagenomic next-generation sequencing (mNGS), as an emerging technique for pathogen detection, has been widely used in clinic. However, reports on the application of mNGS in cancer patients with severe pneumonia remain limited. This study aims to evaluate the diagnostic performance of bronchoalveolar lavage fluid (BALF) mNGS in cancer patients complicated with severe pneumonia. METHODS: A total of 62 cancer patients with severe pneumonia simultaneously received culture and mNGS of BALF were enrolled in this study. We systematically analyzed the diagnostic significance of BALF mNGS. Subsequently, optimization of anti-infective therapy based on the distribution of pathogens obtained from BALF mNGS was also assessed. RESULTS: For bacteria and fungi, the positive detection rate of mNGS was significantly higher than culture method (91.94% versus 51.61%, P < 0.001), especially for poly-microbial infections (70.97% versus 12.90%, P < 0.001). Compared with the culture method, mNGS exhibited a diagnostic sensitivity of 100% and a specificity of 16.67%, with the positive predictive value (PPV) and negative predictive value (NPV) being 56.14% and 100%, respectively. The agreement rate between these two methods was 59.68%, whereas kappa consensus analysis indicated a poor concordance (kappa = 0.171). After receipt of BALF mNGS results, anti-infective treatment strategies in 39 out of 62 cases (62.90%) were optimized. Moreover, anti-tumor therapy was a high-risk factor for mixed infections (87.18% versus 65.22%, P = 0.04). CONCLUSIONS: The present study showed that cancer patients with severe pneumonia, especially those received anti-tumor therapy, were more likely to have poly-microbial infections. BALF mNGS can provide a rapid and comprehensive pathogen distribution of pulmonary infection, making it a promising technique in clinical practice, especially for optimizing therapeutic strategies for cancer patients.

LUCAT1 inhibits ferroptosis in bladder cancer by regulating the mRNA stability of STAT3.

OBJECT: In this study, we aimed to elucidate the role of LUCAT1, a recently identified lncRNA, in ferroptosis within the context of bladder cancer (BC). METHODS: Through a comprehensive array of experimental techniques, including transmission electron microscopy (TEM), RNA pull-down assays, and fluorescence in situ hybridization (FISH), we investigated the molecular interactions and functional consequences associated with LUCAT1 in BC cells. RESULTS: Our findings indicate that LUCAT1 acts as a pivotal regulator in BC, fostering cell proliferation, migration, and invasion, while concurrently impeding ferroptosis. Mechanistically, we unveiled a direct binding between LUCAT1 and insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), which governs the mRNA stability of signal transducer and activator of transcription 3 (STAT3). Intriguingly, ectopic expression of STAT3 counteracted the suppressive effect of LUCAT1 on ferroptosis induction in BC cells. Notably, in an in vivo setting, LUCAT1 emerged as a crucial modulator of ferroptosis inhibition in BC by regulating STAT3 mRNA stability. CONCLUSION: Collectively, our study identifies LUCAT1 as a novel oncogenic player, repressing ferroptosis in BC. These findings shed light on the intricate interplay between lncRNAs and ferroptosis in cancer, implicating LUCAT1 as a promising therapeutic target for patients afflicted with BC. Further investigations into the underlying mechanisms governing LUCAT1-mediated ferroptosis resistance are warranted, with the potential to uncover novel strategies for combating BC progression and improving patient outcomes.

Decidual macrophages derived NO downregulates PD-L1 in trophoblasts leading to decreased Treg cells in recurrent miscarriage.

Introduction: Placental trophoblasts contribute to regulatory T (Treg) function via the programmed cell death-1 (PD-1)/PD-1 ligand 1 (PD-L1) pathway during normal pregnancy. Decreased expression of PD-L1 in trophoblasts was closely associated with Treg deficiency in the development of pregnancy failure. Thus, targeting PD-L1 might be a novel therapy to prevent pregnancy loss. However, the mechanisms for modulating the expression of PD-L1 in trophoblasts are an enigma. Methods: The proportion of decidual Treg cells, and the profile of decidual macrophages (DMs) sampled from women with normal pregnancy (NP) and recurrent miscarriage (RM) were evaluated by flow cytometry. The expression of Yin and Yang 1 protein (YY1) and PD-L1 in human villous were measured by Immunohistochemistry (IHC), qRT-PCR and western blot. The determination of soluble PD-L1 (sPD-L1) in serum from NP and RM, and trophoblast conditioned media (TCM) was performed by the PD-L1 SimpleStep ELISA kit. Knockdown of YY1 was processed in the human trophoblast derived cell lines, HTR-8 and Bewo, with siYY1 transfection. Peripheral naïve CD4+ T cells were isolated from women with NP for the in vitro culture. The percentages of Treg cells differentiated from peripheral naïve CD4+ T cells were measured by flow cytometry. The interaction between YY1 and CD274 was proved by CHIP. The expression of inducible nitric oxide synthase (iNOS) in decidua was evaluated by IHC. The level of NO in serum from women with NP and RM was determined by the Griess reagent system. The effects of NO on YY1 were determined by the in vitro culture of HTR-8 cells with the NO donor, SNAP. The in vivo model comprising twelve pregnant mice and underwent different treatment. The percentages of Treg cells in murine uterus were measured by flow cytometry. Similarly, Western blot and IHC were performed to determine the expression of YY1 and PD-L1 in murine placenta. Results: Decreased expression of YY1 and PD-L1 in trophoblasts and lower proportion of decidual Treg cells were observed in patients with RM. Knockdown of YY1 contributes to a lower expression of YY1 and PD-L1. Soluble PD-L1 in the supernatant from HTR-8 cells was also decreased with siYY1 administration. Lower Treg differentiation was observed in the presence of supernatant from HTR-8 cells treated with siYY1. CHIP analysis revealed that endogenous YY1 directly occupied the promoter region of the CD274 (PD-L1) gene. Accompanied with increased M1 DMs, higher NO was observed in serum sampled from patients with RM. In the presence of Reduced expression of YY1 and PD-L1 was observed in HTR-8 cells with the treatment of SNAP. Furthermore, less Treg differentiation was observed with SNAP treated TCM. Moreover, our in vivo data found that YY1 deficiency was associated with decreased PD-L1, which further resulting in less Treg differentiation and Treg deficiency at the maternal-fetal interface and increased embryo loss. Discussion: Our work found the modulatory capacity of YY1 on PD-L1 in trophoblasts during early pregnancy. Furthermore, reduced YY1 was supposed resulting from higher levels of NO produced from the M1 DMs in RM.

Asymptomatic adult Wilms' tumor: A case report.

Wilms' tumor, also called nephroblastoma, is an extremely uncommon kidney tumor of adulthood. We reported a adult man with a left kidney mass diagnosed as Wilms' tumor. Case presentation: A 25-year-old man was hospitalized due to injury of the anterior cruciate ligament of the right knee. Preoperative imaging accidentally revealed a mass measuring 53 × 46 mm involving the middle and lower segments of the left kidney without evidence supporting the invasion of the surrounding structures or metastasis. The patient didn't show any symptom commonly occurred in Wilms' tumor, such as flank pain or hematuria. After nephrectomy, the diagnosis of adult Wilms' tumor was confirmed based on the tumor morphology and immunohistochemical findings. Conclusion: In adult patients without any clinical manifestations or favorable imaging findings for low-stage renal cell carcinoma, the diagnosis of Wilms' tumor should be taken into consideration.

Clinical characteristics of female patients diagnosed with congenital adrenal hyperplasia and plastic-esthetic related thoughts: a retrospective study.

OBJECTIVES: Congenital adrenal hyperplasia (CAH) is a rare disorder that can cause masculinization of the external genitalia in females, usually evident in neonates. To present a case series of female patients with CAH by summarizing their clinical features and outcomes. DESIGN: This retrospective study analyzed the clinical data of female patients with CAH admitted to the First Affiliated Hospital of Xiamen University from 1995 to 2019. MATERIALS AND METHODS: Clinical characteristics, CAH subtype, treatments, and outcomes were summarized from the medical records and analyzed. Follow-up was conducted after drug therapy and surgical treatment and was censored in 2019. RESULTS: Twenty-one female patients were diagnosed with CAH: 21-hydroxylase deficiency (21-OHD) in 17 patients and 17α-hydroxylase deficiency (17α-OHD) in four patients. The clinical manifestations of 21-OHD were clitoral hypertrophy, pigmentation, male secondary sexual development, genital malformation, sexual precocity, nausea, and vomiting. The clinical manifestations of 17α-OHD were hypertension, feminization, sexual infantilism, and pigmentation. The patients received hormone replacement therapy. When necessary, some patients underwent external genital organ orthomorphia or artificial periodic therapy. Twelve patients were followed up; their sexual development was improved, but seven patients had poor breast development due to late diagnosis and/or poor hormone treatment adherence. CONCLUSION: Female CAH patients are subject to genital deformities, virilizing signs, breast dysplasia, and other appearance defects. The purpose of this report is to improve plastic and esthetic surgeons' understanding of CAH.

INI1/SMARCB1 Rpt1 domain mimics TAR RNA in binding to integrase to facilitate HIV-1 replication.

INI1/SMARCB1 binds to HIV-1 integrase (IN) through its Rpt1 domain and exhibits multifaceted role in HIV-1 replication. Determining the NMR structure of INI1-Rpt1 and modeling its interaction with the IN-C-terminal domain (IN-CTD) reveal that INI1-Rpt1/IN-CTD interface residues overlap with those required for IN/RNA interaction. Mutational analyses validate our model and indicate that the same IN residues are involved in both INI1 and RNA binding. INI1-Rpt1 and TAR RNA compete with each other for IN binding with similar IC50 values. INI1-interaction-defective IN mutant viruses are impaired for incorporation of INI1 into virions and for particle morphogenesis. Computational modeling of IN-CTD/TAR complex indicates that the TAR interface phosphates overlap with negatively charged surface residues of INI1-Rpt1 in three-dimensional space, suggesting that INI1-Rpt1 domain structurally mimics TAR. This possible mimicry between INI1-Rpt1 and TAR explains the mechanism by which INI1/SMARCB1 influences HIV-1 late events and suggests additional strategies to inhibit HIV-1 replication.

Dietary patterns and chronic kidney disease risk: a systematic review and updated meta-analysis of observational studies.

BACKGROUND: A number of studies have reported the association between dietary patterns and the risk of chronic kidney disease (CKD), however a consistent perspective hasn't been established to date. Herein, we conducted this systematic review and meta-analysis of observational studies to assess the association between dietary patterns and CKD. METHODS: MEDLINE, EBSCO and references from eligible studies were searched for relevant articles published up to 9 May 2020 that examined the association of common dietary patterns and CKD. The heterogeneity among studies was assessed by Cochran's Q test and I2 methods. RESULTS: Seventeen eligible studies, involving 149,958 participants, were included in our systematic review and meta-analysis. The highest compared with the lowest category of healthy dietary pattern was significantly associated with a lower risk of CKD (OR=0.69; CI: 0.57, 0.84; P=0.0001). A higher risk of CKD was shown for the highest compared with the lowest categories of Western-type dietary pattern (OR=1.86; CI: 1.21, 2.86; P=0.005). There were evidence of a lower risk of CKD in the highest compared with the lowest categories of light-moderate drinking pattern (OR=0.76; CI: 0.71, 0.81; P< 0.0001) and heavy drinking pattern (OR=0.67; CI: 0.56, 0.80; P< 0.0001). CONCLUSIONS: The results of this systematic review and meta-analysis show that a healthy dietary pattern and alcohol drinking were associated with lower risk of CKD, whereas a Western-type dietary pattern was associated with higher risk of CKD.

Long non-coding RNA in bladder cancer.

Bladder cancer (BC) is the ninth most common malignant disease and ranks fourteenth in cancer mortality worldwide. Moreover, among cancers, the incidence and mortality of BC in males increased to the 6th and 9th place, respectively. The overall survival (OS) declines dramatically as the cancer progresses, especially when urothelial cells transition from noninvasive to invasive. It is well known that epithelial cells can acquire invasive properties and a propensity to metastasize through the epithelial-to-mesenchymal transition (EMT) process in tumourigenesis and progression. However, the potential molecular mechanisms and key pathways are still unclear. As the sequencing technology advances, long non-coding RNAs (lncRNAs) have been proven to play an important role in regulating biological processes and cellular pathways. Here, we reviewed important lncRNAs, such as H19, UCA1 and MALAT1, that participate in the malignant phenotype of BC and regulate EMT signalling networks in the invasion-metastasis cascade during BC development. We further discuss MALAT1, PCAT-1 and SPRY4-IT1, and also urine and blood exosomal H19 and PTENP as potential noninvasive biomarkers. Moreover, antisense oligonucleotides (ASOs) and a double-stranded DNA plasmid (BC-819) have been designed for use in preclinical cancer models and clinical trials in patients. Therefore, the results of investigations have gradually prompted the utility of lncRNAs.

CCL2 mobilizes ALIX to facilitate Gag-p6 mediated HIV-1 virion release.

Cellular ESCRT machinery plays pivotal role in HIV-1 budding and release. Extracellular stimuli that modulate HIV-1 egress are currently unknown. We found that CCL2 induced by HIV-1 clade B (HIV-1B) infection of macrophages enhanced virus production, while CCL2 immuno-depletion reversed this effect. Additionally, HIV-1 clade C (HIV-1C) was refractory to CCL2 levels. We show that CCL2-mediated increase in virus production requires Gag late motif LYPX present in HIV-1B, but absent in HIV-1C, and ALIX protein that recruits ESCRT III complex. CCL2 immuno-depletion sequestered ALIX to F-actin structures, while CCL2 addition mobilized it to cytoplasm facilitating Gag-ALIX binding. The LYPX motif improves virus replication and its absence renders the virus less fit. Interestingly, novel variants of HIV-1C with PYRE/PYKE tetrapeptide insertions in Gag-p6 conferred ALIX binding, CCL2-responsiveness and enhanced virus replication. These results, for the first time, indicate that CCL2 mediates ALIX mobilization from F-actin and enhances HIV-1 release and fitness.

Long noncoding RNA SNHG12 promotes cell proliferation and activates Wnt/ß-catenin signaling in prostate cancer through sponging microRNA-195.

Long noncoding RNAs (lncRNAs) serve critical roles in multiple human malignant tumors, including prostate cancer (PCa). Currently, the biological role of oncogenic lncRNA SNHG12 in PCa remains largely unclear. In the present study, we found that SNHG12 was highly expressed in human PCa tissues and cell lines. In addition, gain-of-function and loss-of-function studies showed that overexpression of SNHG12 promoted, while downregulation suppressed the proliferation, invasion, and migration of PCa cells in vitro. Knockdown of SNHG12 also repressed PCa xenograft tumor growth in vivo. Further in-depth mechanistic studies showed that SNHG12 might serve as a competing endogenous RNA for miR-195 in PCa cells, and miR-195 expression level was negatively associated with the expression of SNHG12 in PCa tissues. Finally, we found that the activity of Wnt/ß-catenin signaling is enhanced by SNHG12 overexpression and rescued by co-transfection with miR-195 mimics in PCa cells. Collectively, the present study indicated the oncogenic function of SNHG12 in PCa and our findings might provide a new target in the treatment of PCa.

Intraoral Prosthetic Chin Augmentation With Vertical Incisions.

To explore a new surgical approach for chin augmentation using a prosthesis with 3 intraoral vertical incisions whereby placement of the prosthesis is more convenient and accurate, with fewer postoperative complications. Following the anatomic characteristics of the chin, a bilateral mucosal vertical incision and a median observation incision are made. The V-shaped mark on the upper side of the prosthesis can be seen through the observation incision after it is placed from the lateral incision into the predesigned compartment. The incision can be sutured if there is no bleeding in the operation area. Surgery performed in all 19 patients with mild microgenia with 6 to 12 months of follow-up resulted in satisfactory chin and face shape without any complications. The application of this novel method can correct McCarthy type I microgenia with more accurate positioning, less possibility of bilateral sideways and/or up/down movement, and fewer complications.

An Essential Role of INI1/hSNF5 Chromatin Remodeling Protein in HIV-1 Posttranscriptional Events and Gag/Gag-Pol Stability.

INI1/hSNF5/SMARCB1/BAF47 is an HIV-specific integrase (IN)-binding protein that influences HIV-1 transcription and particle production. INI1 binds to SAP18 (Sin3a-associated protein, 18 kDa), and both INI1 and SAP18 are incorporated into HIV-1 virions. To determine the significance of INI1 and the INI1-SAP18 interaction during HIV-1 replication, we isolated a panel of SAP18-interaction-defective (SID)-INI1 mutants using a yeast reverse two-hybrid screen. The SID-INI1 mutants, which retained the ability to bind to IN, cMYC, and INI1 but were impaired for binding to SAP18, were tested for their effects on HIV-1 particle production. SID-INI1 dramatically reduced the intracellular Gag/Gag-Pol protein levels and, in addition, decreased viral particle production. The SID-INI1-mediated effects were less dramatic in trans complementation assays using IN deletion mutant viruses with Vpr-reverse transcriptase (RT)-IN. SID-INI1 did not inhibit long-terminal-repeat (LTR)-mediated transcription, but it marginally decreased the steady-state gag RNA levels, suggesting a posttranscriptional effect. Pulse-chase analysis indicated that in SID-INI1-expressing cells, the pr55Gag levels decreased rapidly. RNA interference analysis indicated that small hairpin RNA (shRNA)-mediated knockdown of INI1 reduced the intracellular Gag/Gag-Pol levels and further inhibited HIV-1 particle production. These results suggest that SID-INI1 mutants inhibit multiple stages of posttranscriptional events of HIV-1 replication, including intracellular Gag/Gag-Pol RNA and protein levels, which in turn inhibits assembly and particle production. Interfering INI1 leads to a decrease in particle production and Gag/Gag-Pol protein levels. Understanding the role of INI1 and SAP18 in HIV-1 replication is likely to provide novel insight into the stability of Gag/Gag-Pol, which may lead to the development of novel therapeutic strategies to inhibit HIV-1 late events. IMPORTANCE: Significant gaps exist in our current understanding of the mechanisms and host factors that influence HIV-1 posttranscriptional events, including gag RNA levels, Gag/Gag-Pol protein levels, assembly, and particle production. Our previous studies suggested that the IN-binding host factor INI1 plays a role in HIV-1 assembly. An ectopically expressed minimal IN-binding domain of INI1, S6, potently and selectively inhibited HIV-1 Gag/Gag-Pol trafficking and particle production. However, whether or not endogenous INI1 and its interacting partners, such as SAP18, are required for late events was unknown. Here, we report that endogenous INI1 and its interaction with SAP18 are necessary to maintain intracellular levels of Gag/Gag-Pol and for particle production. Interfering INI1 or the INI1-SAP18 interaction leads to the impairment of these processes, suggesting a novel strategy for inhibiting posttranscriptional events of HIV-1 replication.

Preparation and Identification of a Monoclonal Antibody Against Cystatin C.

Cystatin C (Cys C) has been shown to be an excellent marker of renal function, especially when evaluating the early stages of acute kidney injury. It is less affected by age, gender, muscle mass, and ethnicity. The detection of Cys C is important and has broad application prospects. Therefore, we have developed a panel of monoclonal antibodies against Cys C that can be used to establish an enzyme-linked immunosorbent assay (ELISA) kit and paired for further use in other methods of detecting Cys C. This study describes the preparation, application, and characterization of monoclonal antibodies used in ELISA. The antibodies were developed by PEG fusion of the SP2/0 cells with splenic B cells from Cys C immunized BALB/c mice. Antibody-producing cells were identified by ELISA and Western blot analysis. By way of cloning and screening, four hybridoma cell lines were established. Simultaneously large-scale monoclonal antibodies produced in mice ascites were prepared. The results showed that the cell clone 8D12 could be used in immunohistochemical staining. With the ELISA additivity test, we got a preliminarily finding that the monoclonal antibodies were not on the same epitope. The antibody matching test showed that 5D7 and 7A8 successfully paired with 8D12, and the optimal reaction conditions were initially identified.

Expression and purification of soluble human cystatin C in Escherichia coli with maltose-binding protein as a soluble partner.

Human cystatin C (CYSC) is a 13-kDa endogenous cysteine proteinase inhibitor and was investigated as a replacement for creatinine as a marker of renal function. However, expressing recombinant CYSC is difficult in Escherichia coli because of resulting low yield and insufficient purity and insolubility. Here, we cloned and fused CYSC to the C-terminus of three soluble partners - maltose-binding protein (MBP), glutathione S-transferase (GST) and translation initiation factor 2 domain I (IF2) - to screen for their ability to improve the solubility of recombinant CYSC when expressed in E. coli. MBP was best at enhancing the soluble expression of CYSC, with soluble fractions accounting for 92.8±3.11% of all proteins. For scaled production, we purified the de-tagged CYSC by using a 3C protease-cleaved MBP-T3-CYSC fused protein with immobilized metal affinity chromatography and cation-affinity purification. The molecular weights of the de-tagged CYSC and human natural CYSC were similar, and the former could react specifically with CYSC polyclonal antibody. Moreover, the de-tagged CYSC displayed full biological activity against papain and cathepsin B, which was very similar to that of the human natural CYSC protein standard. We provide a method to produce large amounts of soluble recombinant human CYSC in E. coli.

INI1/hSNF5-interaction defective HIV-1 IN mutants exhibit impaired particle morphology, reverse transcription and integration in vivo.

BACKGROUND: Retroviral integrase catalyzes integration of viral DNA into the host genome. Integrase interactor (INI)1/hSNF5 is a host factor that binds to HIV-1 IN within the context of Gag-Pol and is specifically incorporated into HIV-1 virions during assembly. Previous studies have indicated that INI1/hSNF5 is required for late events in vivo and for integration in vitro. To determine the effects of disrupting the IN-INI1 interaction on the assembly and infectivity of HIV-1 particles, we isolated mutants of IN that are defective for binding to INI1/hSNF5 and tested their effects on HIV-1 replication. RESULTS: A reverse yeast two-hybrid system was used to identify INI1-interaction defective IN mutants (IID-IN). Since protein-protein interactions depend on the surface residues, the IID-IN mutants that showed high surface accessibility on IN crystal structures (K71R, K111E, Q137R, D202G, and S147G) were selected for further study. In vitro interaction studies demonstrated that IID-IN mutants exhibit variable degrees of interaction with INI1. The mutations were engineered into HIV-1(NL4-3) and HIV-Luc viruses and tested for their effects on virus replication. HIV-1 harboring IID-IN mutations were defective for replication in both multi- and single-round infection assays. The infectivity defects were correlated to the degree of INI1 interaction of the IID-IN mutants. Highly defective IID-IN mutants were blocked at early and late reverse transcription, whereas partially defective IID-IN mutants proceeded through reverse transcription and nuclear localization, but were partially impaired for integration. Electron microscopic analysis of mutant particles indicated that highly interaction-defective IID-IN mutants produced morphologically aberrant virions, whereas the partially defective mutants produced normal virions. All of the IID-IN mutant particles exhibited normal capsid stability and reverse transcriptase activity in vitro. CONCLUSIONS: Our results demonstrate that a severe defect in IN-INI1 interaction is associated with production of defective particles and a subsequent defect in post-entry events. A partial defect in IN-INI1 interaction leads to production of normal virions that are partially impaired for early events including integration. Our studies suggest that proper interaction of INI1 with IN within Gag-Pol is necessary for proper HIV-1 morphogenesis and integration.

Recruitment of a SAP18-HDAC1 complex into HIV-1 virions and its requirement for viral replication.

HIV-1 integrase (IN) is a virally encoded protein required for integration of viral cDNA into host chromosomes. INI1/hSNF5 is a component of the SWI/SNF complex that interacts with HIV-1 IN, is selectively incorporated into HIV-1 (but not other retroviral) virions, and modulates multiple steps, including particle production and infectivity. To gain further insight into the role of INI1 in HIV-1 replication, we screened for INI1-interacting proteins using the yeast two-hybrid system. We found that SAP18 (Sin3a associated protein 18 kD), a component of the Sin3a-HDAC1 complex, directly binds to INI1 in yeast, in vitro and in vivo. Interestingly, we found that IN also binds to SAP18 in vitro and in vivo. SAP18 and components of a Sin3A-HDAC1 complex were specifically incorporated into HIV-1 (but not SIV and HTLV-1) virions in an HIV-1 IN-dependent manner. Using a fluorescence-based assay, we found that HIV-1 (but not SIV) virion preparations harbour significant deacetylase activity, indicating the specific recruitment of catalytically active HDAC into the virions. To determine the requirement of virion-associated HDAC1 to HIV-1 replication, an inactive, transdominant negative mutant of HDAC1 (HDAC1(H141A)) was utilized. Incorporation of HDAC1(H141A) decreased the virion-associated histone deacetylase activity. Furthermore, incorporation of HDAC1(H141A) decreased the infectivity of HIV-1 (but not SIV) virions. The block in infectivity due to virion-associated HDAC1(H141A) occurred specifically at the early reverse transcription stage, while entry of the virions was unaffected. RNA-interference mediated knock-down of HDAC1 in producer cells resulted in decreased virion-associated HDAC1 activity and a reduction in infectivity of these virions. These studies indicate that HIV-1 IN and INI1/hSNF5 bind SAP18 and selectively recruit components of Sin3a-HDAC1 complex into HIV-1 virions. Furthermore, HIV-1 virion-associated HDAC1 is required for efficient early post-entry events, indicating a novel role for HDAC1 during HIV-1 replication.

HIV-1 replication in cell lines harboring INI1/hSNF5 mutations.

BACKGROUND: INI1/hSNF5 is a cellular protein that directly interacts with HIV-1 integrase (IN). It is specifically incorporated into HIV-1 virions. A dominant negative mutant derived from INI1 inhibits HIV-1 replication. Recent studies indicate that INI1 is associated with pre-integration and reverse transcription complexes that are formed upon viral entry into the target cells. INI1 also is a tumor suppressor, biallelically deleted/mutated in malignant rhabdoid tumors. We have utilized cell lines derived from the rhabdoid tumors, MON and STA-WT1, that harbor either null or truncating mutations of INI1 respectively, to assess the effect of INI1 on HIV-1 replication. RESULTS: We found that while HIV-1 virions produced in 293T cells efficiently transduced MON and STA-WT1 cells, HIV-1 particle production was severely reduced in both of these cells. Reintroduction of INI1 into MON and STA-WT1 significantly enhanced the particle production in both cell lines. HIV-1 particles produced in MON cells were reduced for infectivity, while those produced in STA-WT1 were not. Further analysis indicated the presence of INI1 in those virions produced from STA-WT1 but not from those produced from MON cells. HIV-1 produced in MON cells were defective for synthesis of early and late reverse transcription products in the target cells. Furthermore, virions produced in MON cells were defective for exogenous reverse transcriptase activity carried out using exogenous template, primer and substrate. CONCLUSION: Our results suggest that INI1-deficient cells exhibit reduced particle production that can be partly enhanced by re-introduction of INI1. Infectivity of HIV-1 produced in some but not all INI1 defective cells, is affected and this defect may correlate to the lack of INI1 and/or some other proteins in these virions. The block in early events of virion produced from MON cells appears to be at the stage of reverse transcription. These studies suggest that presence of INI1 or some other host factor in virions and reverse transcription complexes may be important for early events of HIV-1 replication.