HIV-Associated Hypertension: Risks, Mechanisms, and Knowledge Gaps.

HIV type 1 (HIV-1) is the causative agent of AIDS. Since the start of the epidemic, HIV/AIDS has been responsible for ≈40 million deaths. Additionally, an estimated 39 million people are currently infected with the virus. HIV-1 primarily infects immune cells, such as CD4+ (cluster of differentiation 4+) T lymphocytes (T cells), and as a consequence, the number of CD4+ T cells progressively declines in people living with HIV. Within a span of ≈10 years, HIV-1 infection leads to the systemic failure of the immune system and progression to AIDS. Fortunately, potent antiviral therapy effectively controls HIV-1 infection and prevents AIDS-related deaths. The efficacy of the current antiviral therapy regimens has transformed the outcome of HIV/AIDS from a death sentence to a chronic disease with a prolonged lifespan of people living with HIV. However, antiviral therapy is not curative, is challenged by virus resistance, can be toxic, and, most importantly, requires lifelong adherence. Furthermore, the improved lifespan has resulted in an increased incidence of non-AIDS-related morbidities in people living with HIV including cardiovascular diseases, renal disease, liver disease, bone disease, cancer, and neurological conditions. In this review, we summarize the current state of knowledge of the cardiovascular comorbidities associated with HIV-1 infection, with a particular focus on hypertension. We also discuss the potential mechanisms known to drive HIV-1-associated hypertension and the knowledge gaps in our understanding of this comorbid condition. Finally, we suggest several directions of future research to better understand the factors, pathways, and mechanisms underlying HIV-1-associated hypertension in the post-antiviral therapy era.

Three prime repair exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural, and conformational properties.

HIV-1 integration into the human genome is dependent on 3'-processing of the viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3'-processed DNA remained resistant. Here, we describe the mechanism by which the 3'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3'-processed DNA was significantly lower (approximately 2-2.5-fold) than the unprocessed HIV-1 DNA by TREX1. The kcat values of human TREX1 for the processed U5 and U3 DNA substrates were 3.8 s-1 and 4.5 s-1, respectively. In contrast, the unprocessed U5 and U3 substrates were cleaved at 10.2 s-1 and 9.8 s-1, respectively. The efficiency of degradation (kcat/Km) of the 3'-processed DNA (U5-70.2 and U3-28.05 pM-1s-1) was also significantly lower than the unprocessed DNA (U5-103.1 and U3-65.3 pM-1s-1). Furthermore, the binding affinity (Kd) of TREX1 was markedly lower (∼2-fold) for the 3'-processed DNA than the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3'-processed DNA. These results pinpoint the mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3'-processed HIV-1 DNA.

KLF6 activates Sp1-mediated prolidase transcription during TGF-ß1 signaling.

Prolidase (PEPD) is the only hydrolase that cleaves the dipeptides containing C-terminal proline or hydroxyproline-the rate-limiting step in collagen biosynthesis. However, the molecular regulation of prolidase expression remains largely unknown. In this study, we have identified overlapping binding sites for the transcription factors Krüppel-like factor 6 (KLF6) and Specificity protein 1 (Sp1) in the PEPD promoter and demonstrate that KLF6/Sp1 transcriptionally regulate prolidase expression. By cloning the PEPD promoter into a luciferase reporter and through site-directed deletion, we pinpointed the minimal sequences required for KLF6 and Sp1-mediated PEPD promoter-driven transcription. Interestingly, Sp1 inhibition abrogated KLF6-mediated PEPD promoter activity, suggesting that Sp1 is required for the basal expression of prolidase. We further studied the regulation of PEPD by KLF6 and Sp1 during transforming growth factor ß1 (TGF-ß1) signaling, since both KLF6 and Sp1 are key players in TGF-ß1 mediated collagen biosynthesis. Mouse and human fibroblasts exposed to TGF-ß1 resulted in the induction of PEPD transcription and prolidase expression. Inhibition of TGF-ß1 signaling abrogated PEPD promoter-driven transcriptional activity of KLF6 and Sp1. Knock-down of KLF6 as well as Sp1 inhibition also reduced prolidase expression. Chromatin immunoprecipitation assay supported direct binding of KLF6 and Sp1 to the PEPD promoter and this binding was enriched by TGF-ß1 treatment. Finally, immunofluorescence studies showed that KLF6 co-operates with Sp1 in the nucleus to activate prolidase expression and enhance collagen biosynthesis. Collectively, our results identify functional elements of the PEPD promoter for KLF6 and Sp1-mediated transcriptional activation and describe the molecular mechanism of prolidase expression.

Emerging role of cyclophilin A in HIV-1 infection: from producer cell to the target cell nucleus.

The HIV-1 genome encodes a small number of proteins with structural, enzymatic, regulatory, and accessory functions. These viral proteins interact with a number of host factors to promote the early and late stages of HIV-1 infection. During the early stages of infection, interactions between the viral proteins and host factors enable HIV-1 to enter the target cell, traverse the cytosol, dock at the nuclear pore, gain access to the nucleus, and integrate into the host genome. Similarly, the viral proteins recruit another set of host factors during the late stages of infection to orchestrate HIV-1 transcription, translation, assembly, and release of progeny virions. Among the host factors implicated in HIV-1 infection, Cyclophilin A (CypA) was identified as the first host factor to be packaged within HIV-1 particles. It is now well established that CypA promotes HIV-1 infection by directly binding to the viral capsid. Mechanistic models to pinpoint CypA's role have spanned from an effect in the producer cell to the early steps of infection in the target cell. In this review, we will describe our understanding of the role(s) of CypA in HIV-1 infection, highlight the current knowledge gaps, and discuss the potential role of this host factor in the post-nuclear entry steps of HIV-1 infection.

HIV-1 Preintegration Complex Preferentially Integrates the Viral DNA into Nucleosomes Containing Trimethylated Histone 3-Lysine 36 Modification and Flanking Linker DNA.

HIV-1 DNA is preferentially integrated into chromosomal hot spots by the preintegration complex (PIC). To understand the mechanism, we measured the DNA integration activity of PICs-extracted from infected cells-and intasomes, biochemically assembled PIC substructures using a number of relevant target substrates. We observed that PIC-mediated integration into human chromatin is preferred compared to genomic DNA. Surprisingly, nucleosomes lacking histone modifications were not preferred integration compared to the analogous naked DNA. Nucleosomes containing the trimethylated histone 3 lysine 36 (H3K36me3), an epigenetic mark linked to active transcription, significantly stimulated integration, but the levels remained lower than the naked DNA. Notably, H3K36me3-modified nucleosomes with linker DNA optimally supported integration mediated by the PIC but not by the intasome. Interestingly, optimal intasome-mediated integration required the cellular cofactor LEDGF. Unexpectedly, LEDGF minimally affected PIC-mediated integration into naked DNA but blocked integration into nucleosomes. The block for the PIC-mediated integration was significantly relieved by H3K36me3 modification. Mapping the integration sites in the preferred substrates revealed that specific features of the nucleosome-bound DNA are preferred for integration, whereas integration into naked DNA was random. Finally, biochemical and genetic studies demonstrate that DNA condensation by the H1 protein dramatically reduces integration, providing further evidence that features inherent to the open chromatin are preferred for HIV-1 integration. Collectively, these results identify the optimal target substrate for HIV-1 integration, report a mechanistic link between H3K36me3 and integration preference, and importantly, reveal distinct mechanisms utilized by the PIC for integration compared to the intasomes. IMPORTANCE HIV-1 infection is dependent on integration of the viral DNA into the host chromosomes. The preintegration complex (PIC) containing the viral DNA, the virally encoded integrase (IN) enzyme, and other viral/host factors carries out HIV-1 integration. HIV-1 integration is not dependent on the target DNA sequence, and yet the viral DNA is selectively inserted into specific "hot spots" of human chromosomes. A growing body of literature indicates that structural features of the human chromatin are important for integration targeting. However, the mechanisms that guide the PIC and enable insertion of the PIC-associated viral DNA into specific hot spots of the human chromosomes are not fully understood. In this study, we describe a biochemical mechanism for the preference of the HIV-1 DNA integration into open chromatin. Furthermore, our study defines a direct role for the histone epigenetic mark H3K36me3 in HIV-1 integration preference and identify an optimal substrate for HIV-1 PIC-mediated viral DNA integration.

Human Three Prime Repair Exonuclease 1 Promotes HIV-1 Integration by Preferentially Degrading Unprocessed Viral DNA.

Three prime repair exonuclease 1 (TREX1) is the most abundant 3'→5' exonuclease in mammalian cells. It has been suggested that TREX1 degrades HIV-1 DNA to enable the virus to evade the innate immune system. However, the exact role of TREX1 during early steps of HIV-1 infection is not clearly understood. In this study, we report that HIV-1 infection is associated with upregulation, perinuclear accumulation, and nuclear localization of TREX1. However, TREX1 overexpression did not affect reverse transcription or nuclear entry of the virus. Surprisingly, HIV-1 DNA integration was increased in TREX1-overexpressing cells, suggesting a role of the exonuclease in the post-nuclear entry step of infection. Accordingly, preintegration complexes (PICs) extracted from TREX1-overexpressing cells retained higher levels of DNA integration activity. TREX1 depletion resulted in reduced levels of proviral integration, and PICs formed in TREX1-depleted cells retained lower DNA integration activity. Addition of purified TREX1 to PICs also enhanced DNA integration activity, suggesting that TREX1 promotes HIV-1 integration by stimulating PIC activity. To understand the mechanism, we measured TREX1 exonuclease activity on substrates containing viral DNA ends. These studies revealed that TREX1 preferentially degrades the unprocessed viral DNA, but the integration-competent 3'-processed viral DNA remains resistant to degradation. Finally, we observed that TREX1 addition stimulates the activity of HIV-1 intasomes assembled with the unprocessed viral DNA but not that of intasomes containing the 3'-processed viral DNA. These biochemical analyses provide a mechanism by which TREX1 directly promotes HIV-1 integration. Collectively, our study demonstrates that HIV-1 infection upregulates TREX1 to facilitate viral DNA integration. IMPORTANCE Productive HIV-1 infection is dependent on a number of cellular factors. Therefore, a clear understanding of how the virus exploits the cellular machinery will identify new targets for inhibiting HIV-1 infection. The three prime repair exonuclease 1 (TREX1) is the most active cellular exonuclease in mammalian cells. It has been reported that TREX1 prevents accumulation of HIV-1 DNA and enables the virus to evade the host innate immune response. Here, we show that HIV-1 infection results in the upregulation, perinuclear accumulation, and nuclear localization of TREX1. We also provide evidence that TREX1 promotes HIV-1 integration by preferentially degrading viral DNAs that are incompatible with chromosomal insertion. These observations identify a novel role of TREX1 in a post-nuclear entry step of HIV-1 infection.

The HIV-1 capsid-binding host factor CPSF6 is post-transcriptionally regulated by the cellular microRNA miR-125b.

Cleavage and polyadenylation specificity factor 6 (CPSF6) is a cellular protein involved in mRNA processing. Emerging evidence suggests that CPSF6 also plays key roles in HIV-1 infection, specifically during nuclear import and integration targeting. However, the cellular and molecular mechanisms that regulate CPSF6 expression are largely unknown. In this study, we report a post-transcriptional mechanism that regulates CPSF6 via the cellular microRNA miR-125b. An in silico analysis revealed that the 3'UTR of CPSF6 contains a miR-125b-binding site that is conserved across several mammalian species. Because miRNAs repress protein expression, we tested the effects of miR-125b expression on CPSF6 levels in miR-125b knockdown and over-expression experiments, revealing that miR-125b and CPSF6 levels are inversely correlated. To determine whether miR-125b post-transcriptionally regulates CPSF6, we introduced the 3'UTR of CPSF6 mRNA into a luciferase reporter and found that miR-125b negatively regulates CPSF6 3'UTR-driven luciferase activity. Accordingly, mutations in the miR-125b seed sequence abrogated the regulatory effect of the miRNA on the CPSF6 3'UTR. Finally, pulldown experiments demonstrated that miR-125b physically interacts with CPSF6 3'UTR. Interestingly, HIV-1 infection down-regulated miR-125b expression concurrent with up-regulation of CPSF6. Notably, miR-125b down-regulation in infected cells was not due to reduced pri-miRNA or pre-miRNA levels. However, miR-125b down-regulation depended on HIV-1 reverse transcription but not viral DNA integration. These findings establish a post-transcriptional mechanism that controls CPSF6 expression and highlight a novel function of miR-125b during HIV-host interaction.

Retention of removable complete dentures made with different posterior palatal seal techniques and oral health quality of life: A clinical study.

STATEMENT OF PROBLEM: How different methods of recording posterior palatal seal affect removable complete-denture retention and oral health quality of life is unclear. PURPOSE: The purpose of this clinical study was to compare the retention and oral health quality of life (OHIP-14) between conventional and arbitrary posterior palatal seal techniques in participants with removable complete dentures. MATERIAL AND METHODS: Edentulous patients were recruited according to definitive criteria. The participants were randomly divided into conventional and arbitrary seal. After the delivery of the denture, the retention was evaluated with a force gauge dynamometer and at 3, 6, 9, and 12 months. Denture satisfaction was evaluated with the OHIP-14 questionnaire. Data were statistically analyzed by using the t test and repeated measure ANOVA (α=.05). RESULTS: The mean ±standard deviation values (N) for conventional seal at 0, 3, 6, 9, and 12 months by dynamometer in the anterior region ranged from 4.73 ±0.78 to 4.90 ±0.81 and in the posterior region between 5.07 ±0.84 and 5.31 ±0.99. Dynamometer values for arbitrary seal in the anterior region were from 4.56 ±0.77 to 4.88 ±0.81, and in the posterior, it varied between 4.74 ±0.74 and 5.15 ±0.81. Force gauge values (N) for conventional and arbitrary seal were in the range of 18.35 ±2.84 to 20.69 ±3.89. The general mean ±SD OHIP-14 was higher for the conventional seal at 3.12 ±0.25 than for the arbitrary seal at 2.73 ±0.23 The difference between the conventional and arbitrary seal techniques was not statistically significant (P>.05) CONCLUSIONS: No significant difference in complete denture retention was detected between the 2 posterior palatal seal techniques. Oral health quality of life was higher with the conventional seal technique.

PF74 Inhibits HIV-1 Integration by Altering the Composition of the Preintegration Complex.

The HIV-1 capsid protein (CA) facilitates reverse transcription and nuclear entry of the virus. However, CA's role in post-nuclear entry steps remains speculative. We describe a direct link between CA and integration by employing the capsid inhibitor PF74 as a probe coupled with the biochemical analysis of HIV-1 preintegration complexes (PICs) isolated from acutely infected cells. At a low micromolar concentration, PF74 potently inhibited HIV-1 infection without affecting reverse transcription. Surprisingly, PF74 markedly reduced proviral integration owing to inhibition of nuclear entry and/or integration. However, a 2-fold reduction in nuclear entry by PF74 did not quantitatively correlate with the level of antiviral activity. Titration of PF74 against the integrase inhibitor raltegravir showed an additive antiviral effect that is dependent on a block at the post-nuclear entry step. PF74's inhibitory effect was not due to the formation of defective viral DNA ends or a delay in integration, suggesting that the compound inhibits PIC-associated integration activity. Unexpectedly, PICs recovered from cells infected in the presence of PF74 exhibited elevated integration activity. PF74's effect on PIC activity is CA specific since the compound did not increase the integration activity of PICs of a PF74-resistant HIV-1 CA mutant. Sucrose gradient-based fractionation studies revealed that PICs assembled in the presence of PF74 contained lower levels of CA, suggesting a negative association between CA and PIC-associated integration activity. Finally, the addition of a CA-specific antibody or PF74 inhibited PIC-associated integration activity. Collectively, our results demonstrate that PF74's targeting of PIC-associated CA results in impaired HIV-1 integration.IMPORTANCE Antiretroviral therapy (ART) that uses various combinations of small molecule inhibitors has been highly effective in controlling HIV. However, the drugs used in the ART regimen are expensive, cause side effects, and face viral resistance. The HIV-1 CA plays critical roles in the virus life cycle and is an attractive therapeutic target. While currently there is no CA-based therapy, highly potent CA-specific inhibitors are being developed as a new class of antivirals. Efforts to develop a CA-targeted therapy can be aided through a clear understanding of the role of CA in HIV-1 infection. CA is well established to coordinate reverse transcription and nuclear entry of the virus. However, the role of CA in post-nuclear entry steps of HIV-1 infection is poorly understood. We show that a CA-specific drug PF74 inhibits HIV-1 integration revealing a novel role of this multifunctional viral protein in a post-nuclear entry step of HIV-1 infection.

Analysis of Shade Matching in Natural Dentitions Using Intraoral Digital Spectrophotometer in LED and Filtered LED Light Sources.

PURPOSE: To evaluate the shade matching capabilities in natural dentitions using Vita Toothguide 3D-Master and an intraoral digital spectrophotometer (Vita Easyshade Advance 4.0) in various light sources. MATERIALS AND METHODS: Participants between 20 and 40 years old with natural, unrestored right maxillary central incisors, no history of bleaching, orthodontic treatment, or malocclusion and no rotations were included. According to their shades, subjects were randomly selected and grouped into A1, A2, and A3. A total of 100 participants (50 male and 50 female) in each group were chosen for this study. Shade selection was made between 10 am and 2 pm for all light sources. The same examiner selected the shade of natural teeth with Vita Toothguide 3D-Master under natural light within 2 minutes. Once the Vita Toothguide 3D-Masterwas matched with the maxillary right central incisor, the L*, a*, and b* values, chroma, and hue were recorded with Vita Easyshade Advance 4.0 by placing it on the shade tab under the same light source. The values were statistically analyzed using one-way ANOVA and Tukey's HSD post hoc test with SPSS v22.0 software. RESULTS: The mean ∆E*ab values for shades A1, A2, and A3 for groups 1, 2, and 3 were statistically significantly different from each other (p < 0.001). CONCLUSION: The intraoral digital spectrophotometer showed statistically significant differences in shade matching compared to Vita Toothguide 3D-Master. Incandescent light showed more accurate shade matching than the filtered LED, LED, and daylight.

Arabidopsis miR827 mediates post-transcriptional gene silencing of its ubiquitin E3 ligase target gene in the syncytium of the cyst nematode Heterodera schachtii to enhance susceptibility.

MicroRNAs (miRNAs) are a major class of small non-coding RNAs with emerging functions in biotic and abiotic interactions. Here, we report on a new functional role of Arabidopsis miR827 and its NITROGEN LIMITATION ADAPTATION (NLA) target gene in mediating plant susceptibility to the beet cyst nematode Heterodera schachtii. Cyst nematodes are sedentary endoparasites that induce the formation of multinucleated feeding structures termed syncytia in the roots of host plants. Using promoter:GUS fusion assays we established that miR827 was activated in the initial feeding cells and this activation was maintained in the syncytium during all sedentary stages of nematode development. Meanwhile, the NLA target gene, which encodes an ubiquitin E3 ligase enzyme, was post-transcriptionally silenced in the syncytium to permanently suppress its activity during all nematode parasitic stages. Overexpression of miR827 in Arabidopsis resulted in hyper-susceptibility to H. schachtii. In contrast, inactivation of miR827 activity through target mimicry or by overexpression a miR827-resistant cDNA of NLA produced the opposite phenotype of reduced plant susceptibility to H. schachtii. Gene expression analysis of several pathogenesis-related genes together with Agrobacterium-mediated transient expression in Nicotiana benthamiana provided strong evidence that miR827-mediated downregulation of NLA to suppress basal defense pathways. In addition, using yeast two-hybrid screens we identified several candidates of NLA-interacting proteins that are involved in a wide range of biological processes and molecular functions, including three pathogenesis-related proteins. Taken together, we conclude that nematode-activated miR827 in the syncytium is necessary to suppress immune responses in order to establish infection and cause disease.

The photosystem II oxygen-evolving complex protein PsbP interacts with the coat protein of Alfalfa mosaic virus and inhibits virus replication.

Alfalfa mosaic virus (AMV) coat protein (CP) is essential for many steps in virus replication from early infection to encapsidation. However, the identity and functional relevance of cellular factors that interact with CP remain unknown. In an unbiased yeast two-hybrid screen for CP-interacting Arabidopsis proteins, we identified several novel protein interactions that could potentially modulate AMV replication. In this report, we focus on one of the novel CP-binding partners, the Arabidopsis PsbP protein, which is a nuclear-encoded component of the oxygen-evolving complex of photosystem II. We validated the protein interaction in vitro with pull-down assays, in planta with bimolecular fluorescence complementation assays, and during virus infection by co-immunoprecipitations. CP interacted with the chloroplast-targeted PsbP in the cytosol and mutations that prevented the dimerization of CP abolished this interaction. Importantly, PsbP overexpression markedly reduced virus accumulation in infected leaves. Taken together, our findings demonstrate that AMV CP dimers interact with the chloroplast protein PsbP, suggesting a potential sequestration strategy that may preempt the generation of any PsbP-mediated antiviral state.

HIV-1 capsid and viral DNA integration.

IMPORTANCE: HIV-1 capsid protein (CA)-independently or by recruiting host factors-mediates several key steps of virus replication in the cytoplasm and nucleus of the target cell. Research in the recent years have established that CA is multifunctional and genetically fragile of all the HIV-1 proteins. Accordingly, CA has emerged as a validated and high priority therapeutic target, and the first CA-targeting antiviral drug was recently approved for treating multi-drug resistant HIV-1 infection. However, development of next generation CA inhibitors depends on a better understanding of CA's known roles, as well as probing of CA's novel roles, in HIV-1 replication. In this timely review, we present an updated overview of the current state of our understanding of CA's multifunctional role in HIV-1 replication-with a special emphasis on CA's newfound post-nuclear roles, highlight the pressing knowledge gaps, and discuss directions for future research.

Cyclophilin A Facilitates HIV-1 DNA Integration.

Cyclophilin A (CypA) promotes HIV-1 infection by facilitating reverse transcription, nuclear entry and by countering the antiviral activity of TRIM5α. These multifunctional roles of CypA are driven by its binding to the viral capsid. Interestingly, recent studies suggest that the HIV-1 capsid lattice enters the nucleus of an infected cell and uncoats just before integration. Therefore, we tested whether CypA-capsid interaction regulates post-nuclear entry steps of infection, particularly integration. First, we challenged CypA-expressing (CypA +/+ ) and CypA-depleted (CypA -/- ) cells with HIV-1 particles and quantified the resulting levels of provirus. Surprisingly, CypA-depletion significantly reduced integration, an effect that was independent of CypA's effect on reverse transcription, nuclear entry, and the presence or absence of TRIM5α. Additionally, cyclosporin A, an inhibitor that disrupts CypA-capsid binding, inhibited HIV-1 integration in CypA +/+ cells but not in CypA -/- cells. Accordingly, HIV-1 capsid mutants (G89V and P90A) deficient in CypA binding were also blocked at integration in CypA +/+ cells but not in CypA -/- cells. Then, to understand the mechanism, we assessed the integration activity of HIV-1 preintegration complexes (PICs) extracted from infected cells. The PICs from CypA -/- cells had lower activity in vitro compared to those from CypA +/+ cells. PICs from cells depleted for CypA and TRIM5α also had lower activity, suggesting that CypA's effect on PIC activity is independent of TRIM5α. Finally, addition of CypA protein significantly stimulated the integration activity of PICs extracted from both CypA +/+ and CypA -/- cells. Collectively, these results suggest that CypA promotes HIV-1 integration, a previously unknown role of this host factor. Importance: HIV-1 capsid interaction with host cellular factors is essential for establishing a productive infection. However, the molecular details of such virus-host interactions are not fully understood. Cyclophilin A (CypA) is the first host protein identified to specifically bind to the HIV-1 capsid. Now it is established that CypA promotes reverse transcription and nuclear entry steps of HIV-1 infection. In this report, we show that CypA promotes HIV-1 integration by binding to the viral capsid. Specifically, our results demonstrate that CypA promotes HIV-1 integration by stimulating the activity of the viral preintegration complex and identifies a novel role of CypA during HIV-1 infection. This new knowledge is important because recent reports suggest that an operationally intact HIV-1 capsid enters the nucleus of an infected cell.

Three Prime Repair Exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural and conformational properties.

HIV-1 integration into the human genome is dependent on 3'-processing of the reverse transcribed viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3'-processed DNA remained resistant. Here, we describe the mechanism by which the 3'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3'-processed DNA was significantly lower than the unprocessed HIV-1 DNA by TREX1. The efficiency of degradation (kcat/KM) of the 3'-processed DNA was also significantly lower than the unprocessed DNA. Furthermore, the binding affinity (Kd) of TREX1 was markedly lower to the 3'-processed DNA compared to the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3'-processed DNA. These results pinpoint the biochemical mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3'-processed HIV-1 DNA.

New insights into HIV assembly and trafficking.

Assembly and release of human immunodeficiency virus type 1 (HIV-1) particles is mediated by the viral Gag polyprotein precursor. Gag is synthesized in the cytosol and rapidly translocates to membrane to orchestrate particle production. The cell biology of HIV-1 Gag trafficking is currently one of the least understood aspects of HIV-1 replication. In this review, we highlight the current understanding of the cellular machinery involved in Gag trafficking and virus assembly.

HIV-1 mutants that escape the cytotoxic T-lymphocytes are defective in viral DNA integration.

HIV-1 replication is durably controlled without antiretroviral therapy (ART) in certain infected individuals called elite controllers (ECs). These individuals express specific human leukocyte antigens (HLA) that tag HIV-infected cells for elimination by presenting viral epitopes to CD8+ cytotoxic T-lymphocytes (CTL). In HIV-infected individuals expressing HLA-B27, CTLs primarily target the viral capsid protein (CA)-derived KK10 epitope. While selection of CA mutation R264K helps HIV-1 escape this potent CTL response, the accompanying fitness cost severely diminishes virus infectivity. Interestingly, selection of a compensatory CA mutation S173A restores HIV-1 replication. However, the molecular mechanism(s) underlying HIV-1 escape from this ART-free virus control by CTLs is not fully understood. Here, we report that the R264K mutation-associated infectivity defect arises primarily from impaired HIV-1 DNA integration, which is restored by the S173A mutation. Unexpectedly, the integration defect of the R264K variant was also restored upon depletion of the host cyclophilin A. These findings reveal a nuclear crosstalk between CA and HIV-1 integration as well as identify a previously unknown role of cyclophilin A in viral DNA integration. Finally, our study identifies a novel immune escape mechanism of an HIV-1 variant escaping a CA-directed CTL response.

Reliability of measurements with digital radiographs--a myth.

The purpose of this study was to assess the accuracy of digital radiographs and hence their effectiveness in templating. The methodology involved a retrospective study of post operative radiographs of patients with hemiarthroplasty of the hip. Three observers made observations blinded to each other's measurements. A statistical analysis of the data highlights magnification varying from 6 to 31 percent. There is a statistically significant relationship between the size of the error (size measured on radiograph minus implant size, i.e. magnification) and the implant size (p = 0.005) but the percentage error (error/implant size x 100) is independent of implant size (p = 0.505). It is our impression that digital radiographs and templating on the digital radiographs should not be considered a precise process.

PROLIDASE: A Review from Discovery to its Role in Health and Disease.

Prolidase (peptidase D), encoded by the PEPD gene, is a ubiquitously expressed cytosolic metalloproteinase, the only enzyme capable of cleaving imidodipeptides containing C-terminal proline or hydroxyproline. Prolidase catalyzes the rate-limiting step during collagen recycling and is essential in protein metabolism, collagen turnover, and matrix remodeling. Prolidase, therefore plays a crucial role in several physiological processes such as wound healing, inflammation, angiogenesis, cell proliferation, and carcinogenesis. Accordingly, mutations leading to loss of prolidase catalytic activity result in prolidase deficiency a rare autosomal recessive metabolic disorder characterized by defective wound healing. In addition, alterations in prolidase enzyme activity have been documented in numerous pathological conditions, making prolidase a useful biochemical marker to measure disease severity. Furthermore, recent studies underscore the importance of a non-enzymatic role of prolidase in cell regulation and infectious disease. This review aims to provide comprehensive information on prolidase, from its discovery to its role in health and disease, while addressing the current knowledge gaps.

Activation of proline biosynthesis is critical to maintain glutamate homeostasis during acute methamphetamine exposure.

Methamphetamine (METH) is a highly addictive psychostimulant that causes long-lasting effects in the brain and increases the risk of developing neurodegenerative diseases. The cellular and molecular effects of METH in the brain are functionally linked to alterations in glutamate levels. Despite the well-documented effects of METH on glutamate neurotransmission, the underlying mechanism by which METH alters glutamate levels is not clearly understood. In this study, we report an essential role of proline biosynthesis in maintaining METH-induced glutamate homeostasis. We observed that acute METH exposure resulted in the induction of proline biosynthetic enzymes in both undifferentiated and differentiated neuronal cells. Proline level was also increased in these cells after METH exposure. Surprisingly, METH treatment did not increase glutamate levels nor caused neuronal excitotoxicity. However, METH exposure resulted in a significant upregulation of pyrroline-5-carboxylate synthase (P5CS), the key enzyme that catalyzes synthesis of proline from glutamate. Interestingly, depletion of P5CS by CRISPR/Cas9 resulted in a significant increase in glutamate levels upon METH exposure. METH exposure also increased glutamate levels in P5CS-deficient proline-auxotropic cells. Conversely, restoration of P5CS expression in P5CS-deficient cells abrogated the effect of METH on glutamate levels. Consistent with these findings, P5CS expression was significantly enhanced in the cortical brain region of mice administered with METH and in the slices of cortical brain tissues treated with METH. Collectively, these results uncover a key role of P5CS for the molecular effects of METH and highlight that excess glutamate can be sequestered for proline biosynthesis as a protective mechanism to maintain glutamate homeostasis during drug exposure.