Dysregulation of IFN System Can Lead to Poor Response to Pegylated Interferon and Ribavirin Therapy in Chronic Hepatitis C

Despite being expensive, the standard combination of pegylated interferon (Peg-IFN)- α and ribavirin used to treat chronic hepatitis C (CH) results in a moderate clearance rate and a plethora of side effects. This makes it necessary to predict patient outcome so as to improve the accuracy of treatment. Although the antiviral mechanism of genetically altered IL28B is unknown, IL28B polymorphism is considered a good predictor of IFN combination treatment outcome

Predicting Bevirimat resistance of HIV-1 from genotype

<p>Abstract</p> <p>Background</p> <p>Maturation inhibitors are a new class of antiretroviral drugs. Bevirimat (BVM) was the first substance in this class of inhibitors entering clinical trials. While the inhibitory function of BVM is well established, the molecular mechanisms of action and resistance are not well understood. It is known that mutations in the regions CS p24/p2 and p2 can cause phenotypic resistance to BVM. We have investigated a set of p24/p2 sequences of HIV-1 of known phenotypic resistance to BVM to test whether BVM resistance can be predicted from sequence, and to identify possible molecular mechanisms of BVM resistance in HIV-1.</p> <p>Results</p> <p>We used artificial neural networks and random forests with different descriptors for the prediction of BVM resistance. Random forests with hydrophobicity as descriptor performed best and classified the sequences with an area under the Receiver Operating Characteristics (ROC) curve of 0.93 ± 0.001. For the collected data we find that p2 sequence positions 369 to 376 have the highest impact on resistance, with positions 370 and 372 being particularly important. These findings are in partial agreement with other recent studies. Apart from the complex machine learning models we derived a number of simple rules that predict BVM resistance from sequence with surprising accuracy. According to computational predictions based on the data set used, cleavage sites are usually not shifted by resistance mutations. However, we found that resistance mutations could shorten and weaken the <it>α</it>-helix in p2, which hints at a possible resistance mechanism.</p> <p>Conclusions</p> <p>We found that BVM resistance of HIV-1 can be predicted well from the sequence of the p2 peptide, which may prove useful for personalized therapy if maturation inhibitors reach clinical practice. Results of secondary structure analysis are compatible with a possible route to BVM resistance in which mutations weaken a six-helix bundle discovered in recent experiments, and thus ease Gag cleavage by the retroviral protease.</p

Clathrin Facilitates the Morphogenesis of Retrovirus Particles

The morphogenesis of retroviral particles is driven by Gag and GagPol proteins that provide the major structural component and enzymatic activities required for particle assembly and maturation. In addition, a number of cellular proteins are found in retrovirus particles; some of these are important for viral replication, but many lack a known functional role. One such protein is clathrin, which is assumed to be passively incorporated into virions due to its abundance at the plasma membrane. We found that clathrin is not only exceptionally abundant in highly purified HIV-1 particles but is recruited with high specificity. In particular, the HIV-1 Pol protein was absolutely required for clathrin incorporation and point mutations in reverse transcriptase or integrase domains of Pol could abolish incorporation. Clathrin was also specifically incorporated into other retrovirus particles, including members of the lentivirus (simian immunodeficiency virus, SIVmac), gammaretrovirus (murine leukemia virus, MLV) and betaretrovirus (Mason-Pfizer monkey virus, M-PMV) genera. However, unlike HIV-1, these other retroviruses recruited clathrin primarily using peptide motifs in their respective Gag proteins that mimicked motifs found in cellular clathrin adaptors. Perturbation of clathrin incorporation into these retroviruses, via mutagenesis of viral proteins, siRNA based clathrin depletion or adaptor protein (AP180) induced clathrin sequestration, had a range of effects on the accuracy of particle morphogenesis. These effects varied according to which retrovirus was examined, and included Gag and/or Pol protein destabilization, inhibition of particle assembly and reduction in virion infectivity. For each retrovirus examined, clathrin incorporation appeared to be important for optimal replication. These data indicate that a number of retroviruses employ clathrin to facilitate the accurate morphogenesis of infectious particles. We propose a model in which clathrin contributes to the spatial organization of Gag and Pol proteins, and thereby regulates proteolytic processing of virion components during particle assembly

TRANSFORM (Multicenter Experience With Rapid Deployment Edwards INTUITY Valve System for Aortic Valve Replacement) US clinical trial: Performance of a rapid deployment aortic valve

Background: The TRANSFORM (Multicenter Experience With Rapid Deployment Edwards INTUITY Valve System for Aortic Valve Replacement) trial (NCT01700439) evaluated the performance of the INTUITY rapid deployment aortic valve replacement (RDAVR) system in patients with severe aortic stenosis. Methods: TRANSFORM was a prospective, nonrandomized, multicenter (n 1�4 29), single-arm trial. INTUITY is comprised of a cloth-covered balloon- expandable frame attached to a Carpentier-Edwards PERIMOUNT Magna Ease aortic valve. Primary and effectiveness endpoints were evaluated at 1 year. Results: Between 2012 and 2015, 839 patients underwent RDAVR. Mean age was 73.5 8.3 years. Full sternotomy (FS) was used in 59% and minimally invasive surgical incisions in 41%. Technical success rate was 95%. For isolated RDAVR, mean crossclamp and cardiopulmonary bypass times for FS were 49.3 26.9 minutes and 69.2 34.7 minutes, respectively, and for minimally invasive surgical 63.1 25.4 minutes and 84.6 33.5 minutes, respectively. These times were favorable compared with Society of Thoracic Surgeons data- base comparators for FS: 76.3 minutes and 104.2 minutes, respectively, and for minimally invasive surgical, 82.9 minutes and 111.4 minutes, respectively (P<.001). At 30 days, all-cause mortality was 0.8%; valve explant, 0.1%; throm- boembolism, 3.5%; and major bleeding, 1.3%. In patients with isolated aortic valve replacement, the rate of permanent pacemaker implantation was 11.9%. At 1 year, mean effective orifice area was 1.7 cm2; mean gradient, 10.3 mm Hg; and moderate and severe paravalvular leak, 1.2% and 0.4%, respectively

Dysregulation of IFN System Can Lead to Poor Response to Pegylated Interferon and Ribavirin Therapy in Chronic Hepatitis C

Despite being expensive, the standard combination of pegylated interferon (Peg-IFN)- α and ribavirin used to treat chronic hepatitis C (CH) results in a moderate clearance rate and a plethora of side effects. This makes it necessary to predict patient outcome so as to improve the accuracy of treatment. Although the antiviral mechanism of genetically altered IL28B is unknown, IL28B polymorphism is considered a good predictor of IFN combination treatment outcome

Potent Activity of the HIV-1 Maturation Inhibitor Bevirimat in SCID-hu Thy/Liv Mice

The HIV-1 maturation inhibitor, 3-O-(3',3'-dimethylsuccinyl) betulinic acid (bevirimat, PA-457) is a promising drug candidate with 10 nM in vitro antiviral activity against multiple wild-type (WT) and drug-resistant HIV-1 isolates. Bevirimat has a novel mechanism of action, specifically inhibiting cleavage of spacer peptide 1 (SP1) from the C-terminus of capsid which results in defective core condensation.Oral administration of bevirimat to HIV-1-infected SCID-hu Thy/Liv mice reduced viral RNA by >2 log(10) and protected immature and mature T cells from virus-mediated depletion. This activity was observed at plasma concentrations that are achievable in humans after oral dosing, and bevirimat was active up to 3 days after inoculation with both WT HIV-1 and an AZT-resistant HIV-1 clinical isolate. Consistent with its mechanism of action, bevirimat caused a dose-dependent inhibition of capsid-SP1 cleavage in HIV-1-infected human thymocytes obtained from these mice. HIV-1 NL4-3 with an alanine-to-valine substitution at the N-terminus of SP1 (SP1/A1V), which is resistant to bevirimat in vitro, was also resistant to bevirimat treatment in the mice, and SP1/AIV had replication and thymocyte kinetics similar to that of WT NL4-3 with no evidence of fitness impairment in in vivo competition assays. Interestingly, protease inhibitor-resistant HIV-1 with impaired capsid-SP1 cleavage was hypersensitive to bevirimat in vitro with a 50% inhibitory concentration 140 times lower than for WT HIV-1.These results support further clinical development of this first-in-class maturation inhibitor and confirm the usefulness of the SCID-hu Thy/Liv model for evaluation of in vivo antiretroviral efficacy, drug resistance, and viral fitness

SAMHD1-Deficient CD14+ Cells from Individuals with Aicardi-Goutières Syndrome Are Highly Susceptible to HIV-1 Infection

Myeloid blood cells are largely resistant to infection with human immunodeficiency virus type 1 (HIV-1). Recently, it was reported that Vpx from HIV-2/SIVsm facilitates infection of these cells by counteracting the host restriction factor SAMHD1. Here, we independently confirmed that Vpx interacts with SAMHD1 and targets it for ubiquitin-mediated degradation. We found that Vpx-mediated SAMHD1 degradation rendered primary monocytes highly susceptible to HIV-1 infection; Vpx with a T17A mutation, defective for SAMHD1 binding and degradation, did not show this activity. Several single nucleotide polymorphisms in the SAMHD1 gene have been associated with Aicardi-Goutières syndrome (AGS), a very rare and severe autoimmune disease. Primary peripheral blood mononuclear cells (PBMC) from AGS patients homozygous for a nonsense mutation in SAMHD1 (R164X) lacked endogenous SAMHD1 expression and support HIV-1 replication in the absence of exogenous activation. Our results indicate that within PBMC from AGS patients, CD14+ cells were the subpopulation susceptible to HIV-1 infection, whereas cells from healthy donors did not support infection. The monocytic lineage of the infected SAMHD1 -/- cells, in conjunction with mostly undetectable levels of cytokines, chemokines and type I interferon measured prior to infection, indicate that aberrant cellular activation is not the cause for the observed phenotype. Taken together, we propose that SAMHD1 protects primary CD14+ monocytes from HIV-1 infection confirming SAMHD1 as a potent lentiviral restriction factor

Efficient Production of HIV-1 Virus-Like Particles from a Mammalian Expression Vector Requires the N-Terminal Capsid Domain

It is now well accepted that the structural protein Pr55Gag is sufficient by itself to produce HIV-1 virus-like particles (VLPs). This polyprotein precursor contains different domains including matrix, capsid, SP1, nucleocapsid, SP2 and p6. In the present study, we wanted to determine by mutagenesis which region(s) is essential to the production of VLPs when Pr55Gag is inserted in a mammalian expression vector, which allows studying the protein of interest in the absence of other viral proteins. To do so, we first studied a minimal Pr55Gag sequence called Gag min that was used previously. We found that Gag min fails to produce VLPs when expressed in an expression vector instead of within a molecular clone. This failure occurs early in the cell at the assembly of viral proteins. We then generated a series of deletion and substitution mutants, and examined their ability to produce VLPs by combining biochemical and microscopic approaches. We demonstrate that the matrix region is not necessary, but that the efficiency of VLP production depends strongly on the presence of its basic region. Moreover, the presence of the N-terminal domain of capsid is required for VLP production when Gag is expressed alone. These findings, combined with previous observations indicating that HIV-1 Pr55Gag-derived VLPs act as potent stimulators of innate and acquired immunity, make the use of this strategy worth considering for vaccine development

Analysis of the Initiating Events in HIV-1 Particle Assembly and Genome Packaging

HIV-1 Gag drives a number of events during the genesis of virions and is the only viral protein required for the assembly of virus-like particles in vitro and in cells. Although a reasonable understanding of the processes that accompany the later stages of HIV-1 assembly has accrued, events that occur at the initiation of assembly are less well defined. In this regard, important uncertainties include where in the cell Gag first multimerizes and interacts with the viral RNA, and whether Gag-RNA interaction requires or induces Gag multimerization in a living cell. To address these questions, we developed assays in which protein crosslinking and RNA/protein co-immunoprecipitation were coupled with membrane flotation analyses in transfected or infected cells. We found that interaction between Gag and viral RNA occurred in the cytoplasm and was independent of the ability of Gag to localize to the plasma membrane. However, Gag:RNA binding was stabilized by the C-terminal domain (CTD) of capsid (CA), which participates in Gag-Gag interactions. We also found that Gag was present as monomers and low-order multimers (e.g. dimers) but did not form higher-order multimers in the cytoplasm. Rather, high-order multimers formed only at the plasma membrane and required the presence of a membrane-binding signal, but not a Gag domain (the CA-CTD) that is essential for complete particle assembly. Finally, sequential RNA-immunoprecipitation assays indicated that at least a fraction of Gag molecules can form multimers on viral genomes in the cytoplasm. Taken together, our results suggest that HIV-1 particle assembly is initiated by the interaction between Gag and viral RNA in the cytoplasm and that this initial Gag-RNA encounter involves Gag monomers or low order multimers. These interactions per se do not induce or require high-order Gag multimerization in the cytoplasm. Instead, membrane interactions are necessary for higher order Gag multimerization and subsequent particle assembly in cells