Intracellular interactions between APOBEC3G, RNA, and HIV-1 Gag: APOBEC3G multimerization is dependent on its association with RNA

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Frequent dual initiation of reverse transcription in murine leukemia virus-based vectors containing two primer-binding sites

AbstractRetroviruses package two copies of viral RNA into each virion. Although each RNA contains a primer-binding site for initiation of DNA synthesis, it is unknown whether reverse transcription is initiated on both RNAs. To determine whether a single virion is capable of initiating reverse transcription more than once, we constructed a murine leukemia virus-based vector containing a second primer-binding site (PBS) derived from spleen necrosis virus and inserted the green fluorescent protein gene (GFP) between the two PBSs. Initiation of reverse transcription at either PBS results in a provirus that expresses GFP. However, initiation at both PBSs can result in the deletion of GFP, which can be detected by flow cytometry and Southern blotting analysis. Approximately 22–29% of the proviruses formed deleted the GFP in a single replication cycle, indicating the minimum proportion of virions that initiated reverse transcription on both PBSs. These results show that a significant proportion of MLV-based vectors containing two PBSs have the capacity to initiate reverse transcription more than once

Strain field in doubly curved surface

This paper presents algorithm for development of structural and continuous curved surface into a planar and non planar (radial) shape in 3D space. The development process is modeled by application of strain in certain plane from the curved surface to its planar development. A doubly curved surface has been generated for the purpose of technical studies. Important features of the approach include formulations of the coefficients of first fundamental form, second fundamental form, Gaussian curvature and Serret Frenet curve. The approximate strain field is obtained by solving a constrained linear and nonlinear problem in algorithm

APOBEC3G induces a hypermutation gradient: purifying selection at multiple steps during HIV-1 replication results in levels of G-to-A mutations that are high in DNA, intermediate in cellular viral RNA, and low in virion RNA

<p>Abstract</p> <p>Background</p> <p>Naturally occurring Vif variants that are unable to inhibit the host restriction factor APOBEC3G (A3G) have been isolated from infected individuals. A3G can potentially induce G-to-A hypermutation in these viruses, and hypermutation could contribute to genetic variation in HIV-1 populations through recombination between hypermutant and wild-type genomes. Thus, hypermutation could contribute to the generation of immune escape and drug resistant variants, but the genetic contribution of hypermutation to the viral evolutionary potential is poorly understood. In addition, the mechanisms by which these viruses persist in the host despite the presence of A3G remain unknown.</p> <p>Results</p> <p>To address these questions, we generated a replication-competent HIV-1 Vif mutant in which the A3G-binding residues of Vif, Y⁴⁰RHHY⁴⁴, were substituted with five alanines. As expected, the mutant was severely defective in an A3G-expressing T cell line and exhibited a significant delay in replication kinetics. Analysis of viral DNA showed the expected high level of G-to-A hypermutation; however, we found substantially reduced levels of G-to-A hypermutation in intracellular viral RNA (cRNA), and the levels of G-to-A mutations in virion RNA (vRNA) were even further reduced. The frequencies of hypermutation in DNA, cRNA, and vRNA were 0.73%, 0.12%, and 0.05% of the nucleotides sequenced, indicating a gradient of hypermutation. Additionally, genomes containing start codon mutations and early termination codons within <it>gag </it>were isolated from the vRNA.</p> <p>Conclusion</p> <p>These results suggest that sublethal levels of hypermutation coupled with purifying selection at multiple steps during the early phase of viral replication lead to the packaging of largely unmutated genomes, providing a mechanism by which mutant Vif variants can persist in infected individuals. The persistence of genomes containing mutated <it>gag </it>genes despite this selection pressure indicates that dual infection and complementation can result in the packaging of hypermutated genomes which, through recombination with wild-type genomes, could increase viral genetic variation and contribute to evolution.</p

Methane and carbon dioxide adsorption on edge-functionalized graphene: A comparative DFT study

With a view towards optimizing gas storage and separation in crystalline and disordered nanoporous carbon-based materials, we use ab initio density functional theory calculations to explore the effect of chemical functionalization on gas binding to exposed edges within model carbon nanostructures. We test the geometry, energetics, and charge distribution of in-plane and out-of-plane binding of CO2 and CH4 to model zigzag graphene nanoribbons edge-functionalized with COOH, OH, NH2, H2PO3, NO2, and CH3. Although different choices for the exchange-correlation functional lead to a spread of values for the binding energy, trends across the functional groups are largely preserved for each choice, as are the final orientations of the adsorbed gas molecules. We find binding of CO2 to exceed that of CH4 by roughly a factor of two. However, the two gases follow very similar trends with changes in the attached functional group, despite different molecular symmetries. Our results indicate that the presence of NH2, H2PO3, NO2, and COOH functional groups can significantly enhance gas binding with respect to a hydrogen-passivated edge, making the edges potentially viable binding sites in materials with high concentrations of edge carbons. To first order, in-plane binding strength correlates with the larger permanent and induced dipole moments on these groups. Implications for tailoring carbon structures for increased gas uptake and improved CO2/CH4 selectivity are discussed.Comment: 12 pages, 7 figure

A comparative study of dexmedetomidine versus clonidine in epidural anaesthesia to assess the level of sedation in patients undergoing lower abdominal and lower limb surgery

Background: To compare study of dexmedetomidine versus clonidine in epidural anesthesia to assess the level of sedation in patients undergoing lower abdominal and lower limb surgery.Methods: This was a comparative study conducted on admitted ASA grade I and II patients undergoing lower abdominal and lower limb surgeries. The patients were divided into three groups of 30 patients each, according to the epidural medication they received:-Group A-received 15ml of bupivacaine (0.5%) and dexmedetomidine (1.0µg/kg body weight) in 1ml of normal saline; Group B-received 15ml of bupivacaine (0.5%) and clonidine (2.0µg/kg body weight) in 1ml of normal saline; Group C-received 15ml of bupivacaine (0.5%) with 1ml of normal saline. The heart rate, blood pressure, sensory dermatome level, Motor blocked level, pain and VAS were recorded at different time intervals. The side effects were also noted.Results: The baseline parameters were comparable among the groups. All the hemodynamic parameters and other study parameters were similar at Min. 0. All the hemodynamic parameters such as heart rate, blood pressure and SpO2 were variable at different time intervals. Motor block level   was significantly (p<0.05) lower in Group C than Group A and Group B from Min 50 to Min 90. The sedation score was observed to be nil in Group C. The post-op pain score became higher in Group C than Group A and Group B at subsequent time intervals. A 3 (10%) of the rescue agents was observed in Group C. Atropine (30%) and mephenteramine (10%) were common rescue agents in Group B. The bradycardia was observed in 30% patients of Group B and in 40% of Group A.Conclusions: On addition of dexmedetomidine as adjuvant to bupivacaine in epidural anesthesia provides better anesthesia and sedation than clonidine as adjuvant to bupivacaine or bupivacaine alone with mild hemodynamic changes which are easily manageable

Lack of Detection of Xenotropic Murine Leukemia Virus-Related Virus in HIV-1 Lymphoma Patients

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus reported to be associated with human prostate cancer and chronic fatigue syndrome. Since retroviruses cause various cancers, and XMRV replication might be facilitated by HIV-1 co-infection, we asked whether certain patients with HIV-associated lymphomas are infected with XMRV. Analysis of PMBCs and plasma from 26 patients failed to detect XMRV by PCR, ELISA, or Western blot, suggesting a lack of association between XMRV and AIDS-associated lymphomas

Mechanisms and Factors that Influence High Frequency Retroviral Recombination

With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment, and vaccine development

Crystal structure of the catalytic domain of HIV-1 restriction factor APOBEC3G in complex with ssDNA

The human APOBEC3G protein is a cytidine deaminase that generates cytidine to deoxy-uridine mutations in single-stranded DNA (ssDNA), and capable of restricting replication of HIV-1 by generating mutations in viral genome. The mechanism by which APOBEC3G specifically deaminates 5\u27-CC motifs has remained elusive since structural studies have been hampered due to apparently weak ssDNA binding of the catalytic domain of APOBEC3G. We overcame the problem by generating a highly active variant with higher ssDNA affinity. Here, we present the crystal structure of this variant complexed with a ssDNA substrate at 1.86 A resolution. This structure reveals atomic-level interactions by which APOBEC3G recognizes a functionally-relevant 5\u27-TCCCA sequence. This complex also reveals a key role of W211 in substrate recognition, implicating a similar recognition in activation-induced cytidine deaminase (AID) with a conserved tryptophan