Viral RNA-directed RNA polymerases use diverse mechanisms to promote recombination between RNA molecules

An earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Qbeta phage replicase and the poliovirus 3Dpol protein, to promote RNA recombination through a primer extension mechanism. The substrates of recombination were fragments of complementary strands of a Qbeta phage-derived RNA, such that if aligned at complementary 3'-termini and extended using one another as a template, they would produce replicable molecules detectable as RNA colonies grown in a Qbeta replicase-containing agarose. The results show that while 3Dpol efficiently extends the aligned fragments to produce the expected homologous recombinant sequences, only nonhomologous recombinants are generated by Qbeta replicase at a much lower yield and through a mechanism not involving the extension of RNA primers. It follows that the mechanisms of RNA recombination by poliovirus and Qbeta RdRps are quite different. The data favor an RNA transesterification reaction catalyzed by a conformation acquired by Qbeta replicase during RNA synthesis and provide a likely explanation for the very low frequency of homologous recombination in Qbeta phage.     

Isolation and crystallization of a chimeric Qβ replicase containing <Emphasis Type="Italic">Thermus thermophilus</Emphasis> EF-Ts

Qβ replicase is a protein complex responsible for the replication of the genomic RNA of bacteriophage Qβ. In addition to the phage-encoded catalytic β subunit, it recruits three proteins from the host Escherichia coli cell: elongation factors EF-Tu and EF-Ts and ribosomal protein S1. We prepared a chimeric Qβ replicase in which the E. coli EF-Ts is replaced with EF-Ts from Thermus thermophilus. The chimeric protein is produced in E. coli cells during coexpression of the genes encoding the β subunit and thermophilic EF-Ts. The developed isolation procedure yields a substantially homogeneous preparation of the chimeric replicase. Unlike the wild-type enzyme, the S1-less chimeric replicase could be crystallized. This result facilitates studies on the structure of Qβ replicase and the mechanism of recognition of its templates that can replicate in vitro at a record rate.     

Express hybridization of molecular colonies with fluorescent probes

DNA colonies formed during PCR in a polyacrylamide gel and RNA colonies grown in an agarose gel containing Qβ replicase can be identified using the procedure of transfer of molecular colonies onto a nylon membrane followed by membrane hybridization with fluorescent oligonucleotide probes. The suggested improvements significantly simplify and shorten the procedure. By the example of a chimeric AML1-ETO sequence, a marker of frequently occurring leukemia, the express hybridization method was shown to allow the rapid identification of single molecules and the determination of titers of DNA and RNA targets. Hybridization with a mixture of two oligonucleotide probes labeled with different fluorophores complementary to components of the chimeric molecule ensures the identification of molecular colonies containing both parts of the chimeric sequence and improves the specificity of diagnostics.     

Diminished transmission of drug resistant HIV-1 variants with reduced replication capacity in a human transmission model

Background

Different patterns of drug resistance are observed in treated and therapy naïve HIV-1 infected populations. Especially the NRTI-related M184I/V variants, which are among the most frequently encountered mutations in treated patients, are underrepresented in the antiretroviral naïve population. M184I/V mutations are known to have a profound effect on viral replication and tend to revert over time in the new host. However it is debated whether a diminished transmission efficacy of HIV variants with a reduced replication capacity can also contribute to the observed discrepancy in genotypic patterns.As dendritic cells (DCs) play a pivotal role in HIV-1 transmission, we used a model containing primary human Langerhans cells (LCs) and DCs to compare the transmission efficacy M184 variants (HIV-M184V/I/T) to HIV wild type (HIV-WT). As control, we used HIV harboring the NNRTI mutation K103N (HIV-K103N) which has a minor effect on replication and is found at a similar prevalence in treated and untreated individuals.

Results

In comparison to HIV-WT, the HIV-M184 variants were less efficiently transmitted to CCR5⁺ Jurkat T cells by both LCs and DCs. The transmission rate of HIV-K103N was slightly reduced to HIV-WT in LCs and even higher than HIV-WT in DCs. Replication experiments in CCR5⁺ Jurkat T cells revealed no apparent differences in replication capacity between the mutant viruses and HIV-WT. However, viral replication in LCs and DCs was in concordance with the transmission results; replication by the HIV-M184 variants was lower than replication by HIV-WT, and the level of replication of HIV-K103N was intermediate for LCs and higher than HIV-WT for DCs.

Conclusions

Our data demonstrate that drug resistant M184-variants display a reduced replication capacity in LCs and DCs which directly impairs their transmission efficacy. As such, diminished transmission efficacy may contribute to the lower prevalence of drug resistant variants in therapy naive individuals.

     

Preservation of nucleic acid integrity in guanidine thiocyanate lysates of whole blood

High-molecular-mass RNA and DNA have been shown to retain their integrity for three days at room temperature, no less than two weeks at +4°C, and more than a year at ?20°C when whole blood samples are stored as lysates containing 4 M guanidine thiocyanate. Storage time at room temperature can be prolonged at least up to 14 days if nucleic acids were precipitated by two volumes of isopropanol. This preservation technique allows storage and transportation of samples at ambient temperature and is completely compatible with the procedure of subsequent isolation of nucleic acids.     

Characterization of the mrgRS locus of the opportunistic pathogen Burkholderia pseudomallei: temperature regulates the expression of a two-component signal transduction system

Background  

Burkholderia pseudomallei is a saprophyte in tropical environments and an opportunistic human pathogen. This versatility requires a sensing mechanism that allows the bacterium to respond rapidly to altered environmental conditions. We characterized a two-component signal transduction locus from B. pseudomallei 204, mrgR and mrgS, encoding products with extensive homology with response regulators and histidine protein kinases of Escherichia coli, Bordetella pertussis, and Vibrio cholerae.     

Paradoxes of replication of RNA of a bacterial virus

The extraordinary ability of the bacteriophage Qβ replicase to amplify RNA outside the cell attracted the attention of molecular biologists in the late 1960s-early 1970s. However, at that time, a number of puzzling properties of the enzyme was not explained. Not until recently has Qβ replicase begun to reveal its secrets, promising to give a key not only to understanding the mechanism of replication of the bacterial virus genome but also to the solution of more general fundamental and applied problems.     

Subclinical inflammation on MRI of hand and foot of anticitrullinated peptide antibody–negative arthralgia patients at risk for rheumatoid arthritis

Introduction

It is known that anticitrullinated peptide antibody (ACPA)–positive rheumatoid arthritis (RA) has a preclinical phase. Whether this phase is also present in ACPA-negative RA is unknown. To determine this, we studied ACPA-negative arthralgia patients who were considered prone to progress to RA for local subclinical inflammation observed on hand and foot magnetic resonance imaging (MRI) scans.

Methods

We studied a total of 64 ACPA-negative patients without clinically detectable arthritis and with arthralgia of the small joints within the previous 1 year. Because of the character of the patients’ symptoms, the rheumatologists considered these patients to be prone to progress to RA. For comparisons, we evaluated 19 healthy, symptom-free controls and 20 ACPA-negative RA patients, who were identified according to the 1987 American Rheumatism Association criteria. All participants underwent MRI of unilateral wrist, metacarpophalangeal and metatarsophalangeal joints. Synovitis and bone marrow oedema (BME) were scored according to the OMERACT rheumatoid arthritis magnetic resonance imaging scoring system, and the scores were summed to yield the ‘MRI inflammation score’. Scores were compared between groups. Among the ACPA-negative arthralgia patients, MRI inflammation scores were related to C-reactive protein (CRP) levels and the tenderness of scanned joints.

Results

MRI inflammation scores increased progressively among the groups of controls and ACPA-negative arthralgia and RA patients (median scores = 0, 1 and 10, respectively; P < 0.001). The MRI inflammation scores of ACPA-negative arthralgia patients were significantly higher than those of controls (P = 0.018). In particular, the synovitis scores were higher in ACPA-negative arthralgia patients (P = 0.046). Among the ACPA-negative arthralgia patients, inflammation was observed predominantly in the wrist (53%). The synovitis scores were associated with CRP levels (P = 0.007) and joint tenderness (P = 0.026). Despite the limited follow-up duration, five patients developed clinically detectable arthritis. These five patients had higher scores for MRI inflammation (P = 0.001), synovitis (P = 0.002) and BME (P = 0.003) compared to the other patients.

Conclusion

Subclinical synovitis was observed in the small joints of ACPA-negative arthralgia patients, and especially in patients whose conditions progressed to clinically detectable arthritis. This finding suggests the presence of a preclinical phase in ACPA-negative RA. Further longitudinal studies of these lesions and patients are required to confirm this hypothesis.