Translational Recoding Induced by G-Rich mRNA Sequences That Form Unusual Structures

AbstractWe investigated a herpesvirus mutant that contains a single base insertion in its thymidine kinase (tk) gene yet expresses low levels of TK via a net +1 translational recoding event. Within this mutant gene, we defined a G-rich signal that is sufficient to induce recoding. Unlike other translational recoding events, downstream RNA structures or termination codons did not stimulate recoding, and paused ribosomes were not detected. Mutational analysis indicated that specific tRNAs or codon–anticodon slippage were unlikely to account for recoding. Rather, recoding efficiency correlated with the G-richness of the signal and its ability to form unusual structures. These findings identify a mechanism of translational recoding with unique features and potential implications for clinical drug resistance and other biological systems

Potential Associations between Severity of Infection and the Presence of Virulence-Associated Genes in Clinical Strains of Staphylococcus aureus

BACKGROUND: The clinical spectrum of Staphylococcus aureus infection ranges from asymptomatic nasal carriage to osteomyelitis, infective endocarditis (IE) and death. In this study, we evaluate potential association between the presence of specific genes in a collection of prospectively characterized S. aureus clinical isolates and clinical outcome. METHODOLOGY/PRINCIPAL FINDINGS: Two hundred thirty-nine S. aureus isolates (121 methicillin-resistant S. aureus [MRSA] and 118 methicillin-susceptible S. aureus [MSSA]) were screened by array comparative genomic hybridization (aCGH) to identify genes implicated in complicated infections. After adjustment for multiple tests, 226 genes were significantly associated with severity of infection. Of these 226 genes, 185 were not in the SCCmec element. Within the 185 non-SCCmec genes, 171 were less common and 14 more common in the complicated infection group. Among the 41 genes in the SCCmec element, 37 were more common and 4 were less common in the complicated group. A total of 51 of the 2014 sequences evaluated, 14 non-SCCmec and 37 SCCmec, were identified as genes of interest. CONCLUSIONS/SIGNIFICANCE: Of the 171 genes less common in complicated infections, 152 are of unknown function and may contribute to attenuation of virulence. The 14 non-SCCmec genes more common in complicated infections include bacteriophage-encoded genes such as regulatory factors and autolysins with potential roles in tissue adhesion or biofilm formation

Polyantigenic Interferon-γ Responses Are Associated with Protection from TB among HIV-Infected Adults with Childhood BCG Immunization

Surrogate immunologic markers for natural and vaccine-mediated protection against tuberculosis (TB) have not been identified. HIV-infected adults with childhood BCG immunization entering the placebo arm of the DarDar TB vaccine trial in Dar es Salaam, Tanzania, were assessed for interferon gamma (IFN-γ) responses to three mycobacterial antigen preparations--secreted Mycobacterium tuberculosis antigens 85 (Ag85), early secretory antigenic target 6 (ESAT-6) and polyantigenic whole cell lysate (WCL). We investigated the association between the number of detectable IFN-γ responses at baseline and the subsequent risk of HIV-associated TB. During a median follow-up of 3.3 years, 92 (9.4%) of 979 placebo recipients developed TB. The incidence of TB was 14% in subjects with no detectable baseline IFN-γ responses vs. 8% in subjects with response to polyantigenic WCL (P = 0.028). Concomitant responses to secreted antigens were associated with further reduction in the incidence of HIV-associated TB. Overall the percentage of subjects with 0, 1, 2 and 3 baseline IFN-γ responses to mycobacterial preparations who developed HIV-associated TB was 14%, 8%, 7% and 4%, respectively (P = 0.004). In a multivariate Cox regression model, the hazard of developing HIV-associated TB was 46% lower with each increment in the number of detectable baseline IFN-γ responses (P<0.001). Among HIV-infected adults who received BCG in childhood and live in a TB-endemic country, polyantigenic IFN-γ responses are associated with decreased risk of subsequent HIV-associated TB. ClinicalTrials.gov NCT0052195

Translational Compensation of a Frameshift Mutation Affecting Herpes Simplex Virus Thymidine Kinase Is Sufficient To Permit Reactivation from Latency

Herpes simplex virus thymidine kinase is important for reactivation of virus from its latent state and is a target for the antiviral drug acyclovir. Most acyclovir-resistant isolates have mutations in the thymidine kinase gene; however, how these mutations confer clinically relevant resistance is unclear. Reactivation from explanted mouse ganglia was previously observed with a patient-derived drug-resistant isolate carrying a single guanine insertion within a run of guanines in the thymidine kinase gene. Despite this mutation, low levels of active enzyme were synthesized following an unusual ribosomal frameshift. Here we report that a virus, generated from a pretherapy isolate from the same patient, engineered to lack thymidine kinase activity, was competent for reactivation. This suggested that the clinical isolate contains alleles of other genes that permit reactivation in the absence of thymidine kinase. Therefore, to establish whether thymidine kinase synthesized via a ribosomal frameshift was sufficient for reactivation under conditions where reactivation requires this enzyme, we introduced the mutation into the well-characterized strain KOS. This mutant virus reactivated from latency, albeit less efficiently than KOS. Plaque autoradiography revealed three phenotypes of reactivating viruses: uniformly low thymidine kinase activity, mixed high and low activity, and uniformly high activity. We generated a recombinant thymidine kinase-null virus from a reactivating virus expressing uniformly low activity. This virus did not reactivate, confirming that mutations in other genes that would influence reactivation had not arisen. Therefore, in strains that require thymidine kinase for reactivation from latency, low levels of enzyme synthesized via a ribosomal frameshift can suffice

Percentage of subjects who developed HIV-associated TB during longitudinal follow up according to antigen specificity of baseline IFN-γ responses.

<p>Ag85, antigen 85; ESAT-6, early secreted antigenic target 6; IFN-γ, interferon gamma; TB, tuberculosis; WCL, whole cell lysate.</p

Baseline subject characteristics according to number of detectable baseline IFN-γ responses to mycobacterial antigens.

<p>P values are derived via Mann-Whitney <i>U</i> tests, student's t tests and chi squared tests as appropriate.</p><p>SD, standard deviation; TB, tuberculosis; TST, tuberculin skin test.</p