Identification and functional characterization of Epstein-Barr virus DNA polymerase by in vitro transcription-translation of a cloned gene.

In order to identify the gene encoding the Epstein-Barr virus (EBV) DNA polymerase, a portion of the BamHI-A fragment containing the fifth leftward open reading frame (BALF5) of the EBV genome was cloned into SP6 and T7 promoter-containing vectors for in vitro transcription-translation. The RNA synthesized in vitro was used to program rabbit reticulocyte lysates, which were analyzed for the synthesis of the putative polymerase polypeptide (110 kDa) and assayed directly for EBV DNA polymerase activity. The polypeptide synthesized by the full-length BALF5 genomic fragment had a molecular mass of 110 kDa. 5'-truncated BALF5 with the first and second ATGs deleted produced 95- and 83-kDa polypeptides, respectively. All three translation products were enzymatically active and displayed resistance to high salt concentrations. The identity of the largest polypeptide as the viral polymerase was established by (i) immunoprecipitation with EBV-positive sera from patients with nasopharyngeal carcinoma and by a rabbit polyclonal antiserum prepared with a synthetic peptide derived from the DNA sequence of BALF5; (ii) identification of a polypeptide of identical size (110 kDa) immunoprecipitated from superinfected Raji cell extracts by these antibodies; and (iii) salt-resistant enzymatic activity which was neutralized by the rabbit EBV antiserum. Thus, BALF5 encodes a functional polymerase identical to that induced in superinfected Raji cells

Effect of Universal Testing and Treatment on HIV Incidence - HPTN 071 (PopART).

BACKGROUND: A universal testing and treatment strategy is a potential approach to reduce the incidence of human immunodeficiency virus (HIV) infection, yet previous trial results are inconsistent. METHODS: In the HPTN 071 (PopART) community-randomized trial conducted from 2013 through 2018, we randomly assigned 21 communities in Zambia and South Africa (total population, approximately 1 million) to group A (combination prevention intervention with universal antiretroviral therapy [ART]), group B (the prevention intervention with ART provided according to local guidelines [universal since 2016]), or group C (standard care). The prevention intervention included home-based HIV testing delivered by community workers, who also supported linkage to HIV care and ART adherence. The primary outcome, HIV incidence between months 12 and 36, was measured in a population cohort of approximately 2000 randomly sampled adults (18 to 44 years of age) per community. Viral suppression (<400 copies of HIV RNA per milliliter) was assessed in all HIV-positive participants at 24 months. RESULTS: The population cohort included 48,301 participants. Baseline HIV prevalence was 21% or 22% in each group. Between months 12 and 36, a total of 553 new HIV infections were observed during 39,702 person-years (1.4 per 100 person-years; women, 1.7; men, 0.8). The adjusted rate ratio for group A as compared with group C was 0.93 (95% confidence interval [CI], 0.74 to 1.18; P = 0.51) and for group B as compared with group C was 0.70 (95% CI, 0.55 to 0.88; P = 0.006). The percentage of HIV-positive participants with viral suppression at 24 months was 71.9% in group A, 67.5% in group B, and 60.2% in group C. The estimated percentage of HIV-positive adults in the community who were receiving ART at 36 months was 81% in group A and 80% in group B. CONCLUSIONS: A combination prevention intervention with ART provided according to local guidelines resulted in a 30% lower incidence of HIV infection than standard care. The lack of effect with universal ART was unanticipated and not consistent with the data on viral suppression. In this trial setting, universal testing and treatment reduced the population-level incidence of HIV infection. (Funded by the National Institute of Allergy and Infectious Diseases and others; HPTN 071 [PopArt] ClinicalTrials.gov number, NCT01900977.)