A comprehensive mapping of interactions among Epstein–Barr virus (EBV) proteins and interactions of EBV proteins with human proteins should provide specific hypotheses and a broad perspective on EBV strategies for replication and persistence. Interactions of EBV proteins with each other and with human proteins were assessed by using a stringent high-throughput yeast two-hybrid system. Overall, 43 interactions between EBV proteins and 173 interactions between EBV and human proteins were identified. EBV–EBV and EBV–human protein interaction, or “interactome” maps provided a framework for hypotheses of protein function. For example, LF2, an EBV protein of unknown function interacted with the EBV immediate early R transactivator (Rta) and was found to inhibit Rta transactivation. From a broader perspective, EBV genes can be divided into two evolutionary classes, “core” genes, which are conserved across all herpesviruses and subfamily specific, or “noncore” genes. Our EBV–EBV interactome map is enriched for interactions among proteins in the same evolutionary class. Furthermore, human proteins targeted by EBV proteins were enriched for highly connected or “hub” proteins and for proteins with relatively short paths to all other proteins in the human interactome network. Targeting of hubs might be an efficient mechanism for EBV reorganization of cellular processes.

Epstein-Barr virus (EBV) microRNAs miR-BHRF1-1, -2, and -3 have been detected in latency III-infected lymphoblasts, where they are encoded within EBNA transcripts (X. Cai, A. Schafer, S. Lu, J. P. Bilello, R. C. Desrosiers, R. Edwards, N. Raab-Traub, and B. R. Cullen, PLoS Pathog. 2: e23, 2006). In latency III-infected lymphoblasts, we have also identified a stable 1.3-kb RNA, which begins 3′ to miR-BHRF1-1, includes the BHRF1 open reading frame, and ends near miR-BHRF1-2. This 1.3-kb RNA is the residue of Drosha cleavage of the BHRF1 microRNAs from EBNA transcripts. Early after induction of EBV replication in latency I-infected Akata lymphoblasts, BHRF1 spliced 1.4-kb mRNA accumulated along with low levels of miR-BHRF1-2 and -3 and a 0.9-kb Drosha or miR-BHRF1-2 cleavage product of BHRF1 mRNA. The turning on of latency III infection at 48 to 72 h after induction of EBV replication was associated with higher miR-BHRF1-1, -2, and -3 levels; accumulation of the 1.3-kb RNA residue in the nucleus; abundant BHRF1 spliced 1.4-kb mRNA in the cytoplasm; and more abundant 0.9-kb mRNA cleavage product in the cytoplasm. These findings implicate miR-BHRF1-2 in 3′ cleavage of BHRF1 mRNA in the cytoplasm and Drosha in cleavage of latency III EBNA and EBV replication-associated BHRF1 transcripts in the nucleus.

RNA helicase A (RHA) has been shown to promote HIV-1 replication at both the translation and reverse transcription stages. A prerequisite step for reverse transcription involves the annealing of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} , the primer for reverse transcription, to HIV-1 RNA. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} annealing is a multistep process that is initially facilitated by Gag prior to viral protein processing. Herein, we report that RHA promotes this annealing through increasing both the quantity of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} annealed by Gag and the ability of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} to prime the initiation of reverse transcription. This improved annealing is the result of an altered viral RNA conformation produced by the coordinate action of Gag and RHA. Since RHA has been reported to promote the translation of unspliced viral RNA to Gag protein, our observations suggest that the conformational change in viral RNA induced by RHA and newly produced Gag may help facilitate the switch in viral RNA from a translational mode to one facilitating \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} annealing.

Helicases contribute to diverse biological processes including replication, transcription and translation. Recent reports suggest that unwinding of some helicases display repetitive activity, yet the functional role of the repetitiveness requires further investigation. Using single-molecule fluorescence assays, we elucidated a unique unwinding mechanism of RNA helicase A (RHA) that entails discrete substeps consisting of binding, activation, unwinding, stalling and reactivation stages. This multi-step process is repeated many times by a single RHA molecule without dissociation, resulting in repetitive unwinding/rewinding cycles. Our kinetic and mutational analysis indicates that the two double stand RNA binding domains at the N-terminus of RHA are responsible for such repetitive unwinding behavior in addition to providing an increased binding affinity to RNA. Further, the repetitive unwinding induces an efficient annealing of a complementary RNA by making the unwound strand more accessible. The complex and unusual mechanism displayed by RHA may help in explaining how the repetitive unwinding of helicases contributes to their biological functions.

γ‐Aminobutyric acid (GABA) and glutamate (Glu) are considered as the predominant inhibitory and excitatory neurotransmitters in mammalian central nervous systems (CNS), respectively. The presence of the GABA system and metabotropic glutamate receptors in sperm prompted us to explore the existence of ionotropic glutamate receptors and glutamate transporters in sperm. Immunofluorescent analysis was used to investigate the existence and location of glutamate, glutamate receptor (NR2B), and glutamate transporter (GLT1) in mouse and human sperm. Our present results showed that NR2B was located in the midpiece of sperm, whereas GLT1 mainly existed in the head. Moreover, glutamate uptake activity was detected in mouse sperm and it could be blocked by dihydrokainic acid (DHK, GLT1‐selective inhibitor) and DL‐threo‐beta‐hydroxyaspartic acid (THA, nonselective inhibitor). In addition, reverse transcription‐polymerase chain reaction technique and sequencing analysis revealed that glutamate transporters (GLT1 and EAAC1) and ionotropic glutamate receptors (NR1, NR2B, GluR6, and KA2) existed in mouse sperm as well as in human sperm. The present findings are the first direct evidence for the existence of ionotropic glutamate receptors and glutamate transporters in sperm. It also indicates that, in sperm, glutamate receptors and transporters might have functions other than neurotransmission.