The Novel Nuclear Targeting and BFRF1-Interacting Domains of BFLF2 Are Essential for Efficient Epstein-Barr Virus Virion Release.

Epstein-Barr virus (EBV) genomic DNA is replicated and packaged into procapsids in the nucleus to form nucleocapsids, which are then transported into the cytoplasm for tegumentation and final maturation. The process is facilitated by the coordination of the viral nuclear egress complex (NEC), which consists of BFLF2 and BFRF1. By expression alone, BFLF2 is distributed mainly in the nucleus. However, it colocalizes with BFRF1 at the nuclear rim and in cytoplasmic nuclear envelope-derived vesicles in coexpressing cells, suggesting temporal control of the interaction between BFLF2 and BFRF1 is critical for their proper function. The N-terminal sequence of BFLF2 is less conserved than that of alpha- and betaherpesvirus homologs. Here, we found that BFLF2 amino acids (aa) 2 to 102 are required for both nuclear targeting and its interaction with BFRF1. Coimmunoprecipitation and confocal analysis indicated that aa 82 to 106 of BFLF2 are important for its interaction with BFRF1. Three crucial amino acids (R47, K50, and R52) and several noncontinuous arginine and histidine residues within aa 59 to 80 function together as a noncanonical nuclear localization signal (NLS), which can be transferred onto yellow fluorescent protein (YFP)-LacZ for nuclear targeting in an importin ß-dependent manner. Virion secretion is defective in 293 cells harboring a BFLF2 knockout EBV bacmid upon lytic induction and is restored by trans-complementation of wild-type BFLF2, but not NLS or BFRF1-interacting defective mutants. In addition, multiple domains of BFRF1 were found to bind BFLF2, suggesting multiple contact regions within BFRF1 and BFLF2 are required for proper nuclear egress of EBV nucleocapsids.IMPORTANCE Although Epstein-Barr virus (EBV) BFRF1 and BFLF2 are homologs of conserved viral nuclear egress complex (NEC) in all human herpesviruses, unique amino acid sequences and functions were identified in both proteins. In this study, the nuclear targeting and BFRF1-interacting domains were found within the N terminus of BFLF2. We showed that amino acids (aa) 82 to 106 are the major region required for BFLF2 to interact with BFRF1. However, the coimmunoprecipitation (Co-IP) data and glutathione transferase (GST) pulldown experiments revealed that multiple regions of both proteins contribute to reciprocal interactions. Different from the canonical nuclear localization signal (NLS) in other herpes viral homologs, BFLF2 contains a novel importin-dependent nuclear localization signal, including R47, K50, and R52 and several neighboring discontinuous arginine and histidine residues. Using a bacmid complementation system, we show that both the nuclear targeting and the novel nuclear localization signal within aa 82 to 106 of BFLF2 are required for virion secretion.

Impacts of physician promotion on the online healthcare community: Using a difference-in-difference approach.

In this study, we use a difference-in-difference approach to explore how physician promotion, the advancement of a physician's offline reputation, affects patient behavior toward physicians in online healthcare communities; this allows us to explore how patients interpret the signals created by physician promotion. The study sample was collected from over 140,000 physician online profiles after 25 months of continuous observation, with 280 physicians who were promoted at month 13 as the treatment group and a control group obtained by propensity score matching. Our results show that a physician's promotion causes more patients to choose that physician, makes patients willing to give more psychological rewards, and makes them tend to give that physician a higher online rating. This implies that patient behavior is susceptible to the signal of physician promotion because the quality of the physician is unlikely to have changed significantly in the short term. These findings extend prior research on reputation in online communities and have crucial implications for theory and practice.

Identification and characterization of an extracellular alkaline phosphatase in the marine diatom Phaeodactylum tricornutum.

In phosphorus-deficient conditions, Phaeodactylum tricornutum releases an alkaline phosphatase (PtAPase) to the medium that is readily detectable by activity staining. Nucleic acid and amino acid sequence of this alkaline phosphatase (APase) was identified by performing proteomic analysis and database searches. Sequence alignment suggests that PtAPase belongs to the PhoA family, and it possesses key residues at the Escherichia coli PhoA active site. Quantitative PCR results indicate that the induction of APase mRNA transcription is very sensitive to phosphorus availability and population growth. The molecular mass of native PtAPase (148 kDa) determined by gel filtration chromatography indicates that PtAPase, like most PhoA, is homodimeric. Zn and Mg ions are essential cofactors for most PhoA enzymes; however, PtAPase activity did not require Zn ions. In fact, 5 mM Zn²âº, Mo²âº, Co²âº, Cd²âº, or Cu²âº inhibited its enzymatic activity, whereas 5 mM Mn²âº, Mg²âº, or Ca²âº enhanced its enzymatic activity. The responses of PtAPase to divalent metal ions were different from those of most PhoAs, but were similar to the PhoA in a marine bacterium, Cobetia marina. Phylogenetic analysis shows that homologs of PhoA are also present in other diatom species, and that they clustered in a unique branch away from other PhoA members. PtAPase may represent a novel class of PhoA that helps diatoms to survive in the ocean. Quantification of the PtAPase mRNA may help monitor the physiological condition of diatoms in natural environments and artificial bioreactors.