Beau J. Fenner

Betanodavirus B2 Is an RNA Interference Antagonist That Facilitates Intracellular Viral RNA Accumulation

ABSTRACT Betanodaviruses are small positive-sense bipartite RNA viruses that infect a wide variety of fish species and are notorious for causing lethal outbreaks in juvenile fish hatcheries worldwide. The function of a small nonstructural protein, B2, encoded by the subgenomic RNA3 of betanodaviruses, has remained obscure. Greasy grouper nervous necrosis virus, a betanodavirus model, was used to develop a facile DNA-based reverse genetics system that recapitulated the virus infection cycle, and we used this system to show that B2 is a small nonstructural protein that is essential for high level accumulation of viral RNA1 after RNA transfection of fish, mammalian, and avian cells. The defect in RNA1 accumulation in a B2 mutant was partially complemented by supplying B2 RNA in trans. Confocal analysis of the cellular distribution of B2 indicated that B2 is able to enter the nucleus and accumulates there during the late stages of GGNNV infection. Using human HeLa cells as a cellular RNA interference model, we found that B2 could efficiently antagonize RNA interference, which is a property shared by the distantly related alphanodavirus B2 proteins. This function provides appears to provide an explanation, at least in part, for why B2 mutant RNA1 is severely impaired in its intracellular accumulation.

Expression of hemagglutinin protein from the avian influenza virus H5N1 in a baculovirus/insect cell system significantly enhanced by suspension culture

BackgroundPrevention of a possible avian influenza pandemic necessitates the development of rapid diagnostic tests and the eventual production of a vaccine.ResultsFor vaccine production, hemagglutinin (HA1) from avian influenza H5N1 was expressed from a recombinant baculovirus. Recombinant HA1 was expressed in monolayer or suspension culture insect cells by infection with the recombinant baculovirus. The yield of rHA1 from the suspension culture was 68 mg/l, compared to 6 mg/l from the monolayer culture. Immunization of guinea pigs with 50 μg of rHA1 yielded hemagglutinin inhibition and virus neutralization titers of 1:160 after two times vaccination with rHA1 protein.ConclusionThus, the production of rHA1 using an insect suspension cell system provides a promising basis for economical production of a H5 antigen.

Sequestration and Protection of Double-Stranded RNA by the Betanodavirus B2 Protein

ABSTRACT Betanodavirus B2 belongs to a group of functionally related proteins from the sense-strand RNA virus family Nodaviridae that suppress cellular RNA interference. The B2 proteins of insect alphanodaviruses block RNA interference by binding to double-stranded RNA (dsRNA), thus preventing Dicer-mediated cleavage and the subsequent generation of short interfering RNAs. We show here that the fish betanodavirus B2 protein also binds dsRNA. Binding is sequence independent, and maximal binding occurs with dsRNA substrates greater than 20 bp in length. The binding of B2 to long dsRNA is sufficient to completely block Dicer cleavage of dsRNA in vitro. Protein-protein interaction studies indicated that B2 interacts with itself and with other dsRNA binding proteins, the interaction occurring through binding to shared dsRNA substrates. Induction of the dsRNA-dependent interferon response was not antagonized by B2, as the interferon-responsive Mx gene of permissive fish cells was induced by wild-type viral RNA1 but not by a B2 mutant. The induction of Mx instead relied solely on viral RNA1 accumulation, which is impaired in the B2 mutant. Hyperediting of virus dsRNA and site-specific editing of 5-HT2C mRNA were both antagonized by B2. RNA editing was not, however, observed in transfected wild-type or B2 mutant RNA1, suggesting that this pathway does not contribute to the RNA1 accumulation defect of the B2 mutant. We thus conclude that betanodavirus B2 is a dsRNA binding protein that sequesters and protects both long and short dsRNAs to protect betanodavirus from cellular RNA interference.

RING-H2 Protein WSSV249 from White Spot Syndrome Virus Sequesters a Shrimp Ubiquitin-Conjugating Enzyme, PvUbc, for Viral Pathogenesis

ABSTRACT Modification of proteins by ubiquitin is essential for numerous cellular processes. The RING-H2 finger motif has been implicated in ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Four proteins, WSSV199, WSSV222, WSSV249, and WSSV403, from white spot syndrome virus (WSSV) contain the RING-H2 motif. Here we report that WSSV249 physically interacts with a shrimp ubiquitin-conjugating enzyme, PvUbc, and mediates ubiquitination through its RING-H2 motif in the presence of E1 and PvUbc. Mutations of the putative zinc coordination residues in the RING-H2 domain of WSSV249, however, ablate ubiquitination efficiency. In addition, the RING-H2 domain of WSSV249 is capable of ubiquitination with UbcH1, UbcH2, UbcH5a, UbcH5b, UbcH5c, UbcH6, and UbcH10, respectively, exhibiting a low degree of E2 specificity. Significantly, the expression of WSSV249 and PvUbc increased during infection, as revealed by real-time PCR. Furthermore, in situ hybridization showed that WSSV249 and PvUbc display similar expression patterns in infected shrimps, and immunofluorescence and immunohistochemistry assays showed an increase of PvUbc in infected shrimp cells. These results suggest that the RING-H2 protein WSSV249 from WSSV may function as an E3 ligase via sequestration of PvUbc for viral pathogenesis in shrimp.

White spot syndrome virus open reading frame 222 encodes a viral E3 ligase and mediates degradation of a host tumor suppressor via ubiquitination.

ABSTRACT We have characterized a white spot syndrome virus (WSSV) RING-H2-type protein, WSSV222, which is involved in ubiquitination. WSSV222 exhibits RING-H2-dependent E3 ligase activity in vitro in the presence of the specific conjugating enzyme UbcH6. Mutations in the RING-H2 domain abolished WSSV222-dependent ubiquitination, revealing the importance of this domain in WSSV222 function. Yeast two-hybrid and pull-down analyses revealed that WSSV222 interacts with a shrimp tumor suppressor-like protein (TSL) sharing 60% identity with human OVCA1. To better characterize the interaction of WSSV222 and TSL in vivo, we established a stable TSL-expressing cell line derived from the human ovarian cancer cell line A2780, where we observed a TSL-dependent prolonged G1 phase. Furthermore, we detected WSSV222-mediated ubiquitination and MG132-sensitive degradation of TSL both in shrimp primary cell culture and in the TSL-expressing cell line. Transient expression of TSL in BHK cells leads to apoptosis, which was rescued by WSSV222. Taken together, our data suggest that WSSV222 acts as an antiapoptosis protein by ubiquitin-mediated proteolysis of TSL to ensure successful WSSV replication in shrimp.

Dissection of Double-Stranded RNA Binding Protein B2 from Betanodavirus

ABSTRACT Betanodaviruses are small RNA viruses that infect teleost fish and pose a considerable threat to marine aquaculture production. These viruses possess a small protein, termed B2, which binds to and protects double-stranded RNA. This prevents cleavage of virus-derived double-stranded RNAs (dsRNAs) by Dicer and subsequent production of small interfering RNA (siRNA), which would otherwise induce an RNA-silencing response against the virus. In this work, we have performed charged-to-alanine scanning mutagenesis of the B2 protein in order to identify residues required for dsRNA binding and protection. While the majority of the 19 mutated B2 residues were required for maximal dsRNA binding and protection in vitro, residues R53 and R60 were essential for both activities. Subsequent experiments in fish cells confirmed these findings by showing that mutations in these residues abolished accumulation of both the RNA1 and RNA2 components of the viral genome, in addition to preventing any significant induction of the host interferon gene, Mx. Moreover, an obvious positive correlation was found between dsRNA binding and protection in vitro and RNA1, RNA2, and Mx accumulation in fish cells, further validating the importance of the selected amino acid residues. The same trend was also demonstrated using an RNA silencing system in HeLa cells, with residues R53 and R60 being essential for suppression of RNA silencing. Importantly, we found that siRNA-mediated knockdown of Dicer dramatically enhanced the accumulation of a B2 mutant. In addition, we found that B2 is able to induce apoptosis in fish cells but that this was not the result of dsRNA binding.

Characterization of monoclonal antibody against SARS coronavirus nucleocapsid antigen and development of an antigen capture ELISA.

Abstract This report describes the production of several MAbs against N195 protein, a major immunodomain of SARS CoV nucleocapsid protein [He, Q., Chong, K.H., Chang, H.H., Leung, B., Ling, A.E., Wei, T., Chan, S.W., Ooi, E.E., Kwang, J., 2004. Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. Clin. Diagn. Lab. Immunol. 11 (2) 417–422.]. One representative IgG1 monoclonal antibody (MAb), S-A5D5, was selected and characterized. S-A5D5 reacted specifically react with both recombinant and native nucleocapsid protein of SARS CoV. The reactivity of S-A5D5 with purified N195 protein and utilization of the MAb as a detector antibody to develop an antigen capture ELISA was assessed. As little as 37.5pg of purified N protein and 50 TCID50 of SARS CoV could be detected by the antigen capture ELISA. Specific binding of the MAb S-A5D5 to both purified N195 and SARS CoV nucleocapsid antigen was effectively inhibited by human SARS positive serum and guinea pig anti-N195 serum. The N protein in N195-spike recombinant baculovirus-infected Sf-9 cells could also be identified. N protein was detected in 18 IFA IgM-positive serum samples collected from SARS confirmed patients, but not in nine samples collected from SARS recovery patient. No false positive results were given when 60 samples from healthy individuals were tested, and no cross-reaction occurred when infectious bronchitis virus (IBV), chicken coronavirus, was tested. This monoclonal antibody-based antigen capture ELISA is thus a powerful tool for early diagnosis of SARS CoV infection.

More to stable tears than thickness of the tear film lipid layer.

King-Smith et al. recently published an enlightening piece of work showing that tear film evaporation does not necessarily correlate with thickness of the tear film lipid layer. This has important implications in the field of dry eye research, where the consensus opinion for many years has been that a thicker lipid layer serves to prevent evaporation of the aqueous component of the tear film, helping to prevent the development of dry eye symptoms. We were especially interested in the work of King-Smith et al. because it corroborates our own work on the relationship between lipid layer thicknesses and tear film breakup. A prospective study at our center involving 86 undifferentiated patients (mean age 56.1 6 13.7 years) found that noninvasive tear breakup time (NIBUT) did not significantly correlate with lipid layer thickness (r21⁄4 0.001, slope1⁄4-0.126, P 1⁄4 0.750). In our study, a LipiView interferometer was employed to noninvasively assess lipid layer thickness among the 86 patients (mean thickness 64.9 6 23.5 nm). Optical interference patterns produced by light reflected from the tear film in situ are used to derive lipid layer thickness in this method. An Oculus Keratograph model 5M was used to measure NIBUT among the same patients (mean NIBUT 9.21 6 6.44 seconds) and the data were analyzed using an F test for regression. In light of these findings, we are inclined to agree with the suggestion by King-Smith and coworkers that tear film breakup may be impacted by more than simply lipid layer thickness, and may also be a function of lipid layer composition. Other workers have recently shown that tear film evaporation is dictated by only certain components of the lipid layer. Moreover, some dry eye patients appear to have abnormal lipid compositions that may impact the normal functioning of the tear film. Thus, it remains to be seen how best to interpret tear breakup times in dry eye patients, but it almost certainly requires more information than simply lipid layer thickness data. At the same time, the role and significance of lipid layer thickness in dry eye, previously thought to be functionally important, requires reexamination.

Corneal staining characteristics in limited zones compared with whole cornea documentation for the detection of dry eye subtypes.

PURPOSE To determine the reliability of single- and double-zone corneal fluorescent staining compared with five-zone analysis for the prediction of dry eye disease. METHODS Prospective study of 510 subjects with dry eye disease characterized using corneal fluorescein staining, Schirmer scores, and tear break-up times. Corneal staining was quantified using Baylor scoring with ROC analysis used to assess predictive power of single- and double-zone compared with five-zone analysis for aqueous, evaporative, and mixed dry eye disease. RESULTS Double-zone analysis predicted each subtype of dry eye disease investigated. Aqueous disease was predicted by superior/inferior zones (AUCSup/Inf 0.797 versus AUCTotal 0.816), evaporative disease by inferior/central zones (AUCInf/Cen 0.759 versus AUCTotal 0.778), and mixed disease by superior/inferior, inferior/nasal, and inferior/central zones (AUCSup/Inf 0.765, AUCInf/Nas 0.771, AUCInf/Cen 0.778 versus AUCTotal 0.795). Inferior zone analysis predicted aqueous (AUCInf 0.751 versus AUCTotal 0.750), evaporative (AUCInf 0.756 versus AUCTotal 0.752), and mixed (AUCInf 0.831 versus AUCTotal 0.788) dry eye disease with similar efficacy to complete analysis in diabetic individuals. Inferior zone analysis also predicted aqueous disease in rheumatoid arthritis patients (AUCInf 0.804 versus AUCTotal 0.785), whereas superior zone analysis predicted evaporative disease in thyroid disease patients (AUCSup 0.765 versus AUCTotal 0.752). CONCLUSIONS Double-zone corneal staining predicts the presence of dry eye disease with predictive power similar to complete corneal analysis. Additionally, subtypes of dry eye can be predicted by single-zone analysis among patients with diabetes (inferior zone), rheumatoid arthritis (inferior zone), and thyroid disease (superior zone). Clinical characterization of dry eye can thus be hastened by limiting corneal examination to specific zones.