A DISCOVERY OF DESPITEFUL FB APPS

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Conserved Structural Motifs at the C-Terminus of Baculovirus Protein IE0 are Important for its Functions in Transactivation and Supporting hr5-mediated DNA Replication

IE0 and IE1 are transactivator proteins of the most studied baculovirus, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). IE0 is a 72.6 kDa protein identical to IE1 with the exception of its 54 N-terminal amino acid residues. To gain some insight about important structural motifs of IE0, we expressed the protein and C‑terminal mutants of it under the control of the Drosophila heat shock promoter and studied the transactivation and replication functions of the transiently expressed proteins. IE0 was able to promote replication of a plasmid bearing the hr5 origin of replication of AcMNPV in transient transfections with a battery of eight plasmids expressing the AcMNPV genes dnapol, helicase, lef-1, lef-2, lef-3, p35, ie-2 and lef-7. IE0 transactivated expression of the baculovirus 39K promoter. Both functions of replication and transactivation were lost after introduction of selected mutations at the basic domain II and helix-loop-helix conserved structural motifs in the C-terminus of the protein. These IE0 mutants were unable to translocate to the cell nucleus. Our results point out the important role of some structural conserved motifs to the proper functioning of IE0

Analysis of the orgyia pseudotsugata multiple nucleopolyhedrovirus IE1 acidic activation domain role in viral DNA replication

Baculovirus Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) transient replication assays have previously identified six genes as essential iel, lef-1, lef-2, lef-3, DNA pol, helicase; three genes as stimulatory ie2, p34, iap-1; and two types of replication origins hrs and non-hrs. One of these essential proteins IE1, has an acidic activation domain (AAD) at the N-terminus that is required for transcriptional transactivation but its role in viral DNA replication was unknown. In this study we have determined that the AAD is also essential for DNA replication. Unlike transcriptional activation, the AAD cannot be replaced with the AcMNPV IE1 and herpesvirus VP 16 AADs for replication. Deletion analysis of the OpMNPV IE 1-AAD revealed the presence of separate sub-domains for replication and transcriptional activation. By substituting OpMNPV lef-1, -2, -3 and helicase proteins (part of the putative replisome) with corresponding AcMNPV factors, the inactive OpMNPV IE1 chimeric protein IE 1-Ac AD, which contains the AcMNPV IE1 AAD, was functional for replication activation. This suggests that AADs interact with viral replication factors in order to maintain viral specificity in replication. The IE 1-Ac AD specificity for the AcMNPV replisome was found to be similar to the native AcMNPV IE1, thus demonstrating that by changing the AAD the specificity of the protein changes. Further studies showed that the role of stimulatory factor OpMNPV IE2 in replication was to maintain the specificity between AAD and the replisome. Absence of IE2 allowed replication from non-specific AAD and replisome but presence of IE2 active replication was observed only between specific AAD and replisome. AcMNPV non-hr origin analysis indicated that it was unable to allow for replication with OpMNPV IE1 and OpMNPV replication proteins; however substituting with AcMNPV LEF-3, POL and HELICASE, resulted in replication. This result reveals the presence of specific interactions between origins and replication factors. Our studies on OpMNPV IEO, the only spliced gene of IE1, showed that IEO is functionally active for replication and can replace IE1. We also performed initial functional analysis of OpMNPV EXONO. To date there is no information on OpMNPV EXONO function other than the fact that it contributes 23aa to the N-terminus of IEO. Our study shows EXONO has a conserved novel ring finger and is expressed as a late gene. However, our preliminary research could not identify any function attributable to EXONO.Land and Food Systems, Faculty ofGraduat

The Acidic Activation Domain of the Baculovirus Transactivator IE1 Contains a Virus-Specific Domain Essential for DNA Replication

IE1 is a potent transcriptional transactivator of the baculovirus Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) and has been shown to be essential for viral DNA replication. IE1 contains an acidic activation domain (AAD) at the N terminus that is essential for transcriptional transactivation, but its role in viral DNA replication is unknown. In this study the role of the IE1 AAD in DNA replication is investigated. We have determined that deletion of the AAD eliminates the ability of IE1 to support DNA replication, showing that the AAD is essential for DNA replication as well as transcriptional transactivation. Replacement of the AAD with the archetype domain from herpesvirus VP16 and the evolutionarily related domain from Autographa californica MNPV (AcMNPV) IE1 produces chimeric proteins that are potent transactivators. Surprisingly, however, these chimeric proteins were unable to support DNA replication, indicating that there is a host- or virus-specific replication subdomain in the AAD that was not functionally replaced by the VP16 or AcMNPV AAD. Using N- and C-terminal deletion mutants, the region of the AAD that was essential for DNA replication was mapped to amino acids 1 to 65. AAD deletion mutants also showed that an IE1 that is functional for transcriptional transactivation is not required for viral DNA replication. The IE1 AAD therefore contains an essential replication domain that is separable from the transcriptional activation domains. Our results suggest that IE1 specifically interacts with a component of the viral replication complex, supporting the view that it acts as a nucleating factor by binding to the viral replication origins

Autographa californica Multiple Nucleopolyhedrovirus exon0 (orf141), Which Encodes a RING Finger Protein, Is Required for Efficient Production of Budded Virus

exon0 (orf141) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene that codes for a predicted 261-amino-acid protein. Located in the C-terminal region of EXON0 are a predicted leucine-rich coiled-coil domain and a RING finger motif. The 5′ 114 nucleotides of exon0 form part of ie0, which is a spliced gene expressed at very early times postinfection, but transcriptional analysis revealed that exon0 is transcribed as a late gene. To determine the role of exon0 in the baculovirus life cycle, we used AcMNPV bacmids and generated exon0 knockout viruses (Ac-exon0-KO) by recombination in Escherichia coli. Ac-exon0-KO progressed through the very late phases in Sf9 cells, as evidenced by the development of occlusion bodies in the nuclei of the transfected or infected cells. However, production of budded virus (BV) in Ac-exon0-KO-infected cells was reduced at least 3 orders of magnitude in comparison to that in wild-type virus infection. Microscopy revealed that Ac-exon0-KO was restricted primarily to the cells initially infected, exhibiting a single-cell infection phenotype. Slot blot assays and Western blot analysis indicated that exon0 deletion did not affect the onset or levels of viral DNA replication or the expression of IE1, IE0, and GP64 prior to BV release. These results demonstrate that exon0 is required for efficient production of BV in the AcMNPV life cycle but does not affect late occlusion-derived virus

A Novel Dry-Stabilized Whole Blood Microsampling and Protein Extraction Method for Testing of SARS-CoV-2 Antibody Titers

The COVID-19 pandemic has revealed a crucial need for rapid, straightforward collection and testing of biological samples. Serological antibody assays can analyze patient blood samples to confirm immune response following mRNA vaccine administration or to verify past exposure to the SARS-CoV-2 virus. While blood tests provide vital information for clinical analysis and epidemiology, sample collection is not trivial; this process requires a visit to the doctor’s office, a professionally trained phlebotomist to draw several milliliters of blood, processing to yield plasma or serum, and necessitates appropriate cold chain storage to preserve the specimen. A novel whole blood collection kit (truCOLLECT) allows for a lancet-based, decentralized capillary blood collection of metered low volumes and eliminates the need for refrigerated transport and storage through the process of active desiccation. Anti-SARS-CoV-2 spike (total and neutralizing) and nucleocapsid protein antibody titers in plasma samples obtained via venipuncture were compared to antibodies extracted from desiccated whole blood using Adaptive Focused Acoustics (AFA). Paired plasma versus desiccated blood extracts yields Pearson correlation coefficients of 0.98; 95% CI [0.96, 0.99] for anti-SARS-CoV-2 spike protein antibodies, 0.97; 95% CI [0.95, 0.99] for neutralizing antibodies, and 0.97; 95% CI [0.94, 0.99] for anti-SARS-CoV-2 nucleocapsid protein antibodies. These data suggest that serology testing using desiccated and stabilized whole blood samples can be a convenient and cost-effective alternative to phlebotomy

A new testing platform using fingerstick blood for quantitative antibody response evaluation after SARS-CoV-2 vaccination.

Testing and vaccination have been major components of the strategy for combating the ongoing COVID-19 pandemic. In this study, we have developed a quantitative anti-SARS-CoV-2 spike (S1) IgG antibody assay using a fingerstick dried blood sample. We evaluated the feasibility of using this high-throughput and quantitative anti-SARS-CoV-2 spike (S1) IgG antibody testing assay in vaccinated individuals. Fingerstick blood samples were collected and analyzed from 137 volunteers before and after receiving the Moderna or Pfizer mRNA vaccine. Anti-SARS-CoV-2 S1 IgG antibody could not be detected within the first 7 days after receiving the first vaccine dose, however, the assay reliably detected antibodies from day 14 onwards. In addition, no anti-SARS-CoV-2 nucleocapsid (N) protein IgG antibody was detected in any of the vaccinated or healthy participants, indicating that the anti-SARS-CoV-2 S1 IgG assay is specific for the mRNA vaccine-induced antibodies. The S1 IgG levels detected in fingerstick samples correlated with the levels found in venous blood plasma samples and with the efficacy of venous blood plasma samples in the plaque reduction neutralization test (PRNT). The assay displayed a limit of quantification (LOQ) of 0.59 μg/mL and was found to be linear in the range of 0.51-1000 μg/mL. Finally, its clinical performance displayed a Positive Percent Agreement (PPA) of 100% (95% CI: 0.89-1.00) and a Negative Percent Agreement (NPA) of 100% (95% CI: 0.93-1.00). In summary, the assay described here represents a sensitive, precise, accurate, and simple method for the quantitative detection and monitoring of post-vaccination anti-SARS-CoV-2 spike IgG responses