Subcellular fractions were analyzed by SDS-PAGE and Western blotting using anti-HA tag antibody. Sf9 cells infected with BV-E2/p17 or BV-G2/p17 as indicated on top of the panel were harvested at 48 h pi and processed for cell fractionation into cytosolic compartment (Cy), membranes (Mb), nuclear compartment (Nu) and cytoskeletal-associated proteins (Sk). M, molecular mass markers, with their apparent molecular masses indicated in kilodaltons (kDa) on the right side of the blots. Lysates of mock-infected cells (mo) or BV-E2/p17- or BV-G2/p17-infected cells harvested at 24, 48 and 72 h pi, as indicated on top of the panel were clarified by centrifugation, and soluble fraction (S) and pelletable material (P) analyzed as above. Whole cell lysates were analyzed by SDS-PAGE and Western blotting using anti-HA tag antibody. Sf9 cells were mock-infected (lanes mo), or infected with BV-E2/p17 or BV-G2/p17, and harvested at 24, 48 and 72 h pi, as indicated on top of the panel. Fusion of this peptide to the N-terminus of scFv molecules of interest could be applied as a general method for BV-display of scFv in a GP64- and VSV-G-independent manner.Įxpression, solubility and cellular localization of scFvE2/p17 and scFvG2/p17 in Sf9 cells. The function required for BV envelope incorporation was carried by the N-terminal octadecapeptide of scFvE2/p17, which acted as a signal peptide for BV display. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2) to another scFv recognizing CD147 (scFv-M6-1B9) conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6.Įxpression of scFvE2/p17 in insect cells using a BV vector resulted in baculoviral progeny displaying scFvE2/p17. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP) assembly and egress, and both failed to be encapsidated in VLP. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs) and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17), was found to exert an inhibitory effect on HIV-1 replication. With her instructor thousands of miles away, Nishizaka texted her encouragements to “jump and shake off the tension” before her performance.Īccordion to Chen, the competition was nerve-wracking because it’s been a while since competitions were in-person, but she enjoyed being back on stage and was reminded how much she loves performing in front of a live audience.Ĭhen performed on the Steinway D Grand Piano, and said the keys were light, and the high registers were lovely and took her by surprise, saying, “the Debussy paired really well with that particular Steinway.Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv) antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. Thus, receiving such an award was an absolute honor,” Chen said.Ĭhen said she Googled “how to calm myself down” before walking on stage. “I thought that I would never be able to play such pieces expertly. SCV Signal News Podcast with Aron BenderĮven with the resume of music accomplishments, Chen felt she struggled with “water music,” a form of impressionistic sounds, using nonfunctional harmony and ambiguous key signatures, which Debussy is known for in his compositions.
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