Journal: Microbial Cell Factories

Article Title: A novel, lactase-based selection and strain improvement strategy for recombinant protein expression in Kluyveromyces lactis

doi: 10.1186/1475-2859-11-112

Figure Lengend Snippet: Detection of E2 protein expression in yeast cells by immunocytology. K. lactis strains VAK367 ( A ) and VAK726 ( B and C ), which contains the BVDV-E2 ORF in single copy under control of the LAC4 promoter, were grown under inducing conditions (2% lactose) and analyzed by confocal microscopy after staining with DAPI (panels b ), E2-specific antibody WB-214 (PA2020; c.c.pro, Germany) and a polyclonal secondary antibody (Dylight 488; Dianova) (panels c ) and Dil18 (Invitrogen) for staining of the plasma membrane (panels d ). E2 and nuclear (DAPI) staining is merged in panels a . C ) Overlay of DNA, membrane-, and E2- staining reveals accumulation of E2 in perinuclear regions suggesting localization in the ER.

Article Snippet: After blocking (3% BSA; 0.1% cold fish gelatin, 0.1% Triton X-100; 0.05% Tween20) a 90 min incubation with a monoclonal anti-E2-antibody, [WB-214 (PA2020; c.c.pro, Germany); 1:100 in blocking solution] was performed, followed by 90 min incubation with a secondary antibody (Dylight 488, Dianova; 1:100 in blocking solution).

Techniques: Expressing, Confocal Microscopy, Staining

Journal: Microbial Cell Factories

Article Title: A novel, lactase-based selection and strain improvement strategy for recombinant protein expression in Kluyveromyces lactis

doi: 10.1186/1475-2859-11-112

Figure Lengend Snippet: Quantification of E2 protein levels in different congenic K. lactis strains. A ) Dot blots performed in triplicates with 10, 25 and 50 μg of crude cell lysates from parent strain VAK367, the E2-producing strain VAK726; the Klgal80 mutant derivative of VAK726 (VAK746); and the two derivatives of VAK726 carrying two (VAK834) and five (VAK836) copies of the KlGAL4 gene, respectively. Cell lysates were applied to nitrocellulose membranes and incubated with E2-specific antibody WB-214 (PA2020; c.c.pro, Germany) and polyclonal secondary antibody (Dylight 488; Dianova). B ) Relative fluorescence signal intensities were quantified as described in Material and Methods and are given relative to strain VAK726.

Article Snippet: After blocking (3% BSA; 0.1% cold fish gelatin, 0.1% Triton X-100; 0.05% Tween20) a 90 min incubation with a monoclonal anti-E2-antibody, [WB-214 (PA2020; c.c.pro, Germany); 1:100 in blocking solution] was performed, followed by 90 min incubation with a secondary antibody (Dylight 488, Dianova; 1:100 in blocking solution).

Techniques: Mutagenesis, Incubation, Fluorescence

Journal: mBio

Article Title: Treatment of Sindbis Virus-Infected Neurons with Antibody to E2 Alters Synthesis of Complete and nsP1-Expressing Defective Viral RNAs

doi: 10.1128/mbio.02221-22

Figure Lengend Snippet: SINV RNA forms. (A) Schematic diagram of SINV RNA replication and the forms of SINV RNA made from each type of template strand. “gRNA” represents genomic RNA, “sgRNA” represents subgenomic RNA, and “DVG” represents the defective viral genome. (B) Table of the different SINV RNA forms indicating their sense, genome nucleotide composition, polyadenylation status, and translation to proteins.

Article Snippet: To determine how anti-E2 antibody affects SINV RNA synthesis, Oxford Nanopore Technologies direct long-read RNA sequencing was used to sequence viral RNAs following antibody treatment of infected neurons.

Techniques:

Journal: mBio

Article Title: Treatment of Sindbis Virus-Infected Neurons with Antibody to E2 Alters Synthesis of Complete and nsP1-Expressing Defective Viral RNAs

doi: 10.1128/mbio.02221-22

Figure Lengend Snippet: Nanopore sequencing of SINV-infected differentiated AP-7 cells with or without anti-E2 antibody treatment. Differentiated AP-7 cells were infected with SINV (MOI of 10) and mock treated or treated with anti-E2 antibody (5 μg/mL) 4 h after infection. At 24, 48, and 72 h after infection, total intracellular RNA was collected in triplicate, enriched for poly(A), directly sequenced via nanopore sequencing, and aligned to the rat or SINV genome. (A) Proportion of RNA reads that align to the rat versus SINV genome. Proportions indicate the number of rat or SINV reads divided by the total number of RNA reads at each time point for mock-infected cells. The mock-infected 24-h sample is indicated in purple. (All mock time points had 0 SINV-aligned reads.) SINV-infected no-antibody-treatment samples are indicated in orange, and SINV-infected antibody-treated samples are indicated in green. Each dot indicates a different biological replicate. (B) Averaged sequence coverage of SINV-aligned RNA reads. Coverage plots indicate relative sequencing depth normalized to run yield across the SINV genome with (green) or without (orange) antibody treatment at 24, 48, and 72 h after infection. Coverage data represent the average of 3 biological replicates. (C) Reads were classified into genomic, subgenomic, DVG, or subDVG SINV RNA species using coverage differences at junction locations (see Materials and Methods). Abundance ratios for each species were calculated as a proportion of total SINV reads for each sample. Each point represents 1 biological replicate, and horizontal lines indicate the mean from 3 biological replicates. * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001.

Article Snippet: To determine how anti-E2 antibody affects SINV RNA synthesis, Oxford Nanopore Technologies direct long-read RNA sequencing was used to sequence viral RNAs following antibody treatment of infected neurons.

Techniques: Nanopore Sequencing, Infection, Sequencing

Journal: mBio

Article Title: Treatment of Sindbis Virus-Infected Neurons with Antibody to E2 Alters Synthesis of Complete and nsP1-Expressing Defective Viral RNAs

doi: 10.1128/mbio.02221-22

Figure Lengend Snippet: Anti-E2 antibody increases production of SINV genomic RNA and decreases production of subgenomic RNA. Analysis of newly synthesized viral RNA. Differentiated AP-7 cells were infected with SINV (MOI of 10) and treated with anti-E2 antibody (5 μg/mL) at 4 h after infection. At the indicated time points, cells were labeled with 20 μCi/mL [ 3 H]uridine in the presence of dactinomycin (1 μg/mL) for 2 h. (A) Representative assessment of radioactive viral RNA synthesis by agarose-formaldehyde gel and autoradiography. (Top) Long exposure; (bottom) short exposure of the same gel. (B) Densities of labeled genomic RNA (gRNA [left]) and subgenomic RNA (sgRNA [right]) normalized to SINV-infected, untreated cells at 12 h; (C) ratio of relative subgenomic RNA to genomic RNA density; (D) ratios of qRT-PCR-quantified biotin-captured 5-ethynyl uridine-labeled nascent subgenomic RNA to genomic RNA in SINV-infected dAP-7 cells with and without antibody treatment. Data from three independent experiments are presented as mean ± SD. * * , P < 0.01; *** * , P < 0.0001.

Article Snippet: To determine how anti-E2 antibody affects SINV RNA synthesis, Oxford Nanopore Technologies direct long-read RNA sequencing was used to sequence viral RNAs following antibody treatment of infected neurons.

Techniques: Synthesized, Infection, Labeling, Autoradiography, Quantitative RT-PCR

Journal: mBio

Article Title: Treatment of Sindbis Virus-Infected Neurons with Antibody to E2 Alters Synthesis of Complete and nsP1-Expressing Defective Viral RNAs

doi: 10.1128/mbio.02221-22

Figure Lengend Snippet: Antibody treatment increases SINV genomic RNA and decreases nsP1 defective viral genome RNA levels in dAP-7 cells. (A and B) Differentiated AP-7 cells were infected with SINV (MOI of 10) and treated with anti-E2 antibody (5 μg/mL) or medium (mock) at 4 h after infection. At the indicated time points, total cellular RNA lysates were collected. (A) Semiquantitative RT-PCR for SINV RNA using primers against the 5′ and 3′ ends of the SINV genome (SV-171F, SV-11655R). “gRNA” represents full-length SINV genomic RNA, and “DVG” represents the nsP1 defective viral genome. Asterisks indicate PCR bands excised for DNA extraction and Sanger sequencing. (B) SINV genome sequence alignment of the two defective viral genome sequences identified. (C and D) Differentiated AP-7 cells (dAP-7), undifferentiated cycling AP-7 cells (cAP-7), and BHK-21 cells were infected with SINV (MOI of 10) and treated with medium (mock) or anti-E2 antibody (5 μg/mL) at 4 h after infection. Twenty-four hours after infection, total cellular RNA was reverse transcribed to cDNA for qRT-PCR analysis of nsP1 defective viral genome levels. (C) Primer design used for qRT-PCR assay. Primers for the nsP1 defective viral genome span the deleted SINV region, while primers for the SINV genomic RNA are within the deleted region. (D) qRT-PCR analysis for nsP1 defective genome production. nsP1 RNA levels are expressed as fold regulation relative to SINV gRNA as calculated by ddCT. Data are presented as mean ± SD from two biological replicates. (E) Immunoblot of SINV nsP1, nsP2, and nsP3 expression from untreated and antibody-treated SINV-infected dAP-7 cells. (F) Densitometry was used to determine the ratios of nsP1 to nsP2 in immunoblots and to compare untreated (TE) to antibody-treated (MAb) SINV-infected dAP-7 cells. * , P < 0.05.

Article Snippet: To determine how anti-E2 antibody affects SINV RNA synthesis, Oxford Nanopore Technologies direct long-read RNA sequencing was used to sequence viral RNAs following antibody treatment of infected neurons.

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, DNA Extraction, Sequencing, Reverse Transcription, Quantitative RT-PCR, Western Blot, Expressing

Journal: mBio

Article Title: Treatment of Sindbis Virus-Infected Neurons with Antibody to E2 Alters Synthesis of Complete and nsP1-Expressing Defective Viral RNAs

doi: 10.1128/mbio.02221-22

Figure Lengend Snippet: The nsP1 defective viral genome is packaged and released into viral particles. Differentiated AP-7 cells were infected with SINV (MOI of 10) and treated with medium (mock) or anti-E2 antibody (5 μg/mL) at 4 h after infection. Eighteen hours after infection, RNA from the cells and supernatant fluid was collected. (A) RNA was extracted from the cellular and supernatant samples. RT-PCR analysis for SINV genomic RNA (gRNA) and nsP1 defective viral genome RNA (DVG) was performed. “NTC” represents the no-template control. (B) RNase A protection assay. Culture supernatant was treated with RNase A (10 μg/mL) to digest all nonencapsidated RNAs (+ B [before RNA extraction]) or untreated (−). Following RNase treatment, RNA was extracted from the supernatant and reverse transcribed for RT-PCR analysis of nsP1 defective genome RNA (dgRNA). As a positive control for RNase activity, extracted RNA was also treated with RNase A under the same conditions (+ A [after RNA extraction]).

Article Snippet: To determine how anti-E2 antibody affects SINV RNA synthesis, Oxford Nanopore Technologies direct long-read RNA sequencing was used to sequence viral RNAs following antibody treatment of infected neurons.

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, RNA Extraction, Reverse Transcription, Positive Control, Activity Assay

Journal: mBio

Article Title: Treatment of Sindbis Virus-Infected Neurons with Antibody to E2 Alters Synthesis of Complete and nsP1-Expressing Defective Viral RNAs

doi: 10.1128/mbio.02221-22

Figure Lengend Snippet: The nsP1 defective viral genome RNA can be translated, and expression is associated with increased SINV RNA capping efficiency. (A) The shorter nsP1 defective viral genome RNA (subDVG) was cloned into the pcDNA3.1 DNA expression vector and in vitro transcribed to RNA (dvgRNA). The RNA was capped and polyadenylated in vitro , purified, and transfected into uninfected undifferentiated AP-7 cells (dvgRNA-AAA). As controls, pTE (complete SINV plasmid) and unpolyadenylated defective viral genome RNA (dvgRNA−) were also transfected. Twenty-four hours after transfection, lysates were collected and assessed by immunoblot probing with a polyclonal antibody against SINV nsP1. (B) Proportion of capped genomes in virus particles released from untreated and antibody-treated SINV-infected dAP-7 cells. Differentiated AP-7 cells were infected with SINV (MOI of 10) and treated with medium (mock) or anti-E2 antibody (5 μg/mL) 4 h after infection. Twenty-four hours after infection, virus particles were purified from the supernatant fluid by ultracentrifugation and viral RNA was extracted and treated with XRN-1 exoribonuclease to degrade noncapped RNAs (XRN +) or left untreated (XRN −). Following treatment, the RNA was reverse transcribed to cDNA and assessed by qRT-PCR for SINV nsP2 and E2 transcripts. The y axis indicates the ratio of copy numbers of nsP2 or E2 transcripts in the XRN-treated versus untreated groups. (C) Immunoblot of nsP1 protein expression 24 h after SINV infection or transfection with pGenLenti vectors expressing nsP1 or nsP1 DVG; (D) proportion of capped genomes in virus particles released from untreated (blue) or antibody-treated (red) cells transiently transfected with lentivirus vector plasmids expressing either nsP1 or nsP1 DVG 24 h prior to infection with SINV. Virions were isolated with Viraffinity reagent, and RNA capping was analyzed using XRN degradation, as described above. Data are presented as mean ± SD and are from three biological replicates. * , P < 0.05; * * , P < 0.01; ** * , P < 0.001.

Article Snippet: To determine how anti-E2 antibody affects SINV RNA synthesis, Oxford Nanopore Technologies direct long-read RNA sequencing was used to sequence viral RNAs following antibody treatment of infected neurons.

Techniques: Expressing, Clone Assay, Plasmid Preparation, In Vitro, Purification, Transfection, Western Blot, Virus, Infection, Reverse Transcription, Quantitative RT-PCR, Isolation

Journal: mBio

Article Title: Treatment of Sindbis Virus-Infected Neurons with Antibody to E2 Alters Synthesis of Complete and nsP1-Expressing Defective Viral RNAs

doi: 10.1128/mbio.02221-22

Figure Lengend Snippet: Model of nsP1 defective genome proviral activity (above) and interactions with antiviral anti-E2 antibody (below). nsP1 DVG expression results in increased production of SINV nsP1 protein and improved SINV RNA capping efficiency. The nsP1 DVG is packaged into defective interfering particles (DIPs) that can spread from cell to cell and replicate during coinfection with wild-type virus. Anti-E2 antibody suppresses viral RNA transcription and particularly affects SINV sgRNA and nsP1 DVG production. Anti-E2 antibody also inhibits viral and DIP budding by direct binding of surface E2. Inhibition of spread of the nsP1 DVG by inhibition of budding or neutralization contributes to overall decreased viral replication by decreasing production of nsP1 protein and decreasing SINV RNA capping efficiency during late infection.

Article Snippet: To determine how anti-E2 antibody affects SINV RNA synthesis, Oxford Nanopore Technologies direct long-read RNA sequencing was used to sequence viral RNAs following antibody treatment of infected neurons.

Techniques: Activity Assay, Expressing, Virus, Binding Assay, Inhibition, Neutralization, Infection