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1.
FIG. 2.

FIG. 2. From: Pseudotyping Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV): F Proteins from Group II NPVs Are Functionally Analogous to AcMNPV GP64.

Transfection-infection assay for viral propagation. Sf9 cells were transfected with the indicated bacmids, incubated for 72 h, and stained for GUS activity (A, C, E, and G). Supernatants from the transfected cells (top panels) were used to infect Sf9 cells (B, D, F, and H), which were incubated for 48 h and then stained for GUS activity. Staining of cells in the lower panels (infected cells) indicates that infectious virions were generated in the transfected cells. Bacmids that fail to propagate an infection in Sf9 cells can propagate an infection in cells expressing OpMNPV GP64 (Sf9Op1D). Sf9Op1D cells were transfected with the indicated bacmids (I, K, and M) and incubated for 72 h; then supernatants were transferred to Sf9 cells and stained for GUS activity after 48 h (J, L, and N).

Oliver Lung, et al. J Virol. 2002 June;76(11):5729-5736.
2.
FIG. 4.

FIG. 4. From: Pseudotyping Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV): F Proteins from Group II NPVs Are Functionally Analogous to AcMNPV GP64.

One-step growth curves are shown for wild type AcMNPV, the repair virus vAcgp64−/Acgp64, and AcMNPV gp64-null viruses pseudotyped with VSV-G (vAcgp64−/VSV-G), Se8 (vAcgp64−/Se8), or Ld130 (vAcgp64−/Ld130). Sf9 cells were infected at a multiplicity of infection of 5, and supernatants were harvested at the indicated times postinfection and titered on Sf9Op1D cells. Each data point represents the average from three separate infections (error bars, standard deviations), except for the data point for the Se8-pseudotyped virus at 168 h postinfection, which represents a single infection.

Oliver Lung, et al. J Virol. 2002 June;76(11):5729-5736.
3.
FIG. 5.

FIG. 5. From: Pseudotyping Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV): F Proteins from Group II NPVs Are Functionally Analogous to AcMNPV GP64.

Western blot analysis of pseudotyped virions. A series of Western blots of purified virions from pseudotyped viruses vAcgp64−/VSV-G (lane 1), vAcgp64−/Se8 (lane 2), and vAcgp64−/Ld130 (lane 3) were probed with antibodies α-Se8 (against SeMNPV F1 protein, the 60-kDa Se8 cleavage product) (33), α-VSV-G (20), and α-GP64 (monoclonal antibody AcV5) (14). An anti-nucleocapsid antibody (α-VP39) was used as an internal control for each preparation of purified budded virions (20). GP64 was not detected from pseudotyped viruses but was detected in a positive control shown on the right (Sf9 cell lysates infected with the repair virus, vAcgp64−/Acgp64). Positions of uncleaved and cleaved SeMNPV F are indicated.

Oliver Lung, et al. J Virol. 2002 June;76(11):5729-5736.
4.
FIG. 6.

FIG. 6. From: Pseudotyping Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV): F Proteins from Group II NPVs Are Functionally Analogous to AcMNPV GP64.

(A) Diagram of the furin cleavage site in the SeMNPV F protein (Se8) and the Se8K149 mutant. RSKR represents the furin cleavage site in the wild-type SeMNPV F protein (Se8), and RSKK represents the furin cleavage site containing a single amino acid substitution (Se8K149). (B) Western blot analysis of gp64-null budded virions pseudotyped with Se8 or Se8K149. Virion preparations (lanes 1 to 3) were probed with antibodies directed against the SeMNPV F protein (α-Se8), GP64 (α-GP64), and VP39 (α-VP39). Virions of vAcgp64−/Se8 were generated in infected Sf9 cells (lane 1), whereas virions of vAcgp64− (lane 2) and vAcgp64−/Se8K149 (lane 3) were generated in infected Sf9Op1D cells. The latter virions therefore contain Op MNPV GP64 (lanes 2 and 3). The VP39 capsid protein was included as an internal control. Positions of cleaved and uncleaved forms of Se8 are indicated.

Oliver Lung, et al. J Virol. 2002 June;76(11):5729-5736.
5.
FIG. 1.

FIG. 1. From: Pseudotyping Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV): F Proteins from Group II NPVs Are Functionally Analogous to AcMNPV GP64.

(A) Strategy for generation of a gp64-null AcMNPV bacmid by homologous recombination in E. coli. The gp64 locus of the AcMNPV bacmid (bMON14272) is shown above the EcoRI-HindIII fragment that was used as a transfer vector to replace the gp64 locus with a chloramphenicol resistance gene (cat). Sequences included for homologous recombination (107325 to 108039 and 109761 to 112049) are indicated (1). (B) Strategy for insertion of envelope protein gene constructs into the polyhedrin locus of the gp64-null AcMNPV bacmid. Inserts include a p6.9 promoter-GUS reporter and no envelope protein gene (top) and a p6.9 promoter-GUS reporter plus envelope protein genes (ENV) under the control of either the gp64 promoter (pGP64) or the polyhedrin promoter (pPolh) (center). Envelope protein gene cassettes were inserted into the att b site (indicated by right and left insertion sites, Tn7R and Tn7L) in the polyhedrin locus by Tn 7-based transposition. Envelope proteins and genes (italicized) are listed on the right.

Oliver Lung, et al. J Virol. 2002 June;76(11):5729-5736.
6.
FIG. 3.

FIG. 3. From: Pseudotyping Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV): F Proteins from Group II NPVs Are Functionally Analogous to AcMNPV GP64.

Thogoto virus GP75 protein expression, detection in AcMNPV budded virions, and membrane fusion activity. (A) Western blot analysis of GP75 expression in Sf9 cells that were either mock transfected (lane 1) or transfected with the vAcgp64−/Thogp75 (lane 2) or vAcgp64− (lane 3) bacmid and examined at 72 h posttransfection. GP75 was detected by using an affinity-purified antibody generated against a GP75 peptide (N-KERAHEKSKDLPFGNK-C). (B) Western blot detection of GP75 protein in purified budded virions (vAcgp64− or vAcgp64−/Thogp75) generated in Sf9Op1D cells. An anti-capsid protein (VP39) antibody was used as an internal control (lower panel). (C and D) Syncytium formation assay. CHO cells were transfected with a plasmid (pRC-THO) expressing GP75 under the control of a cytomegalovirus early promoter. At 24 h posttransfection, cells were incubated in phosphate-buffered saline at pH 7.0 (C) or pH 5.0 (D) for 15 min; then they were examined for syncytium formation after 90 min. Large syncytial masses containing many nuclei are indicated by arrowheads. Cells transfected with the empty vector (pRC-CMV) did not show syncytium formation (data not shown).

Oliver Lung, et al. J Virol. 2002 June;76(11):5729-5736.

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