Journal: International Journal of Molecular Sciences

Article Title: Hsp90 Activity Is Necessary for the Maturation of Rabies Virus Polymerase

doi: 10.3390/ijms23136946

Figure Lengend Snippet: Antiviral activity of 17-AAG against RABV (MOI = 0.1) in N2a, NAC1300, and HEK293 cell monolayers demonstrated by ( A ) a reduction of RABV titre, expressed as TCID 50 /mL, and ( B ) a reduction of N gene copy numbers in the infected cell supernatant 48 h after infection. ( C ) The relative number of the uninfected cells cultured for 48 h in the presence of 17-AAG compared to the control cultures without 17-AAG. The means and ranges of the experiment (performed in duplicate) are plotted.

Article Snippet: The rabies virus strain CVS-11 (laboratory strain of Challenge Virus Standard) was obtained from the European Reference Laboratory for Rabies (ANSES, Malzeville, Nancy, France) and was propagated in NAC1300 and N2a cell lines.

Techniques: Activity Assay, Infection, Cell Culture, Control

Journal: International Journal of Molecular Sciences

Article Title: Hsp90 Activity Is Necessary for the Maturation of Rabies Virus Polymerase

doi: 10.3390/ijms23136946

Figure Lengend Snippet: Hsp90 inhibition does not influence the expression of RABV proteins. HEK293 cells were transfected with plasmids expressing myc-tagged N, G, M, and P ( left panel) and Flag-L ( right panel). We added 500 nM 17-AAG 2 h after transfection. Expression was tested using anti-myc and anti-Flag antibodies 24 h later. The experiment was repeated twice, with similar results.

Article Snippet: The rabies virus strain CVS-11 (laboratory strain of Challenge Virus Standard) was obtained from the European Reference Laboratory for Rabies (ANSES, Malzeville, Nancy, France) and was propagated in NAC1300 and N2a cell lines.

Techniques: Inhibition, Expressing, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Hsp90 Activity Is Necessary for the Maturation of Rabies Virus Polymerase

doi: 10.3390/ijms23136946

Figure Lengend Snippet: Soluble RABV L protein is expressed without P protein present. ( A ) HEK293 cells were transfected with plasmids that expressed the RABV Flag-L and P-myc proteins, as indicated. The cells were lysed with IP buffer 48 h after transfection. The insoluble fraction was separated by centrifugation and solubilized with 8 M of urea. Proteins were detected with antibodies specific for Flag and myc. HEK293 cells were transfected with plasmids that expressed the Flag-L proteins of the RABV and MeV virus ( B ) or with the plasmid that expressed VSV Flag-L ( C ). We added 0.5 μM 17-AAG 8 h after transfection. Cells were lysed 48 h after transfection. The results of one of two experiments are presented.

Article Snippet: The rabies virus strain CVS-11 (laboratory strain of Challenge Virus Standard) was obtained from the European Reference Laboratory for Rabies (ANSES, Malzeville, Nancy, France) and was propagated in NAC1300 and N2a cell lines.

Techniques: Transfection, Centrifugation, Virus, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Hsp90 Activity Is Necessary for the Maturation of Rabies Virus Polymerase

doi: 10.3390/ijms23136946

Figure Lengend Snippet: Right after infection, CVS-11 RNA synthesis is not affected by Hsp90 inhibition. NAC1300 cells pre-incubated for 2 h with 500 nM 17-AAG and control cells not treated with the inhibitor were infected on ice with CVS-11 at MOI = 100. After 1 h, unbound virus was washed off with cold PBS. The washed cells were incubated in a medium with or without 17-AAG. RNA was isolated at the indicated time and RABV RNA copies for the N gene were measured using RT-qPCR. The means and SD values of the four repeats of the experiment are plotted. The Student’s t -test was used to calculate the p values.

Article Snippet: The rabies virus strain CVS-11 (laboratory strain of Challenge Virus Standard) was obtained from the European Reference Laboratory for Rabies (ANSES, Malzeville, Nancy, France) and was propagated in NAC1300 and N2a cell lines.

Techniques: Infection, Inhibition, Incubation, Control, Virus, Isolation, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Hsp90 Activity Is Necessary for the Maturation of Rabies Virus Polymerase

doi: 10.3390/ijms23136946

Figure Lengend Snippet: Primers used for cloning. The KpnI and NotI sites used for plasmid construction are underlined.

Article Snippet: The rabies virus strain CVS-11 (laboratory strain of Challenge Virus Standard) was obtained from the European Reference Laboratory for Rabies (ANSES, Malzeville, Nancy, France) and was propagated in NAC1300 and N2a cell lines.

Techniques: Cloning, Plasmid Preparation