Editing MuLV Psueduvirus (section)

=== 6. Data Analysis ===

#'''Calculation and plotting of relative luciferase units' averages and standard deviations'''
##Use a graph plotting software to calculate luciferase assay measurement averages and standard deviations of experimental and biological replicates.
##Plot data as bar chart with standard deviations.

'''NOTE:&nbsp;'''When performing statistical analyses on data, make sure to include at least three biological replicates in data sets.

VSV G pseudotyped positive control particles (VSV-Gpp) gave very high average infectivity in the 106 to 107 relative luciferase units (RLU) range respectively. For SARS-CoV S pseudotyped particles (Figure 1A) infection of susceptible Vero-E6 cells, a strong average infectivity was measured at around 9.8 x 104 RLU. This value is almost 3 orders of magnitude higher than the values measured for the non-infected control (1.1 x 102 RLU), or the Δenv particles (1.5 x 102 RLU), which do not harbor any viral envelope glycoproteins at their surface. Similarly, for MERS-CoV S pseudotyped particles (Figure 1B) infection of Huh-7 cells, a high average infectivity was measured at around 1.0 x 106 RLU. This is almost 4 orders of magnitude higher than the values measured for the non-infected control (0.8 x 102 RLU), or the Δenv particles (2.0 x 102 RLU).

one 10-cm-diameter dish of subconfluently grown 293T or Anjou65 cells was transfected with 7.5 μg of pMP71-eGFP-pre (37), 12.5 μg of pSV-Mo-MLVgagpol, and 1 to 4 μg of env expression plasmids by using a calcium phosphate transfection kit (Sigma, Taufkirchen, Germany). The medium was replaced 6 to 8 h after transfection. Pseudotype vector-containing supernatants were harvested after an additional 24, 39, and 48 h; filtered through a 0.45 μm-pore-size Millex-HV filter (Millipore, Schwalbach, Germany); and used for transduction.

Deletion mutant CΔ19 (with 19 amino acids deleted from the C terminus) yielded about 100-fold-higher pseudotype titers than the wt S protein. Independently prepared pseudotype particles showed variations in titers, ranging from 4 × 103 to 1 × 104 TU/ml. Further truncations involved highly conserved cysteine residues and resulted in decreased pseudotype titers (Fig. 3A). Particles without envelope protein failed to infect Vero cells (not shown), whereas VSV and LCMV glycoprotein pseudotypes yielded two- to sevenfold-higher titers than the SARS S CΔ19 construct.
[[Category:Virus]]