Editing Spike Protein VLP (section)

=== Representative Results ===
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Representative results of infectivity assays of SARS-CoV S and MERS-CoV S pseudotyped particles are shown in '''Figure 1'''. As expected, for both'''''<i>Figure 1'</i>'''A and 1B''', the VSV G pseudotyped positive control particles (VSV-Gpp) gave very high average infectivity in the 10<sup>6</sup> to 10<sup>7</sup> relative luciferase units (RLU) range respectively. For SARS-CoV S pseudotyped particles ('''Figure 1''''''A''') infection of susceptible Vero-E6 cells, a strong average infectivity was measured at around 9.8 x 10<sup>4</sup> RLU. This value is almost 3 orders of magnitude higher than the values measured for the non-infected control (1.1 x 10<sup>2</sup> RLU), or the Δenv particles (1.5 x 10<sup>2</sup> RLU), which do not harbor any viral envelope glycoproteins at their surface. Similarly, for MERS-CoV S pseudotyped particles ('''Figure 1''''''B''') infection of Huh-7 cells, a high average infectivity was measured at around 1.0 x 10<sup>6</sup> RLU. This is almost 4 orders of magnitude higher than the values measured for the non-infected control (0.8 x 10<sup>2</sup> RLU), or the Δenv particles (2.0 x 10<sup>2</sup> RLU). An additional infectivity assay was performed in which the SARS-Spp and MERS-Spp were serially diluted and used to infect Vero-E6 cells ('''Figure 2''''''A'''). This assay confirms that the luciferase activity measured is dependent on the concentration of the particles used to infect cells. To confirm the role of angiotensin converting enzyme 2 (ACE2) and dipeptidyl peptidase 4 (DPP4), the receptors of SARS-CoV and MERS-CoV, respectively, in mediating attachment and entry of the pseudotyped particles, we used poorly-permissive HEK-293T cells and transfected them to express either ACE2 or DPP4 ('''Figure 2''''''B'''). The transfected cells were then used for an infectivity assay. This analysis demonstrates that upon overexpression of ACE2 and DPP4, there is a ~4-log and ~2-log increase for SARS-Spp and MERS-Spp infectivity, respectively, confirming that receptor usage of pseudovirions is similar to that of native viruses.

The examples shown here demonstrate the importance of including negative (non-infected, Δenv particles) as well as positive control (VSV-Gpp) conditions when producing pseudotyped particles. Indeed, the positive control VSV-Gpp particles allow us to assess whether a particular batch of pseudotyped particles was successful in yielding functional and infective pseudovirions. Expected results of typical infection by VSV-Gpp particles in most mammalian cell lines are in the 10<sup>6</sup>−10<sup>7</sup> RLU range. Problems with HEK-293T/17 producer cells (high passage number, issues with cell densities) or poor transfection efficiencies can impact overall pseudotyped particle production and infectivity. Furthermore, the negative control conditions are also important as they allow us to assess the baselines of RLU measurements in a particular cell line (non-infected condition) and non-specific internalization of particles (Δenv infection) which is not mediated by a viral envelope protein. Ideally, for a given type of particle pseudotyped with a viral envelope protein of interest, it is recommended to obtain values that are a few orders of magnitude higher than the negative control values, as shown here in '''Figure 1''''''A,B'''. However, if for a given cell type a pseudotyped virus infection gives very little infectivity (i.e.'','' close to negative controls such as in '''Figure 2''''''B'''in non-transfected N.T. conditions) it does not necessarily mean that the pseudotyped particle production was faulty. It could well be that the particular cell line used is not or poorly permissive to infection. It is recommended to check whether a given cell type is expected to be susceptible to infection by the virus being investigated. Transfecting poorly permissive cells with plasmid(s) expressing the viral receptor(s) can allow more efficient viral entry and infection to occur, as shown in '''Figure 2''''''B''', where upon transfection of ACE2 and DPP4 receptors in target HEK-293T cells, there is a ~4- and ~2-log increase in infectivity, respectively.

{|
|-
| '''Plasmid/reagent'''
| '''Quantity'''
|-
| pCMV-MLVgagpol MLV gag and pol encoding plasmid
| 300 ng
|-
| pTG-Luc transfer vector with luciferase reporter
| 400 ng
|-
| pcDNA-SARS-S, pcDNA-MERS-S or empty vector
| 300 ng
|-
| Reduced serum cell culture medium
| To 50 µL
|}

'''Table 1: Quantities of plasmids and reduced serum cell culture medium required to transfect one well of a 6-well plate of HEK-293T/17 cells for pseudotyped particle production. '''From the concentration of plasmid preparations, calculate the required volume to reach the required amount of plasmid DNA. If more than one well is transfected with the same plasmids, multiply volumes by required number of wells to transfect, and include an extra well in calculations to avoid running out of mix in later steps. The total amount of DNA being transfected is 1 µg/well. Plasmids are available upon request to authors.

{|
|-
| '''Reagent'''
| '''Quantity'''
|-
| Transfection reagent
| 3 µL
|-
| Reduced serum cell culture medium
| 47 µL
|}

'''Table 2: Quantities of transfection reagent and reduced serum cell culture medium required to transfect one well of a 6-well plate of HEK-293T/17 cells for pseudotyped particle production. '''Multiply volumes by required number of wells to transfect and include extra wells in calculations to avoid running out of mix in later steps. The transfection reagent: plasmid DNA ratio used is 3:1.

&lt;img src="//cloudflare2.jove.com/files/ftp_upload/59010/59010fig1.jpg" alt="Figure 1" /&gt;<br/>'''Figure 1'''''': Coronavirus S-pseudotyped particle infectivity assays in susceptible host cells using a murine leukemia virus (MLV) backbone and luciferase reporter gene.'''(''''''<b>A</b>) SARS-CoV S pseudotyped particle infectivity assay in Vero-E6 cells. ('''B''') MERS-CoV S pseudotyped particle infectivity assay in Huh-7 cells. For both ('''A''') and ('''B'''), plotted data corresponds to the average relative luciferase units from three independent experiments, with error bars corresponding to standard deviation (s.d.). Data plotted in log<sub>10</sub> scale on y-axis. N.I.: non-infected control; Δenv: infection with pseudotyped particles lacking viral envelope glycoproteins and VSV-Gpp: infection with pseudotyped particles bearing positive control VSV G envelope glycoprotein. Other abbreviation used, SARS-Spp: infection with SARS-CoV S pseudotyped particles, MERS-Spp: infection with MERS-CoV S pseudotyped particles. <a href="https://www.jove.com/files/ftp_upload/59010/59010fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.&lt;/a&gt;

&lt;img src="//cloudflare2.jove.com/files/ftp_upload/59010/59010fig2.jpg" alt="Figure 2" /&gt;<br/>'''Figure 2'''''': Concentration-dependence of CoV S-pseudovirion infectivity and role of ACE2 and DPP4 receptors in SARS-Spp and MERS-Spp entry.'''(''''''<b>A</b>) Concentration-dependence of SARS-Spp and MERS-Spp infectivity measured by luciferase activity assay. Pseudovirions were serially diluted and used to infect Vero-E6 cells. ('''B''') Infectivity assay of SARS-Spp and MERS-Spp in poorly permissive HEK-293T target cells transfected to express ACE2, and DPP4 receptors, respectively. Data plotted in log<sub>10</sub> scale on y-axis as in '''Figure 1''' from duplicate experiments, with error bars corresponding to standard deviation (s.d.). N.T.: non-transfected control HEK-293T cells; ACE2: angiotensin converting enzyme 2 (SARS-CoV receptor) and DPP4: dipeptidyl peptidase 4 (MERS-CoV receptor). Other abbreviations used are the same as in '''Figure 1'''. <a href="https://www.jove.com/files/ftp_upload/59010/59010fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.&lt;/a&gt;
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