Editing Spike Protein VLP (section)

=== Introduction ===
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Host cell entry constitutes the initial steps of the viral infectious life cycle. For enveloped viruses, this involves binding to a single host cell receptor or several receptors, followed by fusion of viral and cellular membranes. These essential functions are carried out by viral envelope glycoproteins<sup>1</sup><sup>,</sup><sup>2</sup>. The coronavirus envelope glycoprotein is called the spike (S) protein and is a member of the class I viral fusion proteins<sup>2</sup><sup>,</sup><sup>3</sup><sup>,</sup><sup>4</sup><sup>,</sup><sup>5</sup><sup>,</sup><sup>6</sup>. Studying viral envelope glycoproteins is critical for understanding many important characteristics of a given virus, such as: lifecycle initiation, its host and cellular tropism, interspecies transmission, viral pathogenesis, as well as host cell entry pathways. Viral pseudotyped particles, also named pseudovirions, are powerful tools that enable us to easily study the function of viral fusion proteins. Pseudotyped particles or pseudovirions are chimeric virions that consist of a surrogate viral core with a heterologous viral envelope protein at their surface. The protocol's main purpose is to show how to obtain coronavirus spike pseudotyped particles that are based on a murine leukemia virus (MLV) core and contain a luciferase reporter gene. As examples, the method to produce pseudotyped particles with the spike proteins of the highly pathogenic severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses are presented. The protocol describes the transfection procedure involved, how to infect susceptible target cells, and infectivity quantification by luciferase assay.

Since the entry steps of the pseudovirions are governed by the coronavirus S at their surface, they enter cells in a similar fashion to native counterparts. As such, they are excellent surrogates of functional infectivity assays. Pseudotyped particles are usually derived from parental model viruses such as retroviruses (MLV<sup>7</sup><sup>,</sup><sup>8</sup><sup>,</sup><sup>9</sup><sup>,</sup><sup>10</sup><sup>,</sup><sup>11</sup><sup>,</sup><sup>12</sup><sup>,</sup><sup>13</sup><sup>,</sup><sup>14</sup><sup>,</sup><sup>15</sup><sup>,</sup><sup>16</sup><sup>,</sup><sup>17</sup><sup>,</sup><sup>18</sup><sup>,</sup><sup>19</sup><sup>,</sup><sup>20</sup><sup>,</sup><sup>21</sup><sup>,</sup><sup>22</sup> and the lentivirus human immunodeficiency virus — HIV<sup>23</sup><sup>,</sup><sup>24</sup><sup>,</sup><sup>25</sup><sup>,</sup><sup>26</sup><sup>,</sup><sup>27</sup><sup>,</sup><sup>28</sup><sup>,</sup><sup>29</sup><sup>,</sup><sup>30</sup><sup>,</sup><sup>31</sup><sup>,</sup><sup>32</sup><sup>,</sup><sup>33</sup><sup>,</sup><sup>34</sup><sup>,</sup><sup>35</sup>) or rhabdoviruses (vesicular stomatitis virus — VSV<sup>36</sup><sup>,</sup><sup>39</sup><sup>,</sup><sup>40</sup><sup>,</sup><sup>41</sup><sup>,</sup><sup>42</sup><sup>,</sup><sup>43</sup><sup>,</sup><sup>44</sup><sup>,</sup><sup>45</sup><sup>,</sup><sup>46</sup><sup>,</sup><sup>47</sup>). When used in pseudotyping, the parental viruses' genomes are modified to remove essential genes, rendering them defective for accomplishing a complete replication cycle. This feature allows them to be used in intermediate biosafety level facilities (BSL-2) and is an important advantage over using highly pathogenic native viruses that require higher biosafety facilities (BSL-3, BSL-4 which are not as readily available) when conducting virus entry studies. Here, the S proteins of risk group 3 pathogens SARS-CoV and MERS-CoV are used as examples of viral envelope proteins being incorporated into MLV pseudotyped particles, generating SARS-CoV S and MERS-CoV S pseudovirions (SARS-Spp and MERS-Spp, respectively). These pseudovirions have been successfully used in studies focusing on entry events of these viruses<sup>48</sup><sup>,</sup><sup>49</sup><sup>,</sup><sup>50</sup><sup>,</sup><sup>51</sup>. Another advantage is that the technique described here is not limited to pseudotyping coronavirus S proteins: it is very flexible and can be used to incorporate representatives of all three classes of viral fusion proteins. Examples include influenza hemagglutinin (HA, class I)<sup>52</sup>, Ebola virus glycoprotein (GP class I), E1 protein of the alphavirus Semliki Forest virus (SFV, class II), and VSV glycoprotein (G, class III)<sup>53</sup>. In addition, more than one kind of viral glycoprotein can be co-incorporated into a pseudotyped particle, as in the case of influenza HA- and NA- pseudotyped particles<sup>51</sup>.

Based on the work performed by Bartosch et al.<sup>20</sup>, this protocol describes the generation of MLV pseudotyped particles with a three-plasmid co-transfection strategy using the widely available and highly transfection-competent HEK-293T cell line<sup>54</sup>. The first plasmid encodes the MLV core genes ''gag'' and ''pol'' but lacks the MLV envelope ''env'' gene. The second plasmid is a transfer vector that encodes a firefly luciferase reporter gene, an MLV Ψ-RNA packaging signal, along with 5'- and 3'-flanking MLV long terminal repeat (LTR) regions. The third plasmid encodes the fusion protein of interest, in this case either the SARS-CoV S or MERS-CoV S protein. Upon co-transfection of the three plasmids using a transfection reagent, viral RNA and proteins get expressed within transfected cells allowing generation of pseudotyped particles. Since MLV is used as pseudovirion backbone, this occurs at the plasma membrane: the RNAs containing the luciferase gene reporter and packaging signal get encapsulated into nascent particles that also incorporate plasma membrane-expressed coronavirus spike proteins. The particles that bud out from cells contain the coronavirus S protein at their surface and are harvested for use in infectivity assays. Because pseudotyped particles harbor the coronavirus S protein and not the MLV envelope protein, when used for infecting cells, they behave like their native coronavirus counterparts for entry steps. The viral RNA containing the luciferase reporter and flanking LTRs is then released within the cell and the retroviral polymerase activities enable its reverse transcription into DNA and integration into the host cell genome. Quantification of the infectivity of viral pseudotyped particles in infected cells is then performed with a simple luciferase activity assay. Because the DNA sequence that gets integrated into the host cell genome only contains the luciferase gene and none of the MLV or coronavirus protein-encoding genes, they are inherently safer to use than replication-competent native viruses.

In addition to being safer surrogates and highly adaptable to allow incorporation of various kinds of envelope glycoproteins, the pseudotyped particles described here are also highly versatile and can be used to study many aspects of virus entry. This includes but is not limited to: testing host cell susceptibility to virus infection, analyzing the cellular entry pathways an enveloped virus uses, studying the effects of pharmacological inhibitors and drug screenings, conducting neutralization antibody assays, characterizing host cell entry of enveloped viruses that cannot be cultured, and generating viral vectors for gene delivery, stable cellular expression of genes of interest, or gene silencing.
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