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. 2018 Oct 11;9:2406. doi: 10.3389/fmicb.2018.02406

Table 1.

Experimental approaches for evaluating key steps in lytic HSV replication cycles.

Step or process Commonly used methodology (outline) Limitations and advantages Critical considerations References
Binding Western blot targeting structural viral proteins on samples obtained from cells incubated with HSV at different MOI at low temperature. This assay allows determining the amount of surface-bound virus. Samples need to be incubated and processed under conditions that reduce the chances of virus internalization (low temperature). Cells need to be washed properly before protein extraction to remove excess unbound virus. Another experimental approach consists on the use of GFP-tagged virus.
It can be done proceeding with infection at 4°C, washing and then performing confocal microscopy or flow cytometry.		 Western blot is a routine technique in most laboratories. However, more qualitative than quantitative.

  • - Usually uses variable MOI to titer cell-surface bound virus (e.g., MOI 10, MOI 1, MOI 0.1).

  • - Temperature: 4°C.

  • - Incubation time: >1 h

  • - Wash to remove unbound virus.

 Atanasiu et al., 2010; Cheshenko et al., 2013; Ibáñez et al., 2017
Capsid entry into the cytoplasm The number of internalized capsids is assessed using a recombinant virus that has a structural protein fused to a reporter (e.g., GFP). Infection is performed at low temperature. Cells are then washed and transferred at 37°C for 1–2 h. Finally, cells are trypsinized to remove any surface-bound virus and fixed prior to analysis. Virus-derived fluorescence can be measured by flow cytometry or by laser confocal microscopy. Requires sophisticated equipment (flow cytometer or confocal microscope) and trained staff.

  • - High MOI (e.g., 100).

  • - Structural reporter virus (e.g., HSV K26GFP) fluorescently labeled.

  • - Temperature 4°C, then 37°C.

  • - Wash, trypsinize cells to remove unbound and surface-bound virus.

  • - Time: analyze at 1–2 hpi.

 Desai and Person, 1998; Nicola and Straus, 2004; Wang et al., 2016; Ibáñez et al., 2017
Capsid transport to the nucleus Cells are infected with a fluorescently-labeled virus (structural, e.g., K26GFP virus) at 37°C and viral capsids followed in live or fixed cells by laser confocal microscopy at 1–3 h post-infection. Membrane and nucleus stain inform about the relative distribution of the capsids within the cells. Alternatively, an approach using qPCR could be used (see text). Requires access to confocal microscopy or TEM with trained staff. Cell fractioning (nucleus and cytoplasm) easy to perform. Western blot is a routine technique in most laboratories.

  • - High MOI 100–400.

  • - Structural reporter virus (e.g., HSV K26GFP) fluorescently labeled.

  • - Time: analyze at 1–3 hpi.

  • - Stain cell membrane and nucleus for relative capsid position.

 Desai and Person, 1998; Dohner et al., 2002; Petro et al., 2015
Viral gene transcription Cells are infected at a MOI that infects 100% of cells. Extract RNA and analyze by RT-qPCR viral transcripts. Optimal time-point for analysis depends on the viral gene analyzed (α, β and γ genes) and may depend on the cell type infected. Must choose correct timing. qPCR is nowadays a routine technique.

  • - MOI for 100% cell infection (>3).

  • - Time: analyze gene expression at:

    1. 2–4 h for α genes

    2. 6–12 h for β genes

    3. 10–16 h for γ genes

 Honess and Roizman, 1975; Roizman et al., 2013; Du et al., 2015
Viral gene translation Western blot or flow cytometry analyses are performed on the infected cells to determine the expression of viral proteins. Optimal time-point for analysis depends on the viral gene analyzed (α, β, and γ genes) and may depend on the cell type infected. Western blot is a routine technique in most laboratories. Flow cytometry requires equipment and trained staff.

  • - MOI for 100% cell infection (>3).

  • - Time: analyze gene expression at:

    1. 2–4 h for α genes.

    2. 6–12 h for β genes.

    3. 10–16 h for γ genes.

 Loret et al., 2008; Pasieka et al., 2008; Conway and Homa, 2011; Ma et al., 2017
Viral genome replication Genome replication can be assessed by qPCR on total DNA extracted from infected cells at 8–24 h post-infection. Must choose correct timing. qPCR considered a routine technique.

  • - MOI for 100% cell infection (>3).

  • - Time: analyze 18–24 hpi.

 Nystrom et al., 2004; Ibáñez et al., 2017
Capsid assembly Transmission electron microscopy allows determining the presence and phenotypes of viral capsids (A and B without viral DNA and C with virus genome). Cells are infected and visualized at 6–8 h post-infection. Sucrose density gradients generated by ultracentrifugation can be performed when it is desired to detect capsid proteins by Western blot or assess the amount of viral DNA encapsidated by qPCR. Sucrose density gradients requires ultracentrifuge. Western blot is a routine technique in most laboratories. qPCR considered a routine technique.

  • - MOI for 100% cell infection (>3).

  • - Time: Analyze at 6–8 h.p.i. Multiple time-points are recommended.

 Gibson and Roizman, 1972; Preston et al., 1983; Gao et al., 1994; McNab et al., 1998; Spencer et al., 1998; Dasgupta and Wilson, 1999; Taddeo et al., 2003; Turcotte et al., 2005; Sugimoto et al., 2008; Loret et al., 2012
Viral protein glycosylation Western blot in viral proteins that undergo post-translational modifications. Gel-migration profiles are analyzed and can be compared with untreated cells. Western blot is a routine technique in most laboratories.

  • - Viral glycoproteins (e.g., gB, gC, gD).

  • - Time: 12 hpi.

 Komuro et al., 1989; Pagano et al., 1989; Ladinsky and Howell, 1992; Futter et al., 1996; Turcotte et al., 2005; Calistri et al., 2007; Henaff et al., 2012
Glycoprotein export to the cell surface Co-localization between TGN, MVB markers (with fluorescently-labeled antibodies) and viral glycoproteins can be determined by confocal laser microscopy 12 h. Requires access to confocal microscopy with trained staff.

  • - Reported MOI 5.

  • - Time: 12 hpi.

  • - Organelle markers: TGN, MVB.

 Avitabile et al., 2004; Calistri et al., 2007
Endocytosis of glycoproteins Endocyted viral glycoproteins can be determined by electron microscopy by marking the cell surface with HRP and then localizing viral proteins with labeled antibodies. Cells are infected, labeled 12 h with HRP and then fixed. Antibody staining of viral glycoproteins can contribute determining the localization of these proteins. Requires access to TEM with trained staff.

  • - Reported MOI 2.

  • - Mark the cell Surface with HRP.

  • - Antibodies against viral glycoproteins.

 Foster et al., 2004; Hollinshead et al., 2012; Albecka et al., 2016
Capsid envelopment Capsid envelopment can be assessed by confocal laser microscopy or transmission electron microscopy, using antibodies against host and virus proteins to determine the localization and assess whether they are located within early endosomes in the cytoplasm. Requires access to confocal laser microscopy or transmission electron microscopy with trained staff.

  • - Reported MOI 2.

  • - Time: analyzed at 12–24 hpi.

  • - Organelle/Cell. compartment markers.

    Antibodies against viral glycoproteins.

 Albecka et al., 2016
Virion release Infective virus release can be assessed by plaque assays by performing serial dilutions of recovered supernatants, as well as lysed-cell preparations (to recover virus within the cells and unable to undergo exit) over HSV-susceptible cells. Sample collection can be done within 18–36 h post-infection. Routine technique in most laboratories.

  • - Cell lysis strategy (must not damage the viral particles).

  • - Time: analyze within 18–36 hpi.

 Arens et al., 1988; Sugimoto et al., 2008; Fabiani et al., 2017
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