Cell Type Differences in HSV-1 Infection and HSV-1's Dependency on ICP27

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Introduction

Herpes simplex virus type 1 may affect up to 90% of the adult population and is known to infect 64% of the human population globally.1 Viral research into the infection, efficiency of infection, and outcome of infection of herpes simplex virus type-1 has allowed for treatments to be derived for extreme outbreaks which specialize in managing the infection and preventing further spread of the virus. Yet, these viral studies utilized different permissive cell types which best benefit the needs of their study, whether alleviating cost or cell type availability or using cells which are best for the specific aspect of HSV-1 they intend to study. Numerous cell types are used in HSV-1 studies, including African green monkey kidney epithelial cells (Vero)2, human lung fibroblasts (MRC-5)3, human foreskin fibroblasts (HFF)4, human diploid keratinocytes (N/TERT-2G)5, and human cervical cancer epithelial cells (HeLa)6. This lack of standardization can affect the outcomes of these viral studies. Here, Sabrina L. Rutan and Jill A. Dembowski of the Dembowski Lab at Duquesne University explored how different cell types can vary the infection, efficiency of infection, and outcome of infection in the aforementioned cells in their paper titled Cell type-specific differences in herpes simplex virus type 1 infection and dependency on ICP27, published in the Journal of Virology by the American Society for Microbiology. Furthermore, Sabrina and Jill aimed to investigate how reliant different cell types are on the infected cell protein (ICP27), a cell-type specific essential viral immediate early gene product which promotes efficient processing and transport of viral mRNAs. ICP27 helps to regulate viral gene expression through mRNA export and 3’ end processing.7 

Initial Findings

Initially, the kinetics of viral DNA replication between cell types was explored by infection of Vero, MRC-5, N/TERT-2G, HFF, and HeLa cells with HSV-1 strain KOS at a multiplicity of infection (MOI) of 10 plaque-forming units (PFU)/cell. Infected cells were harvested before the qPCR was analyzed, allowing for calculation of the number of viral genomes per cell. Through this process, HeLa cells were noted as having the number of infecting viral genomes decrease shortly after infection, with a delay in viral DNA replication. Replication kinetics for Vero, MRC-5, and HFF cells were similar, whereas N/TERT-2G cells were noted for having viral replication begin earlier and at a faster rate. All cell types had a similar maximum viral capacity. (Figure 1: seen below). Furthermore, HeLa cells were found to produce less infectious particles, despite producing similar quantities of viral genomes per cell as 24 hours past infection. On the other hand, HFF cells produced a notable larger quantity of infectious particles at 24 hpi, and Vero, MRC-5, N/TERT-2G, and HFF cells produces 1,000 PFU/cell by 96 hpi, whereas HeLa cells only produced 4 PFU/cell. 

Fig 1: Characterization of HSV-1 DNA replication and infectious virus output in several cell types. A) Comparative chart describing the quantities of cellular and viral genomes calculated using qPCR for each cell type. B) Comparison of viral yield between cell types after high MOI infection (10 PFU/cell). C) Comparison of viral yield after low MOI infection (0.01 PFU/cell). D) Coomparison of levels of deffective virus particles between cell types after high MOI infection after 24 hours. All values were obtain via a one-way ANOVA with a Tukey’s multiple comparisons test. 

HeLa cells produced more defective viral particles

    These findings were further explored, as HeLa cells were noted for producing more defective viral particles than all other cell types. By subjecting an aliquot of supernatant media from infected cells to proteinase K digestion, followed by DNA isolation and viral genome quantification through qPCR, the number of defective particles per PFU could be measured by cell type. Here, Sabrina and Jill found that Vero cells produced, on average, 60 defective particles per PFU (dp/PFU), HFF cells produced 27 dp/PFU, MRC-5 and N/TERT-G2 cells produced 82 dp/PFU, and HeLa cells produced an astonishing 170 dp/PFU. (Figure 2)

Fig 2) Comparison of HSV-1 KOS protein expression between cell types after infection at an MOI of 10 PFU/cell. Total protein was collected 2, 4, 6, and 8 hpi.

Viral Protein Expression Timing

Next, the researchers observed the timing of viral protein expression through observation of whole cell lysates at 2, 4, 6, and 8 hpi via western blotting. This method allowed for the observation of immediate early (IE), early (E), leaky late (LL), and late (L) viral proteins. ICP27 and ICP8 were found to have highly variable timing of expression between cell types. LL viral proteins were similar among all cell types except for HeLa cells, which saw a delay in LL protein expression. Lastly, L proteins were observed at 6 hpi for HFF and N/TERT-G2 cells and consistently at 8 hpi for all cell types. From these findings, the researchers concluded that despite relative temporal expression of viral protein consistency across cell types, the LL and L gene products expressed after the onset of viral DNA replication were unique enough that they may contribute to differences in observed viral DNA replication and viral particle production. 
Viral gene expression was then investigated through the quantification of expression of representative viral transcripts for each gene class. Total RNA was isolated at 2, 4, 6, and 8 hpi and after DNAse treatment, reverse transcription, and qPCR, mRNA copies of each gene per ug total RNA were documented. Viral mRNA levels were observed to be the highest in N/TERT-2G cells compared to other cell types at early times after infection. By 8 hpi, mRNA levels were similar for HFF, MRC-5, and N/TERT-2G cells for all HSV-1 gene classes. For N/TERT-2G cells, IE and E genes were observed until 4 hpi, whereas in HFF, MRC-5, and HeLa cells IE and E genes plateaued at 4 hpi. Also, in N/TERT-2G cells, LL gene expression peaked at 4 hpi and decreased onwards, whereas in all other cell types, LL genes steadily increased throughout the experiment. Consistently, HeLa cells displayed fewer E, LL, and L viral transcripts than the other cell types. (Figure 3) 

Fig 3) Viral gene expression kinetics varying between cell types during strain KOS infection. A) Immediate Early (IE) genes. B) Early (E) genes. C) Leaky Late (LL) genes. D) Late (L) genes. E) 18S rRNA and GAPDH controls for comparison.

Differences in the absence of ICP27

With differences in gene and protein expression noted for each cell type and different times past infection, the researchers then moved to explore the cell type-specific differences in infection in the absence of ICP27, focusing first on viral DNA replication. When ICP27 is absent, viral replication compartments still form in MRC-5 cells, where in Vero cells incoming viral genomes form small punctate structures which never grow to full compartments, indicating that without ICP27, viral replication cannot occur in Vero cells, which was proven through Edc-labeled DNA. Replication was also found to vary dependent on cell type, showing a cell type dependence on ICP27 for DNA replication. Viral gene and protein expression were roughly consistent across cell types during infection with ICP27 absent. 

Fig 5) Viral Gene expression kinetics during 5dl1.2 (ICP27 absent) infection. mRNA levels obtained at 2-8hpi for A) IE, B) E, C) LL, D), L genes with E) 18S rRNA as a control. All mRNA levels were quantified through qPCR and differences were noted through unpaired two-tailed Student’s t-test.

Conclusion

    Overall, Sabrina and Jill found that HeLa cells had a lower number of infecting viral genomes and that genome amplification was delayed. Although viral genomes produced across cell types were similar at 24 hpi, HeLa cells produced the least infectious particles, whereas N/TERT-2G cells produced the most infectious particles per PFU. N/TERT-2G cells were found to have viral DNA replication and gene expression begin earlier, but plateau earlier as well. These findings indicate that there are distinct differences between cell types for the kinetics of viral DNA replication. In addition, cell cycle-specific differences were observed for the infectious cycle of HSV-1 mutants who lacked ICP27, where HFF cells see nearly normal DNA replication, MRC-5 and N/TERT-2G see reduced DNA replication, and HeLa and Vero cells see no DNA replication. Their results highlight the importance of consistency and standardization in viral studies, as selection of cell type should be considerate of differences in viral infection kinetics. Despite these findings, no cell type is noted as the particular best for investigation of HSV-1 infection study. Instead, the authors go out of their way to highlight that there are advantages and disadvantages to each cell type, and that these must be considered when selecting cell type and comparing studies. Thus, viral infection kinetics vary depending on cell type, and viral DNA replication is cell type-dependent during HSV-1 infection in the absence of ICP27.

References:
1) Organization WH. 2023. Implementing the global health sector strategies on HIV, viral hepatitis and sexually transmitted infections, 2022–2030: report on progress and gaps 2024, Vol. 2, p 1–70.

2) Huleihel M, Shufan E, Zeiri L, Salman A. Detection of Vero Cells Infected with Herpes Simplex Types 1 and 2 and Varicella Zoster Viruses Using Raman Spectroscopy and Advanced Statistical Methods. PLoS One. 2016 Apr 14;11(4):e0153599. doi: 10.1371/journal.pone.0153599. PMID: 27078266; PMCID: PMC4831712.

3) Gleaves CA, Wilson DJ, Wold AD, Smith TF. Detection and serotyping of herpes simplex virus in MRC-5 cells by use of centrifugation and monoclonal antibodies 16 h postinoculation. J Clin Microbiol. 1985 Jan;21(1):29-32. doi: 10.1128/jcm.21.1.29-32.1985. PMID: 2981901; PMCID: PMC271574.

4) Pan X, Xie J, Zhang Z, Guo X, Li J, Lin D, Qian Y, Xu J, Hu Y, Shi J. Serotype-specific host proteome remodeling in human foreskin fibroblasts during lytic HSV-1 and HSV-2 infection. Virol J. 2025 Jul 14;22(1):239. doi: 10.1186/s12985-025-02803-w. PMID: 40660299; PMCID: PMC12257817.

5) Kite J, Russell T, Jones J, Elliott G. 2021. Cell-to-cell transmission of HSV1 in human keratinocytes in the absence of the major entry receptor, nectin1. PLoS Pathog 17:e1009631.

6)Wang, X., Diao, C., Yang, X. et al. ICP4-induced miR-101 attenuates HSV-1 replication. Sci Rep 6, 23205 (2016). https://doi.org/10.1038/srep23205

7) Smith RWP, Malik P, Clements JB. 2005. The herpes simplex virus ICP27 protein: a multifunctional post-transcriptional regulator of gene expression. Biochem Soc Trans 33:499–501.