The Cyclin-Dependent Kinase 5 Inhibitor Peptide Inhibits Herpes Simplex Virus Type 1 Replication

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Author(s)
Man, Adrian
Slevin, Mark
Petcu, Eugen
Fraefel, Cornel
Griffith University Author(s)
Year published
2019
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In order to evaluate the influence of CDK5 inhibitory peptide (CIP) on Human alphaherpesvirus 1 (HSV-1) replication, we constructed two recombinant adeno-associated-virus 2 (rAAV2) vectors encoding CIP fused with cyan-fluorescent-protein (CFP), with or without nuclear localization signal. A third vector encoding non-fused CIP and CFP was also constructed. HeLa and HEK 293T cells were infected with the rAAV-CIP vectors at multiplicity of infection (MOI) of 5000, in the absence or presence of a recombinant HSV-1 that encodes a yellow-fluorescent-protein (rHSV48Y; MOI = 1). Cells co-infected with rHSV48Y and rAAV vectors that ...
View more >In order to evaluate the influence of CDK5 inhibitory peptide (CIP) on Human alphaherpesvirus 1 (HSV-1) replication, we constructed two recombinant adeno-associated-virus 2 (rAAV2) vectors encoding CIP fused with cyan-fluorescent-protein (CFP), with or without nuclear localization signal. A third vector encoding non-fused CIP and CFP was also constructed. HeLa and HEK 293T cells were infected with the rAAV-CIP vectors at multiplicity of infection (MOI) of 5000, in the absence or presence of a recombinant HSV-1 that encodes a yellow-fluorescent-protein (rHSV48Y; MOI = 1). Cells co-infected with rHSV48Y and rAAV vectors that did not express the CIP gene (rAAV-CFP-Neo) served as controls. At 24 h after infection, the effect of CIP on rHSV48Y replication was assessed by PCR, qRT-PCR, Western-blot, flow-cytometry, epifluorescence and confocal microscopy. We show that in cultures co-infected with rAAV-CFP-Neo, 27% of the CFP-positive cells present rHSV48Y replication compartments. By contrast, in cultures co-infected with CIP-encoding rAAV2 vectors and rHSV48Y only 6–20% of the cells positive for CIP showed rHSV48Y replication compartments, depending on the CIP variant. Flow-cytometry showed that less than 40% of the rHSV48Y/rAAV-CIP, and more than 75% of rHSV48Y/rAAV-CFP-Neo co-infected cells were positive for both transgene products. The microscopy and flow-cytometry data support the hypothesis that CIP is inhibiting HSV-1 replication.
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View more >In order to evaluate the influence of CDK5 inhibitory peptide (CIP) on Human alphaherpesvirus 1 (HSV-1) replication, we constructed two recombinant adeno-associated-virus 2 (rAAV2) vectors encoding CIP fused with cyan-fluorescent-protein (CFP), with or without nuclear localization signal. A third vector encoding non-fused CIP and CFP was also constructed. HeLa and HEK 293T cells were infected with the rAAV-CIP vectors at multiplicity of infection (MOI) of 5000, in the absence or presence of a recombinant HSV-1 that encodes a yellow-fluorescent-protein (rHSV48Y; MOI = 1). Cells co-infected with rHSV48Y and rAAV vectors that did not express the CIP gene (rAAV-CFP-Neo) served as controls. At 24 h after infection, the effect of CIP on rHSV48Y replication was assessed by PCR, qRT-PCR, Western-blot, flow-cytometry, epifluorescence and confocal microscopy. We show that in cultures co-infected with rAAV-CFP-Neo, 27% of the CFP-positive cells present rHSV48Y replication compartments. By contrast, in cultures co-infected with CIP-encoding rAAV2 vectors and rHSV48Y only 6–20% of the cells positive for CIP showed rHSV48Y replication compartments, depending on the CIP variant. Flow-cytometry showed that less than 40% of the rHSV48Y/rAAV-CIP, and more than 75% of rHSV48Y/rAAV-CFP-Neo co-infected cells were positive for both transgene products. The microscopy and flow-cytometry data support the hypothesis that CIP is inhibiting HSV-1 replication.
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Journal Title
Scientific Reports
Volume
9
Issue
1
Copyright Statement
© The Author(s) 2019. This article is licensed under a Creative Commons Attribution 4.0 International
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Subject
Medical and Health Sciences