Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2019 The cyclin-dependent kinase 5 inhibitor peptide Inhibits herpes simplex virus type 1 replication Man, Adrian ; Slevin, Mark ; Petcu, Eugen ; Fraefel, Cornel Abstract: In order to evaluate the influence of CDK5 inhibitory peptide (CIP) on Human alphaher- pesvirus 1 (HSV-1) replication, we constructed two recombinant adeno-associated-virus 2 (rAAV2) vec- tors 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. DOI: https://doi.org/10.1038/s41598-018-37989-3 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-167395 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at: Man, Adrian; Slevin, Mark; Petcu, Eugen; Fraefel, Cornel (2019). The cyclin-dependent kinase 5 inhibitor peptide Inhibits herpes simplex virus type 1 replication. Scientific Reports, 9(1):1260. DOI: https://doi.org/10.1038/s41598-018-37989-3 www.nature.com/scientificreports OPEN The Cyclin-Dependent Kinase 5 Inhibitor Peptide Inhibits Herpes Simplex Virus Type 1 Replication Received: 16 May 2018 Adrian Man 1,2, Mark Slevin3,4, Eugen Petcu5 & Cornel Fraefel1 Accepted: 18 October 2018 In order to evaluate the infuence of CDK5 inhibitory peptide (CIP) on Human alphaherpesvirus 1 (HSV-1) Published: xx xx xxxx replication, we constructed two recombinant adeno-associated-virus 2 (rAAV2) vectors encoding CIP fused with cyan-fuorescent-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-fuorescent-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 efect of CIP on rHSV48Y replication was assessed by PCR, qRT-PCR, Western-blot, fow-cytometry, epifuorescence 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 fow-cytometry data support the hypothesis that CIP is inhibiting HSV-1 replication. Human alphaherpesvirus 1, also known as herpes simplex virus type 1 (HSV-1), is a species in the genus Simplexvirus, family Herpesviridae, order Herpesvirales. It is one of the most common human pathogens, largely spread due to its oral to oral dissemination, with an estimated prevalence of 67%, an incidence of 118 million and a prevalence of 3.7 billion in 20121. Te herpetic lesions are located in mostly in perioral area, but ocular, skin mucous membrane lesions are also frequent. Severe complications such as recurrent infections, aseptic men- ingitis, keratitis, encephalitis, neonatal herpes, visceral involvement, including HSV-1 pneumonia can occur, especially in immune-compromised patients. Common therapeutic options include acyclic nucleoside analogues (Acyclovir, Famcyclovir) that act as viral DNA polymerase inhibitors. Acyclovir resistant strains are described since 1980s2 and is still a problem3, so alternative treatment is required in these cases, but with higher costs and toxicity risks4. Prevention against HSV-1 infection is not yet possible in humans, though experimental vaccines are developed and tested on animals5. New therapeutic and prophylactic measures against HSV-1 have to be developed, such as gene-therapy using viral vectors. Recombinant Adeno-associated virus 2 (rAAV2) vectors, in contrast with the wild-type Adeno-associated viruses, were found to be safe and efective in preclinical and clini- cal settings, as they cannot replicate, do not contain any virulence genes and do not integrate into host genomes. Instead, the encoded transgenes can form circular concatemers that persist as episomes in the nucleus of the infected cells. In addition, rAAV2 vectors present a broad tissue tropism, which makes them usable in multiple pathologies. Tese vectors can be easily engineered to include a DNA sequence of interest of up to 5 kb6. CDK5 and Its Activators and Suppressors CDK5 is a 32 kDa protein composed of 292 amino-acids with ubiquitous expression, but with proline-directed serine/threonine kinase activity mainly in post-mitotic neurons. CDK5 is present also in other cell types, but its activity in neurons is directly linked with the high level of p35 and p397. It is an atypical kinase, as compared to CDK1-4 and -6 that regulate cell cycle progression, CDK5 acts as a regulatory kinase in several post-mitotic 1Institute of Virology, University of Zurich, Zurich, Switzerland. 2Department of Microbiology, University of Medicine and Pharmacy of Tîrgu Mureș, Târgu Mureș, Romania. 3University of Medicine and Pharmacy of Tîrgu Mureș, Târgu Mureș, Romania. 4School of Healthcare Science, Manchester Metropolitan University, Manchester, UK. 5Grifth University, Gold Coast, Brisbane, Australia. Correspondence and requests for materials should be addressed to M.S. (email: [email protected]) SCIENTIFIC REPORTS | (2019) 9:1260 | https://doi.org/10.1038/s41598-018-37989-3 1 www.nature.com/scientificreports/ Figure 1. Splice products of p35. P25 is a CDK5 hyperactivator, while p21, p16, CIP and p5 are CDK5 inhibitors. processes such as neuronal activity, neuronal migration during development and neurite outgrowth8. Compared to the other cyclin-dependent kinases, CDK5 does not need to be phosphorylated in order to express its kinase activity9. Tough CDK5 is present in higher amount in the cytoplasm, its kinase activity is higher in the nucleus, probably due to the higher amounts of nuclear p35. One of the main activities is the Retinoblastoma (Rb) phos- phorylation at several C-terminus sites (780, 788, 795, 807, 811, 821, 826) as proved by mass spectrometry, behav- ing similarly to Cdk2/Cyclin E (phospho-specifc antibodies of Rb are limited to Ser780, Ser795 and Ser807/811). Dephosphorylated Rb protein binds to E2F1 and turns it “OFF,” while phosphorylated Rb protein dissociates from E2F1 and turns it “ON.” In normal condition, CDK5 does not afect the activity of E2F1 even though it phospho- rylates Rb; more than this, CDK5 suppresses the neuronal cell cycle by disrupting the E2F1-DP1 complex and arrest the cell cycle in G1 state. Instead, the overexpression of CDK5 increases the Rb phosphorylation, followed by the initiation of E2F1 activity, stimulating the neuronal cell cycle, leading to neuron apoptosis10,11. CDK5 is activated by p35 (CDK5R1) and p39 (CDK5R2 – a 367 aa isoform of p35), hyperactivated by p25, and inhibited by Cyclin-dependent kinase inhibitor 1 (p21), CIP, p5 or several chemical compounds (roscovitine, resveratrol, AT-7519 and olomoucine)12. Te role of CDK5 in other tissues than neurons remains poorly described. It was shown that CDK5 may have a role in tumorogenesis, or that the inhibition of CDK5 may have antitumoral activity by altering the expression of several cell-cycle related proteins13,14. p35 (CDK5R1) is a 35 kDa protein expressed mostly in CNS, in post-mitotic neurons, with regulatory function (activates CDK5) and present high specifcity for CDK5, but not for other kinases as CDK2, CDK3, CDK4, DCK6 or CDC215,16. It has a short half-life of 20–30 minutes. p35 is produced afer the cleavage of the initiator methio- nine from Cyclin-dependent kinase 5 activator 1, which is encoded by CDK5R1 on 17q11.217,18. It is non-soluble, being associated with membrane/cytoskeleton. Te phosphorylation of p35 by CDK5 is a signal for ubiquiti- nation/degradation. p35 is found mostly in phosphorylated state in fetal brain, while the non-phosphorylated state is found mostly in adult brain7. In addition, P21/WAF1 is an inhibitor of cyclin-dependant kinases 2,3,4,6, with a lower activity on CDK519. Its binding to cyclin/CDK complexes leads to the inhibition of Rb protein phosphorylation. CDK5 inhibitory peptide (CIP) is a 125 aa (154–279), 12.5 KDa peptide that is generated afer the C- and N-terminal truncation of p35 (Supplemental Fig. 1), with a high afnity for CDK5 and a longer half-life20.