Examination of herpes simplex virus protease mutant capsids to define the role of the protease in capsid assembly and maturation

Herpes simplex virus type 1 (HSV-1) capsids are assembled with an internal protein scaffold. Scaffold proteins, encoded by overlapping in-frame UL26 and UL26.5 transcripts, are essential for formation and efficient maturation of capsids. UL26 encodes an N-terminal protease domain and shares identical C-terminal oligomerization and capsid protein-binding domains with UL26.5. The UL26 protease cleaves itself (releasing VP24 and VP21) and the more abundant UL26.5 protein (releasing VP22a). Unlike VP21 and VP22a, which are removed from capsids upon DNA packaging, we demonstrate that VP24 is quantitatively retained. To investigate factors controlling UL26 capsid incorporation and retention we used a virus that fails to express UL26.5 (ΔICP35 virus). Purified ΔICP35 B-capsids showed altered sucrose gradient sedimentation and lacked the dense scaffold core seen in micrographs of wild-type B-capsids. ΔICP35 capsids did not contain compensatory high levels of UL26 proteins. In contrast to UL26.5, increased expression of UL26 did not complement Δ ICP35 virus growth. These findings suggest that despite identical C-termini, UL26 products are restricted from occupying UL26.5 binding sites within the capsid. This restriction is not controlled by expression levels. Furthermore, capsids can be maintained and/or assembled on a minimal scaffold containing only wild-type levels of UL26 proteins. ^ Internal scaffold release appears tightly linked to viral DNA packaging since no mutations have separated these events. However, the mechanisms underlying this linkage are unclear. Stable intracellular capsids are known to contain several proteins required for cleavage and packaging of viral DNA. To test whether scaffold cleavage and accompanying changes in the capsid shell affected association of packaging proteins, we isolated unstable precursor procapsids from protease mutant virus infected cells. Procapsids contained unprocessed scaffold and lacked one capsid shell protein, VP26. The complement of packaging proteins associated with procapsids resembled those of B-capsids containing processed scaffold. Notably, however, amounts of the UL25 protein, essential for stable packaging of DNA, were reduced relative to B-capsids. In contrast, the ΔICP35 capsids contained increased levels of UL25. DNA-containing C-capsids lacking scaffold had the most UL25. We suggest a model whereby the UL25 protein associates with and seals capsids in response to cleavage and loss of internal scaffold proteins.

Physical and Functional Interactions between the Herpes Simplex Virus UL15 and UL28 DNA Cleavage and Packaging Proteins

Herpes simplex virus (HSV) DNA is cleaved from concatemers and packaged into capsids in infected cell nuclei. This process requires seven viral proteins, including UL15 and UL28. UL15 expressed alone displays a nuclear localization, while UL28 remains cytoplasmic. Coexpression with UL15 enables UL28 to enter nuclei, suggesting an interaction between the two proteins. Additionally, UL28 copurified with UL15 from HSV-infected cells after ion-exchange and DNA affinity chromatography, and the complex sedimented as a 1:1 heterodimer upon sucrose gradient centrifugation. These findings are evidence of a physical interaction of UL15 and UL28 and a functional role for UL15 in directing UL28 to the nucleus

A Small Molecule Inhibitor Selectively Induces Apoptosis in Cells Transformed by High Risk Human Papilloma Viruses

<div><p>A phenotypic high-throughput cell culture screen was performed to identify compounds that prevented proliferation of the human Papilloma virus type 16 (HPV-16) transformed cell line Ca Ski. A series of quinoxaline compounds exemplified by Compound 1 was identified. Testing against a panel of cell lines demonstrated that Compound 1 selectively inhibited replication of all HPV-16, HPV-18, and HPV-31 transformed cell lines tested with 50% Inhibitory Concentration (IC₅₀) values of 2 to 8 μM relative to IC₅₀ values of 28 to 73 μM in HPV-negative cell lines. Treatment with Compound 1 resulted in a cascade of multiple apoptotic events, including selective activation of effector caspases 3 and 7, fragmentation of cellular DNA, and PARP (poly(ADP-ribose) polymerase) cleavage in HPV-positive cells relative to HPV-negative cells. Unregulated proliferation of HPV transformed cells is dependent on the viral oncogenes, E6 and E7. Treatment with Compound 1 resulted in a decrease in HPV E7 protein in Ca Ski cells. However, the timing of this reduction relative to other effects of compound treatment suggests that this was a consequence, rather than a cause, of the apoptotic cascade. Likewise, compound treatment resulted in no obvious effects on the E6- and E7- mediated down regulation of p53 and Rb, or their downstream effectors, p21 or PCNA. Further investigation of apoptotic signals induced by Compound 1 revealed cleavage of Caspase-8 in HPV-positive cells as early as 2 hours post-treatment, suggesting the compound initiates apoptosis through the extrinsic, death receptor-mediated, pathway of cell death. These studies provide proof of concept that cells transformed by oncogenic Papillomaviruses can be selectively induced to undergo apoptosis by compound treatment.</p></div

Effect of Compound 1 on markers of apoptosis in treated cells.

<p>(A) HPV-positive and HPV-negative cell lines were treated with increasing concentrations of Compound 1 for 6 hours, and then tested for Caspase-3/7 activity. (B) Ca Ski cells were treated with the indicated concentrations of Compound 1 and tested for Caspase-3/7 activity at various times. The graph shows mean values of percent Caspase-3/7 activity relative to DMSO-treated controls. Each point represents the mean of triplicates, with error bars indicating standard deviation. (C) Ca Ski cells were treated with DMSO or the indicated concentrations of Compound 1 for 6 or 24 hours, and then stained with Annexin-V and 7-AAD prior to flow cytometry. (D) HPV-positive Ca Ski or HPV-negative C33a cells treated for 24 hours with DMSO, 18 μM Compound 1, or the control inhibitors 5 μM nocodazole or 1 μM camptothecin. Cells were fixed and stained with either the DNA stain Hoechst or by TUNEL, as indicated.</p