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Definition
Human Herpes Virus 8 (HHV-8) or Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) has been linked to Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. A genomic clone encoding the protease has been characterized and its substrate is found to be essential for antiviral therapy.

Discovery
In 1997, Uenal et al., isolated and sequenced a genomic clone encoding the Pr and the assembly protein (AP) of Kaposi’s sarcoma-associated herpesvirus (KSHV) or human herpesvirus. As with other herpesviruses, the Pr and AP coding regions are present within a single long open reading frame. The coding strategy for the Pr and AP is similar in all well-characterized herpesvirus family members. The Pr and AP coding sequences are found in a single large open reading frame (ORF) encoding a polyprotein (Pr/AP) whose amino- and carboxy-terminal domains represent Pr and AP, respectively. Active Pr excises itself from Pr/AP by cleavage at the so-called release site (R-site) and can then cleave AP at the M-site. Most AP, however, is not generated by Pr/AP cleavage; rather, a separate mRNA initiated within the ORF directs AP translation from an internal AUG codon. For virion structural components, Pr/AP and AP transcripts are expressed as late viral genes in the lytic cycle and presumably are not expressed during latent infection 1,2.

Structural Characteristics
The mature KSHV Pr and AP polypeptides are predicted to contain 230 and 283 residues, respectively. The amino acid sequence of KSHV Pr has 56% identity with that of herpes virus saimiri, the most similar virus by phylogenetic comparison. Pr is expressed in infected human cells as a late viral gene product, as suggested by RNA analysis of KSHV-infected BCBL-1 cells. Expression of the Pr domain in Escherichia coli yields an enzymatically active species, as determined by cleavage of synthetic peptide substrates, while an active-site mutant of this same domain yields minimal proteolytic activity. Sequence comparisons with human cytomegalovirus (HCMV) Pr permitted the identification of the catalytic residues, Ser114, His46, and His134, based on the known structure of the HCMV enzyme. The amino acid sequences of the release site of KSHV Pr (Tyr-Leu-Lys-Ala*Ser-Leu- Ile-Pro) and the maturation site (Arg-Leu-Glu-Ala*Ser-Ser-Arg-Ser) show that the extended substrate binding pocket differs from that of other members of the family. The conservation of amino acids known to be involved in the dimer interface region of HCMV Pr suggests that KSHV Pr assembles in a similar fashion 1.

Mode of Action
Viral capsids assemble in the nucleus late in infection. Immature capsids lack DNA but contain an abundant internal polypeptide, the AP that is not found in the mature, DNA-containing particles 3. This protein is known to interact, through its carboxy-terminal domain, with the major capsid protein 4. This interaction is required for nuclear transport of the major capsid protein and has also been proposed to act as a scaffold to facilitate the assembly of the capsid shell. Following immature capsid assembly, AP undergoes proteolytic processing at the so-called maturation site (M-site) near its carboxy terminus, which removes the last 25 amino acids. This proteolysis is mediated by a virally encoded serine Pr. Herpesvirus Pr molecules show clear homology, ranging from 90% amino acid sequence identity among closely related viruses to 30% identity between distantly related viruses. These sequences are unrelated to the two major classes of presently characterized cellular serine Pr’s, the chymotrypsin and subtilisin families. The viral enzymes display similar substrate specificities, preferring an Ala residue at P1, Tyr at P4, and Ser at P19. Cleavage of AP by the viral Pr is essential for viral growth; HSV mutants bearing an inactive Pr accumulate capsids lacking viral DNA. Presumably, cleavage of substrate AP by protease is required to allow its release from the capsid and permit the packaging of newly replicated viral DNA 1,5,6.

Functions

Anti viral therapy, defining the protease target of HHV-8 and its natural substrates is an essential step in achieving anti viral therapy for HHV8 virus 1.

Substrate specificities, various Pr members of the herpes virus family are highly similar in their primary structure, but there are several differences, especially with regard to their individual substrate specificities. This can be seen in the kinetic analysis of various synthetic substrates. This property of protease exploited to interfere replication of different Herpes virus family 7.

Dominant negative inhibitors, small-molecule or dominant negative inhibitors of dimerization, has been proven to be effective in inhibiting the HIV Pr. Such inhibitors can be used to dissect the role of the Pr in the HHV-8 viral life cycle and to examine the impact of antiviral therapy on the natural history of HHV-8  infection 8.

References

1.     Unal A, Pray TR, Lagunoff M, Pennington MW, Ganem D, Craik CS (1997). The protease and the assembly protein of kaposi’s sarcoma- associated herpesvirus (human herpesvirus 8). J. Virol., 71:7030-7038.

2.     Albrecht JC, Nicholas J, Biller D, Cameron KR, Biesinger B, Newman C, Wittmann S, Craxton MA, Coleman H, Fleckenstein B (1992). Primary structure of the herpesvirus saimiri genome. J. Virol., 66:5047–5058.

3.     Gibson W, Roizman B (1972). Proteins specified by herpes simplex virus. VIII. Characterization and composition of multiple capsid forms of subtypes 1 and 2. J. Virol., 10:1044-1052.

4.     Hong Z, Beaudet-Miller M, Durkin J, Zhang R, Kwong AD (1996). Identification of a minimal hydrophobic domain in the herpes simplex virus type 1 scaffolding protein which is required for interaction with the major capsid protein. J. Virol., 70: 533–540.

5.     Sherman G, Bachenheimer SL (1988). Characterization of intranuclear capsids made by ts morphogenic mutants of HSV-1. Virology, 163:471-480.

6.     Perona J, Craik CS (1995). Structural basis of substrate specificity in the serine proteases. Protein Sci., 4:337-360.

7.     Tigue NJ, Matharu PJ, Roberts NA, Mills JS, Kay J, Jupp R (1996). Cloning, expression and characterization of the proteinase from human herpesvirus 6. J. Virol., 70:4136-4141.

8.     McPhee F, Good AC, Kuntz ID, Craik CS (1996). Engineering HIV-1 protease heterodimers as macromolecular inhibitors of viral maturation. PNAS., 93:11477-11481.

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