Gene Therapy and Molecular Biology Vol 3, page 75
Gene Ther Mol Biol Vol 3, 75-78. August 1999.
Human cytomegalovirus (HCMV) nuclease:
implications for new strategies in gene therapy
Institute of Virology, Robert-Koch-Str. 17, 35037 Marburg, Germany
Correspondence : Phone: +49-6421-285362; Fax: +49-6421-285482; E- mail: email@example.com
Received 9 October 1998 and in revised version 14 October; accepted 17 October 1998
Human cytomegalovirus (HCMV), one of eight human herpesviruses, can cause serious illness in neonates as well as in immunocompromised adults (Alford and Britt, 1993). Transplant and AIDS patients, e.g. may develop life- threatening diseases as a consequence of primary infection or reactivation of latent infection. Additionally, HCMV infections are also associated with congenital neurological complications in approximately 7,500 newborns annually (Alford and Britt, 1993). The current drugs are toxic and cause additional complications including drug resistance. Since present therapeutical approaches are limited new strategies are needed that may result from a better understanding of viral molecular biology.
The initial step of production of new virions is the packaging of newly synthesized, concatenated viral DNA into procapsids. As a consequence, blocking of this step will prevent production of viral progeny. Recently a new highly conserved gene product of ORF UL56, p130 (pUL56), was identified and partially characterized (Bogner et al., 1993). The homologous proteins of herpes simplex virus 1 (HSV-1), ICP 18.5 (UL28), and of pseudorabies virus (PrV) were reported to play an important role in DNA packaging. Viral mutants failed to cleave concatenated viral DNA which leads to an accumulation of naked nucleocapsid and uncleaved concatenated DNA in the nucleus (Addison et al., 1990; Mettenleiter et al., 1993). These reports suggested that UL56 may also play a role in virus assembly. The mechanism of DNA translocation into the procapsid and that of end formation by concatemer cutting at packaging sites (pac) are not well understood. Recently it was shown that HCMV p130 has the ability to interact with specific HCMV DNA packaging motifs and to cleave DNA bearing this motifs. Possible implications of this finding, its relation to the function of another HCMV
protein, pUL89, the so-called terminase, and to the bacteriophage system are discussed in this review.
II. HCMV p130 (pUL56) is a sequence specific nuclease
Viral DNA-replication results in the formation of large head-to-tail DNA concatemers (Ben-Porat and Rixon, 1979), and maturation into unit-length molecules involves site- specific cleavage at sequences (pac motifs) located within the a sequence (Spaete and Mocarski, 1985). Unit-length DNA is encapsidated in the nucleus and the DNA- containing C-capsids bud into the cytoplasm through the nuclear membranes. The final event is the envelopment in the TGN and the release into the extracellular space.
The process where newly synthesized viral DNA is cleaved and packaged into preformed capsids has long been of interest, because this is the initial step in viral assembly. The HCMV genomic a sequence is a short sequence located at both termini of the genome and in inverted orientation at the L-S junction (Mocarski et al., 1987; Tamashiro and Spector, 1986). The a sequence plays a key role in replication as a cis-acting signal for cleavage and packaging (Chou and Roizman, 1985). There is evidence that cleavage and packaging of DNA are linked processes (Ladin et al.,
1980). The HCMV a sequence contains two conserved motifs, pac 1 and pac 2. These packaging motifs have an AT-rich core flanked by a GC-rich sequence. In the case of HCMV, pac sites are located on one side of the cleavage site, whereas in other herpesvirus genomes the cleavage site is between these motifs (Marks and Spector, 1988). We sugggest that during the initial step of viral packaging, the capsid-associated protein p130 may bind to the pac sequences and promote cleavage of the concatemer.
Electrophoretic mobility shift assays with DNA probes
Bogner: HCMV nuclease in gene therapy
spanning the region of the cis-acting pac elements demonstrated that recombinant baculovirus-infected HCMV p130 formed specific DNA-protein complexes. These data suggested that p130, as a putative cleavage and packaging protein, attaches to the pac sequence. Furthermore it is proposed that viral DNA is taken up into the capsid being scanned along the complex from the bound a sequence across the L- and S-component until an a sequence in an identical orientation is found. The final step requires nicking of both strands at signals on opposite sites of the sequence.
Interestingly, by using circular plasmid DNA bearing a single a sequence as a substrate, purified baculovirus expressed HCMV p130 has an enzymatic activity that converts supercoiled plasmid DNA into open circular as well as linear molecules (Bogner et al., 1998; Fig.1 ). This observation is comparable with the notion that HCMV p130 may be involved in cleavage of viral DNA. By using Apyrase, evidence was provided that in contrast to the adeno-associated virus origin-binding protein Rep68 (Im and Muzyczka, 1990) the reaction is independent of ATP. Interestingly, baculovirus-UL28 cell extracts, containing the HSV-1 homolog ICP18.5 (pUL28), also cleaved supercoiled plasmid DNA molecules bearing the a sequence (Fig.1 ). It was then suggested that p130 and its herpesviral homologs are involved in cleaving of concatenated viral DNA and packaging into procapsids. It is currently under investigation, whether p130 operates with another viral protein, UL89.
III. Comparison with the bacteriophage system
Herpesviruses share common features with respect to
DNA maturation with dsDNA bacteriophages. DNA
replication results in high molecular weight concatenated DNA and procapsids are assembled around a protein scaffold (Black, 1989; Murialdo and Becker, 1978). Translocation of the DNA to the procapsid bacteriophages is an ATP- dependent process carried out by terminases (Feiss and Becker, 1983). Terminases are ATP-binding proteins which also bind and cleave concatenated DNA at cohesive (cos; e.g. phage ) or packaging (pac; e.g. phage P22) sites (Gold et al., 1983). In the case of bacteriophage T4, procapsids are apparently filled without sequence specificic cleavage by a
âheadful mechanismâ (Kalinski and Black, 1986; Streisinger et al., 1967). The HCMV UL89 gene product has some homology to the phage T4 gp17 terminase subunit. T4 terminase has two subunits. The large subunit, gp17, contains the ATP binding sites, the small subunit, gp16, is required for packaging of concatenated DNA (Rao and Black,
1988). There is preliminary evidence that baculovirus-UL89 cell extracts exhibit endonuclease activity (Fig.2 ). HCMV UL89 is also able to nonspecifically cleave DNA in a manner reminiscent of phage T4 terminase (Black, 1986). Terminase can use either concatemeric or monomeric DNA of any sequence and prefers nonspecific ends in monomeric DNA over pac-containing concatemers (Serwer; 1986).
Recently, Krosky et al. (1998) and Underwood et al. (1998) reported on a new drug, which is a derivative of benzimidazole ribonucleosides. HCMV mutants were selected by treatment of infected cultures with increasing amount of the drug. The mutations were mapped to the UL56 gene (Krosky et al., 1998) and to the gene product of pUL89 (Underwood et al., 1998). Based on the identical phenotypes of the drug resistant mutants, it is speculated that these viral proteins form the putative HCMV terminase.
Fig.1 : Nuclease activity of the p130 homolog of HSV ICP18.5 (pUL28). Nuclease reactions were incubated for 1 h at 37Â°C and samples were treated with proteinase K for an additional hour at 37Â°C. Lane 1, plasmid pON205 in the absence of protein;
2, pON205 treated with the restriction enzyme HindIII; 3, pON205 incubated with rp130; 4, pON205 incubated with HSV UL28; 5, pON205 plus wild-type infected extracts; 6, pON205 incubated with mock-infected extracts. The arrows indicate three different plasmid DNA forms: open circular molecules (a); linear forms (b); and supercoiled molecules (c).
Gene Therapy and Molecular Biology Vol 3, page 77
Fig.2 : Nuclease activity of the T4 terminase homolog HCMV pUL89. Lanes: 1, pON205 alone;
2, pON205 treated with HindIII; 3, pON205 incubated with extract containing p130; 4, pON205 incubated with extracts containing HCMV UL89; 5, pON205 incubated with wild-type infected extracts; 6, pON205 incubated with mock- infected extracts. Open circular DNA molecules (a), linear (b) and supercoiled molecules (c) are indicated.
IV. Questions for the future
Considering that the current drugs (ganciclovir, cidofovir and foscarnet) have limited effects and dose- dependent toxicity, new antiviral therapeutics are needed. The mechanism of the current drugs is the inhibition of viral replication through an interaction with viral DNA polymerase (Erriksson et al., 1982; Ho et al., 1992; Mar et al., 1985). The inhibition of the cleavage and packaging of the viral DNA by a nuclease inhibitor may offer a potentially alternative therapy.
Taken together, the reports by Addison et al. (1990), Tengelsen et al (1993) and Mettenleiter et al. (1993), demonstrating that the HSV-1 ICP18.5 (pUL28) gene product and the PRV homolog are necessary for cleavage and packaging of concatenated viral DNA, and the observation that HCMV p130 (pUL56) can interact with specific DNA packaging motifs and is able to cleave DNA bearing these motifs, provide a basis for understanding the herpesvirus DNA packaging process at the molecular level. Identification of the structure of the proteins involved is needed as a prerequisite for the development of new antivirals. Knowledge of the three dimensional protein structure is pertinent in revealing the catalytic domain for the enzymatic activity prior to anti-viral drug-design. Regarding that mammalian cell DNA replication does not involve cleavage of concatemeric DNA, drugs targeted to the viral nucleases should be safe and selective. Therefore, our findings may help to develop new nontoxic anti-HCMV
reagents for treatment of the immunocompromised patient population.
I thank Fred Homa for kindly providing recombinant baculoviurs expressing HSV-1 UL28, Edward Mocarski for the a sequence containing plasmid pON205 and Mark Underwood for the baculovirus plasmid containing HCMV UL89. I am grateful to Klaus Radsak for critically reading the manuscript and to lab members for helpful comments. This work was supported by Deutsche Forschungsgesellschaft, Sonderforschungsbereich 286, Teilprojekt A3.
Addison, C., F.J. Rixon and V.G. Preston (1990) Herpes simplex virus type 1 UL28 gene product is important for the formation of mature capsids. J Gen Virol 71, 2377-
Alford, C.A. and W.J. Britt (1993 ) Cytomegalovirus, p 227-
255. IN B.Roizman, RlJ. Whiteley and C. Lopez et al. (ed.), The human herpesviruses . Raven Press, Ltd., New York.
Ben-Porat, T. and F.J.Rixon (1979) Replication of herpesvirus DNA. IV: Analysis of concatemers. Virology
Black, L.W. (1986) In vitro packaging of bacteriophage T4
Bogner: HCMV nuclease in gene therapy
DNA. Virology 113, 336-344.
Black, L.W. (1988) DNA packaging in dsDNA bacteriophages. In: The Bacteriophages , ed. R. Calendar, 2, 321-273. New York: Plenum.
Black, L.W. (1989) DNA packaging in dsDNA
bacteriophages. Annu Rev Microbiol 43, 267-292.
Bogner, E., M. Reschke, B. Reis, T. Mockenhaupt and K.
Radsak (1993) Identification of the gene product encoded by ORF UL56 of human cytomegalovirus genome. Virology 196, 290-293.
Bogner, E., K. Radsak and M.F. Stinski (1998) The gene product of human cytomegalovirus open reading frame UL56 binds the pac motif and has specific nuclease activity. J Virol 72 , 2259-2264.
Chou, J. and B. Roizman (1985) The isomerization of the herpes simplex virus 1 genome: identification of the cis- acting and recombination sites within the domain of the a sequence. Cell 41, 803-811.
Erriksson, D., B. Oberg and B. Wahren (1982) Pyrohphosphate analogs as inhibitors of DNA polymerases of cytomegalovirus, herpes simplex virus and cellular origin. Biochim Biophys Acta 669, 115-123.
Feiss, M. and A. Becker (1983) DNA packaging and cutting, p
305-330. IN R.W. Hendrix, J.W. Roberts, F.W. Stahl and R.A. Weisberg (ed.), Lambda II. Cold Spring Harbor, New York.
Gold, M. and A. Becker (1983) The baceriophage terminase: partial purification and preliminary characterization of properties. J Biol Chem 258, 14619-14625.
Ho, H.-T., K.L. Woods, J.J. Bronson, H. DeBoeck, J.C. Martin and M.J.M. Hitchcock (1992) Intracellular metabolism of the antiherpes agent (S)-1-[3-hydroxy-2- (phosphonylmethoxy)propyl] cytosine. Mol Pharmacol 41, 197-202.
Kalinski, A. and L.W. Black (1986) End structure and mechanism of packaging of bacteriophage T4 DNA. J Virol 58, 951-954.
Krosky, P.M., M.R. Underwood, S.R. Turk, K. W.-H. Feng, R.K. Jain, R.G. Ptak, A.C. Westerman, K.K. Biron, L.B. Townsend and J.C. Drach (1998) Resistance of human cytomegalovirus to benzimidazole ribonucleosides maps to two open reading frames: UL89 and UL56. J Virol 72,
Ladin, B.F., M.L. Blankenship and T. Ben-Porat (1980) Replication of herpesviurs DNA. V. The maturation of concatemeric DNA of pseudorabies virus to genome length is related to capsid formation. J Virol 33, 1151-1164.
Mar, E., J. Chiou, Y. Cheng and E. Huang (1985) Inhibition
of cellular DNA polymerase alpha and human cytomegalovirus-induced DNA polymerase by triphosphates of 9-(2-hydroxymethyl)guanine and 9-(1,3- dihydroxy-2-propoxymethyl)guanine. J Virol 53, 776-
Marks, J.R. and D.H. Spector (1988) Replication of the murine cytomegalovirus genome: structure and role of the termini the generation and cleavage of concatemers. Virology 162, 98-107.
Mettenleiter, T.C., A. SaalmÃ¼ller and F.Weiland (1993) Pseudorabies virus protein homologous to herpes simplex virus type1 ICP 18.5 is necessary for capsid maturation. J Virol 67, 1236-1245.
Mocarski, E.S., A.C. Liu and R.R. Spaete (1987) Structure and variability of the a sequence in the genome of human cytomegalovirus (Towne strain). J Gen Virol 68, 2223-
Murialdo, H. and A. Becker (1978) Head morphogenesis of complex double-stranded deoxyribonucleic acid bacteriophages. Microbiol Rev 42, 529-576.
Rao, V.B. and L.W. Black (1988) Cloning, overexpression and purification of the terminase proteins gp16 and gp17 of bacteriophage T4: construction of a defined in vitro DNA packaging system using purified terminase proteins. J Mol Biol 200, 475-488.
Serwer, P. (1986) Arrangement of double-stranded DNA
packaged in bacteriophage capsids. J Mol Biol 190,
Spaete, R.R. and E.S. Mocarski (1985) The a sequence of the cytomegalovirus genome functions as a cleavage/packagung signal for herpes simplex virus defective genomes. J Virol 54, 817-824.
Streisinger, G., J. Emrich and M.M. Stahl (1967) Chromosome structure in phage T4. III. Terminal redundancy and lenght determination. Proc Natl Acad Sci USA 57, 292-295.
Tamashiro, J.C. and D.H. Spector (1986) Terminal structure and heterogeneity in human cytomegalovirus strain AD
169. J Virol 59, 591-604.
Tengelsen LA, Pederson NE, Shaver PR, Wathen MW, Homa FL (1993 ) Herpes simplex virus type 1 DNA cleavage and encapsidation require the product of the UL28 gene: isolation and characterization of two UL28 deletion mutants. J Virol 67, 3470-3480.
Underwood, M.R., R.J. Harvey, S.C. Stanat, M.L. Hemphill, T.
Miller, J.C. Drach, L.B. Townsend and K.K. Biron (1998) Inhibition of human cytomegalovirus DNA maturation by a benzimidazole ribonucleoside is mediated through the UL89 gene product. J Virol 72, 717-725.