Human cytomegalovirus (HCMV) is a major human pathogen belonging to the b-herpesvirinae subfamily. Most of the worldwide population has been infected with HCMV, and given its ability to undergo latency, it will be infected for the rest of their lives. Under normal circumstances, being latently infected with HCMV will not have any consequences.

However, HCMV infection is extremely problematic in newborns following congenital infection, and in immunocompromised individuals such as those who underwent transplantation or developed AIDS (1). Replication of its double stranded DNA (dsDNA) genome occurs in the nuclei of infected cells via a rolling-circle process mediated by 11 virally encoded proteins (2), including a viral DNA polymerase holoenzyme, comprising a catalytic subunit, UL54, and a proposed processivity factor, UL44 (3) which is an essential factor for viral replication and therefore represents a potential therapeutic target to fight HCMV infection (4). It is a multifunctional protein capable of self-associating (5), as well as interacting with a plethora of viral factors, in addition to the catalytic subunit pUL54, such as the viral kinase UL97 (6), the viral transactivating protein UL84 (7) and the viral uracil DNA glycosylase UL114.

In the past year we have focused our efforts on the characterization of protein-protein interactions regulating UL44 function during viral infection. Our efforts lead to the identification of the key cellular and viral molecular determinants responsible for the transport of UL44 into the nucleus during viral DNA replication, for its ability to dimerize, and to bind viral DNA (8-13). In addition we have identified key residues located at the very C-terminus of the protein as target of extensive post translational modifications regulating its subcellular localization, which include phosphorylation and SUMOylation (10, 14-17).  We are now using these data as a starting point to design new, highly needed, extremely specific anti viral compounds targeting UL44 function

 

REFERENCES

 

1.      Steininger, C. (2007) Clinical relevance of cytomegalovirus infection in patients with disorders of the immune system, Clin Microbiol Infect 13, 953-963.

2.      Pari, G. S., and Anders, D. G. (1993) Eleven loci encoding trans-acting factors are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA replication, J. Virol. 67, 6979-6988.

3.      Ertl, P. F., and Powell, K. L. (1992) Physical and functional interaction of human cytomegalovirus DNA polymerase and its accessory protein (ICP36) expressed in insect cells, J. Virol. 66, 4126-4133.

4.      Ripalti, A., Boccuni, M. C., Campanini, F., and Landini, M. P. (1995) Cytomegalovirus-mediated induction of antisense mRNA expression to UL44 inhibits virus replication in an astrocytoma cell line: identification of an essential gene, J. Virol. 69, 2047-2057.

5.      Appleton, B. A., Loregian, A., Filman, D. J., Coen, D. M., and Hogle, J. M. (2004) The cytomegalovirus DNA polymerase subunit UL44 forms a C clamp-shaped dimer, Mol. Cell 15, 233-244.

6.      Marschall, M., Freitag, M., Suchy, P., Romaker, D., Kupfer, R., Hanke, M., and Stamminger, T. (2003) The protein kinase pUL97 of human cytomegalovirus interacts with and phosphorylates the DNA polymerase processivity factor pUL44, Virology 311, 60-71.

7.      Gao, Y., Colletti, K., and Pari, G. S. (2008) Identification of human cytomegalovirus UL84 virus- and cell-encoded binding partners by using proteomics analysis, J. Virol. 82, 96-104.

8.      Alvisi, G., Jans, D. A., Guo, J., Pinna, L. A., and Ripalti, A. (2005) A protein kinase CK2 site flanking the nuclear targeting signal enhances nuclear transport of human cytomegalovirus ppUL44, Traffic 6, 1002-1013.

9.      Alvisi, G., Jans, D. A., and Ripalti, A. (2006) Human cytomegalovirus (HCMV) DNA polymerase processivity factor ppUL44 dimerizes in the cytosol before translocation to the nucleus, Biochemistry (USA). 45, 6866-6872.

10.    Alvisi, G., Rawlinson, S. M., Ghildyal, R., Ripalti, A., and Jans, D. A. (2008) Regulated nucleocytoplasmic trafficking of viral gene products: a therapeutic target?, Biochim. Biophys. Acta 1784, 213-227.

11.    Alvisi, G., Ripalti, A., Ngankeu, A., Giannandrea, M., Caraffi, S. G., Dias, M. M., and Jans, D. A. (2006) Human cytomegalovirus DNA polymerase catalytic subunit pUL54 possesses independently acting nuclear localization and ppUL44 binding motifs, Traffic 7, 1322-1332.

12.    Alvisi, G., Roth, D. M., Camozzi, D., Pari, G. S., Loregian, A., Ripalti, A., and Jans, D. A. (2009) The flexible loop of the human cytomegalovirus DNA polymerase processivity factor ppUL44 is required for efficient DNA binding and replication in cells, J. Virol. 83, 9567-9576.

13.    Sinigalia, E., Alvisi, G., Mercorelli, B., Coen, D. M., Pari, G. S., Jans, D. A., Ripalti, A., Palu, G., and Loregian, A. (2008) Role of homodimerization of human cytomegalovirus DNA polymerase accessory protein UL44 in origin-dependent DNA replication in cells, J. Virol. 82, 12574-12579.

14.    Alvisi, G., Marin, O., Pari, G., Mancini, M., Avanzi, S., Loregian, A., Jans, D. A., and Ripalti, A. (2011) Multiple phosphorylation sites at the C-terminus regulate nuclear import of HCMV DNA polymerase processivity factor ppUL44, Virology 417, 259-267.

15.    Fulcher, A. J., Roth, D. M., Fatima, S., Alvisi, G., and Jans, D. A. (2010) The BRCA-1 binding protein BRAP2 is a novel, negative regulator of nuclear import of viral proteins, dependent on phosphorylation flanking the nuclear localization signal, FASEB J. 24, 1454-1466.

16.    Alvisi, G., Jans, D. A., Camozzi, D., Avanzi, S., Loregian, A., Ripalti, A., and Palu, G. (2013) Regulated transport into the nucleus of herpesviridae DNA replication core proteins, Viruses 5, 2210-2234.

17.    Sinigalia, E., Alvisi, G., Segre, C. V., Mercorelli, B., Muratore, G., Winkler, M., Hsiao, H. H., Urlaub, H., Ripalti, A., Chiocca, S., Palu, G., and Loregian, A. (2012) The human cytomegalovirus DNA polymerase processivity factor UL44 is modified by SUMO in a DNA-dependent manner, PLoS One 7, e49630.

 

READ MORE FROM OUR GROUP

1.         Alvisi, G., et al., A protein kinase CK2 site flanking the nuclear targeting signal enhances nuclear transport of human cytomegalovirus ppUL44. Traffic, 2005. 6(11): p. 1002-1013.

2.         Alvisi, G., D. Jans, and A. Ripalti, Human cytomegalovirus (HCMV) DNA polymerase processivity factor ppUL44 dimerizes in the cytosol before translocation to the nucleus. Biochemistry, 2006. 45(22): p. 6866-6872.

3.         Alvisi, G., et al., Human cytomegalovirus DNA polymerase catalytic subunit pUL54 possesses independently acting nuclear localization and ppUL44 binding motifs. Traffic, 2006. 7(10): p. 1322-32.

4.         Alvisi, G., et al., An importin alpha/beta-recognized bipartite nuclear localization signal mediates targeting of the human herpes simplex virus type 1 DNA polymerase catalytic subunit pUL30 to the nucleus. Biochemistry, 2007. 46(32): p. 9155-63.

5.         Alvisi, G., et al., Nuclear import of HSV-1 DNA polymerase processivity factor UL42 is mediated by a C-terminally located bipartite nuclear localization signal. Biochemistry, 2008. 47(52): p. 13764-77.

6.         Alvisi, G., et al., Regulated nucleocytoplasmic trafficking of viral gene products: a therapeutic target?Biochim Biophys Acta, 2008. 1784(1): p. 213-27.

7.         Sinigalia, E., et al., Role of homodimerization of human cytomegalovirus DNA polymerase accessory protein UL44 in origin-dependent DNA replication in cells. J Virol, 2008. 82(24): p. 12574-9.

8.         Alvisi, G., et al., The flexible loop of the human cytomegalovirus DNA polymerase processivity factor ppUL44 is required for efficient DNA binding and replication in cells. J Virol, 2009. 83(18): p. 9567-76.

9.         Fulcher, A.J., et al., The BRCA-1 binding protein BRAP2 is a novel, negative regulator of nuclear import of viral proteins, dependent on phosphorylation flanking the nuclear localization signal. FASEB J, 2010. 24(5): p. 1454-66.

10.       Alvisi, G., et al., Multiple phosphorylation sites at the C-terminus regulate nuclear import of HCMV DNA polymerase processivity factor ppUL44. Virology, 2011. 417(2): p. 259-267.

11.       Sinigalia, E., et al., The human cytomegalovirus DNA polymerase processivity factor UL44 is modified by SUMO in a DNA-dependent manner. PLoS One, 2012. 7(11): p. e49630.

12.       Alvisi, G. and G. Palu, Reprogramming the host: Modification of cell functions upon viral infection.World J Virol, 2013. 2(2): p. 16-7.

13.       Avanzi, S., et al., Susceptibility of human placenta derived mesenchymal stromal/stem cells to human herpesviruses infection. PLoS One, 2013. 8(8): p. e71412.

14.       Gualtiero, A., et al., Regulated transport into the nucleus of herpesviridae DNA replication core proteins. Viruses, 2013. 5(9): p. 2210-34.

15.       Raza, S., et al., Ivermectin Inhibits Bovine Herpesvirus 1 DNA Polymerase Nuclear Import and Interferes with Viral Replication. Microorganisms, 2020. 8(3).

16.       Di Antonio, V., G. Palu, and G. Alvisi, Live-Cell Analysis of Human Cytomegalovirus DNA Polymerase Holoenzyme Assembly by Resonance Energy Transfer Methods. Microorganisms, 2021. 9(5).

17.       Ghassabian, H., et al., Divide et impera: An In Silico Screening Targeting HCMV ppUL44 Processivity Factor Homodimerization Identifies Small Molecules Inhibiting Viral Replication. Viruses, 2021. 13(5).