Mechanisms of human cytomegalovirus capsid maturation
Final Report Abstract
Infection with human cytomegalovirus (HCMV) can elicit severe complications in immunosuppressed patients, and is the leading viral cause of birth defects. Medication currently used to treat HCMV disease has one common target, namely the viral DNA polymerase, and is often afflicted with serious side effects. Inhibition of protein-proteininteractions between essential viral proteins is a promising strategy that is considered for the development of novel antivirals with a different mode of action. A highly virus-specific process suitable for this approach is the packaging of HCMV genomes into capsids, which is accomplished by the viral terminase proteins. Of note, a recently developed drug candidate targeting the HCMV terminase, Prevymis (a.k.a. Letermovir), was lately licensed in the US. We have recently described a third HCMV protein being part of the viral terminase, namely pUL51, which forms a stable complex with the known terminase subunits pUL56 and pUL89, and is required for cleavage and encapsidation of HCMV genomes. We now thoroughly characterized the UL51 protein and its function within the terminase complex, and found that pUL51 is necessary to ensure appropriate UL56 and UL89 protein levels, to mediate nuclear targeting of pUL89, and to promote the pUL56/pUL89 interaction. Remarkably, it then turned out that these functions are not specific for pUL51, but that a similar scenario applies to all three terminase constituents, because in the absence of either subunit the others underwent proteasomal degradation, their interaction was severely diminished, and nuclear localization was impaired. Our data are indicative of a folding-upon-binding mechanism governing HCMV terminase assembly, where each subunit is stabilized and acquires its correct conformation through loading into the final terminase complex. Mutational analysis of pUL51 revealed that its C-terminal part, presumably consisting of α-helical structures, is not only necessary, but also sufficient for terminase complex formation, whereas its putatively disordered N-terminal region seems to be dispensable in this respect. Our results further establish pUL51 as a third component of the HCMV terminase complex, and advance the knowledge about terminase assembly, which altogether will help to reveal the precise mechanism of action of drugs targeting HCMV genome cleavage-packaging. Another auspicious drug target is the interaction network driving HCMV capsid assembly, and particularly the roles of viral proteins pUL77 and pUL93 were unclear. By generating and analyzing suitable HCMV mutants we showed that both proteins are incorporated into capsids as well as into extracellular virions, and that viral genomes remain uncut and only empty capsids (B capsids) are detected when either protein is missing. This indicates that genome packaging is not even initiated, and hence suggests that the function of pUL77 differs from that described for its ortholog in α-herpesviruses. These data for the first time assign a crucial role to both pUL77 and pUL93 in HCMV genome cleavage-packaging. Furthermore, the HCMV recombinant expressing pUL77 fused to monomeric EGFP is the first HCMV mutant with a fluorescent capsid, which will be a valuable future tool to investigate capsid trafficking in living cells. Additional understanding of the interfaces mediating association of pUL77 and pUL93 with each other and with capsid shells may allow the development of custom-tailored inhibitors interfering with these interactions.
Publications
- Analysis of essential viral gene functions after highly efficient adenofection of cells with cloned human cytomegalovirus genomes. Viruses. 2014 Jan 23;6(1):354-70
Elbasani E, Gabaev I, Steinbrück L, Messerle M, Borst EM
(See online at https://doi.org/10.3390/v6010354) - The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation. J Virol. 2016 Jun 10;90(13):5860-75
Borst EM, Bauerfeind R, Binz A, Stephan TM, Neuber S, Wagner K, Steinbrück L, Sodeik B, Lenac Roviš T, Jonjić S, Messerle M
(See online at https://doi.org/10.1128/JVI.00384-16) - Mutual Interplay between the Human Cytomegalovirus Terminase Subunits pUL51, pUL56, and pUL89 Promotes Terminase Complex Formation. J Virol. 2017 May 26;91(12). pii: e02384-16
Neuber S, Wagner K, Goldner T, Lischka P, Steinbrueck L, Messerle M, Borst EM
(See online at https://doi.org/10.1128/JVI.02384-16) - The C-terminal part of the human cytomegalovirus terminase subunit pUL51 is central for terminase complex assembly. J Gen Virol. 2017 Dec 5
Neuber S, Wagner K, Messerle M, Borst EM
(See online at https://doi.org/10.1099/jgv.0.000984)
