Full-length human cytomegalovirus terminase pUL89 adopts a two-domain structure specific for DNA packaging

Abstract

A key step in replication of human cytomegalovirus (HCMV) in the host cell is the generation and packaging of unit-length genomes into preformed capsids. The enzymes involved in this process are the terminases. The HCMV terminase complex consists of two terminase subunits, the ATPase pUL56 and the nuclease pUL89. A potential third component pUL51 has been proposed. Even though the terminase subunit pUL89 has been shown to be essential for DNA packaging and interaction with pUL56, it is not known how pUL89 mechanistically achieves sequence-specific DNA binding and nicking. To identify essential domains and invariant amino acids vis-a-vis nuclease activity and DNA binding, alanine substitutions of predicted motifs were analyzed. The analyses indicated that aspartate 463 is an invariant amino acid for the nuclease activity, while argine 544 is an invariant aa for DNA binding. Structural analysis of recombinant protein using electron microscopy in conjunction with single particle analysis revealed a curvilinear monomer with two distinct domains connected by a thinner hinge-like region that agrees well with the predicted structure. These results allow us to model how the terminase subunit pUL89’s structure may mediate its function.

A pentameric protein ring with novel architecture is required for herpesviral packaging

“…Herpesviral DNA packaging requires both sequence-specific and nonspecific DNA binding. Site-specific binding and cleavage within the terminal repeats is required to ensure full-length genomes are packaged; however, this role is thought to be fulfilled by the terminase ( Adelman et al, 2001 ; Theiß et al, 2019 ). Packaging also depends on nonsequence-specific interactions with various factors, such as HSV-1 UL25 (KSHV ORF19) that binds DNA and has recently been shown to be the ‘portal cap’ that prevents DNA escape after packaging has been completed ( Gong et al, 2019 ; Liu et al, 2019 ; Ogasawara et al, 2001 ).…”

“…Herpesviral DNA packaging requires both sequence specific and nonspecific DNA binding. Site-specific binding and cleavage within the terminal repeats is required to ensure fulllength genomes are packaged; however, this role is thought to be fulfilled by the terminase 19,20 .…”

Structures and Divergent Mechanisms in Capsid Maturation and Stabilization Following Genome Packaging of Human Cytomegalovirus and Herpesviruses

“…(6) Finally, the complex is ready for the next DNA-packaging step ( Figure 1 and Figure 2 ). In general, terminase complexes are hetero-oligomers composed of three core proteins (pUL56, pUL89 and pUL51 for HCMV and pUL28, pUL15 and pUL33 for HSV-1), each carrying a different function required for the packaging process [ 30 , 31 , 32 ]. At least three other proteins (pUL52, pUL77 and pUL93 for HCMV and pUL32, pUL25 and pUL17 for HSV-1) seem to be part of the terminase complex and/or participate in the DNA cleavage/packaging process [ 32 , 33 , 34 , 35 , 36 ].…”

“…pUL89 is a homolog of the HSV-1 protein pUL15. This protein of approximately 75 kDa possesses a putative nuclease domain and a putative DNA binding domain, implying that the aspartate 463 is involved in nuclease activity and the arginine 544 in DNA binding [ 31 ]. Different conserved patterns were highlighted in silico, comprising an adenine binding site ( 156 EPFQ 159 in HCMV pUL89), a Walker A motif ( 213 PRRHGKT 219 in HCMV pUL89), a Walker B motif ( 305 LLLLVDEAHFI 314 in HCMV pUL89) and pattern III ( 337 SST 339 in HCMV pUL89) [ 47 ] ( Figure 6 ).…”

Small Molecules—Prospective Novel HCMV Inhibitors

“…Recently, the terminase inhibitor letermovir ( Figure 1 ) was approved for HCMV prophylaxis for hematopoietic stem cell recipients [ 9 ]. However, mutations in the HCMV terminase subunit pUL56 [ 10 , 11 ] and, to a lesser extent, in pUL89 [ 12 , 13 ] or pUL51 [ 14 ] lead to resistance [ 6 , 15 , 16 , 17 ]. Due to the multiple problems caused by the currently available drugs, new antiviral targets with different modes of action are needed.…”