Chinese researchers reveal replication mechanism of monkeypox virus DNA polymerase holoenzyme

The World Health Organization declared monkeypox a public health emergency of international concern in July 2022.  

In a study recently published in Science, a research team from the Institute of Microbiology of the Chinese Academy of Sciences revealed the working mechanism of monkeypox virus (MPXV) DNA polymerase. 

MPXV belongs to the Orthopoxvirus genus of the Poxviridae family. It is a large double-stranded DNA virus that replicates exclusively in the cytoplasm of infected cells. After entry, the virus initiates early gene transcription events, and viral DNA synthesis occurs at perinuclear sites called viral factories. The MPXV replicative holoenzyme contains catalytic polymerase F8 as well as a heterodimeric processivity factor consisting of A22 and uracil-DNA glycosylase E4. The holoenzyme is an ideal target for drug development. 

Although previous genetic, biochemical, and structural studies of the core replication machinery of the Vaccinia virus (VACV) have advanced the understanding of poxvirus DNA replication, a reliable high-resolution structure of the replicating state of the Orthopoxvirus polymerase holoenzyme, and the mode of operation of the processivity factor, had not been elucidated. 

The researchers first determined the structure of the MPXV DNA polymerase holoenzyme in its replicating state. The structure of the holoenzyme/DNA complex contains one F8, one A22, one E4, and the primer/template DNA, as well as an incoming dTTP substrate in a closed conformation. 

The F8 and E4 resemble the counterparts of VACV. A22 can be divided into three domains: the A22 NTD, middle domain (Mid), and C-terminal domain (CTD), with the NTD and CTD binding with E4 and F8, respectively. The Mid displays high structural similarities to DNA ligases but does not possess ordinary ligase activity like T4 ligase.  

Compared with the structures of adenylation domains from the T4 and ASFV ligases, the putative ligase active site of the A22 Mid is replaced by hydrophobic and negatively charged residues, which may prevent the binding of ATP substrate. Therefore, A22 may comprise a degenerative ligase domain acting simply as a flexible linker. 

In addition, the researchers revealed that MPXV polymerase has a DNA binding mode that is similar to other B-family DNA polymerases from different species. 

Compared with the structure of VACV polymerase in the apo state, the finger domain rotates toward the palm domain by approximately 17° in the replicating state. This rotation drags the positively charged Arg634 and Lys661 of the fingers domain closer to the active site, where they can interact with the triphosphate group of incoming dNTP.  

The thumb domain also makes a significant rotation to wrap around the primer-template DNA duplex on its minor groove side. The DNA duplex is accommodated in a positively charged groove of the thumb domain, as observed in other B-family DNA polymerases. 

For B-family DNA polymerases, proliferating cell nuclear antigen (PCNA) or PCNA-like proteins are required for high processivity. However, for poxviruses, including MPXV and VACV, no homologous PCNA-like proteins have been identified in the viral genome. Instead, the poxvirus-specific A22-E4 heterodimer is responsible for the high processivity of DNA replication. 

The researchers in this study also demonstrated that F8 polymerase alone dissociated from the primer-template DNA after incorporating less than 14 nt, while the addition of the A22-D4 heterodimer conferred processivity to F8 in a concentration-dependent manner. 

The E4 cofactor interacts with the Exo of F8 polymerase, and together with the NTD of F8, they form a closed-ring channel to encircle the single-stranded template DNA and prevent dissociation from the holoenzyme complex, suggesting a “forward sliding clamp” mechanism.  

In contrast, in the yeast DNA polymerase complex, the Exo and NTD form an open semicircular channel to accommodate the single-stranded template DNA, and the trimeric PCNA ring encircles the template-product DNA duplex, a process known as the “backward sliding clamp.” This architectural difference between MPXV and yeast polymerase complexes is responsible for the different processivity mechanisms during DNA replication events. 

In conclusion, the findings of this study reveal the mechanism of MPXV genome replication and may guide the development of anti-poxvirus drugs. 

This article is based on a press release from the Chinese Academy of Sciences Headquarters.