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Arenaviruses

Arenaviruses are a group of enveloped and segmented negative-sense RNA viruses (sNSVs), comprising the family Arenaviridae. All human-infecting arenaviruses belong to the genus mammarenavirus, exemplified by Lassa virus (LASV) and Machupo virus (MACV). LASV is endemic in west Africa which causes approximately 100,000 to 300,000 human infections annually, with a case fatality rate as high as 20%. Unfortunately, there are no specific drugs or vaccines available for most arenavirus infections, posing great challenges for the treatments of related diseases. Arenaviruses encode their own RNA-dependent RNA polymerase (RdRp), namely the L protein, for replication and transcription of the viral genome, and this molecule is quite conserved among different viral species, which indicates a very important antiviral therapeutic target. However, the structure of intact arenavirus polymerase is unknown and its underlying mechanism for RNA synthesis remains poorly understood. Therefore, elucidating the working mechanism of arenavirus polymerase is of great significance for developing specific and broadspectrum antiviral drugs.

To achieve a comprehensive understanding in the mechanisms of arenavirus replication, we selected LASV and Machupo virus (MACV) as the representative species of the two groups respectively for our investigation. We determined their near-atomic resolution structures individually by cryo-electron microscopy (cryo-EM) single-particle reconstruction. These structures reveal that arenavirus polymerase adopts a similar overall architecture to bunyavirus and influenza virus polymerases, suggesting the conservation of these proteins in evolution. Intriguingly, the active site of arenavirus polymerase is inherently switched-on, independent of the activation by the 5’-viral RNA (5’-vRNA) as demonstrated in influenza virus and bunyavirus polymerases. Besides, we identified an arenavirus-specific insertion domain within the L polymerase, and confirmed its regulatory roles for RNA synthesis by biochemical assays. 

To further understand the mechanism of RNA recognition by arenavirus L polymerase, we determined the structure of MACV L protein in complex with vRNA promoter. The binding site of 3’-vRNA in arenavirus polymerase is similar to that observed in bunyavirus as well as influenza virus polymerases, suggesting a common mechanism for 3’-vRNA recognition by sNSVs. This site may represent a promising candidate target for developing broad-spectrum antiviral drugs against various sNSVs. Besides, we found the binding of 3’-vRNA significantly promoted dimerization of MACV L polymerase and mutations of the key residues at the dimeric interface severely inhibited the replication and transcription activities of both LASV and MACV L polymerases. According to sequence alignment, the interacting residues at the dimeric interface are moderately conserved for all mammarenaviruses, implying this dimerization might be a universal regulatory mechanism for arenavirus polymerases. (Nature, 2020).

Overall structure of LASV and MACV L proteins

In addition, we also investigated the mechanism of arenavirus matrix protein Z regulating the polymerase activity. Previous studies have revealed that the Z protein could interact with the polymerase to inhibit RNA synthesis at the late stage of viral replication, thus initiating the assembly of progeny virions. In this study, we determined the structures of LASV and MACV L proteins in complex with their cognate Z proteins, and the ternary complexes of L-Z-vRNA. These structures reveal that the Z protein binds to the periphery of palm domain of RdRp, far away from the binding site of vRNA. Therefore, the binding of Z protein does not prevent vRNA recruitment by the L polymerase. Despite no catalytic residues being directly engaged by Z protein, the distal ends of two catalytic motifs, D and E, are involved in interactions with Z protein outside the catalytic center. Moreover, these binding motifs are located at the interface between multiple domains. The binding of Z protein would thus restrict the conformational changes of key catalytic motifs required for catalysis, resulting in inactivation of the polymerase. Looking into the L-Z contacting interface, we found that Z protein binds to the L protein through its central domain, in which a highly conserved hydrophobic loop dominates the interaction with the L polymerase. Because of the conservation of the dominant binding motif, we observed that LASV and MACV Z proteins could cross-inhibit the activity of heterologous L polymerases. This evidence demonstrates a universal mechanism of L polymerase regulation by Z proteins, and indicates a novel strategy for developing broad-spectrum antiviral drugs against different arenaviruses.


Catalysis of L polymerase and its modulation by Z protein

Monkeypox Virus

Monkeypox Virus (MPXV), a member of the Orthopoxvirus genus within the Chordopoxvirinae subfamily of Poxviridae, shares its genus with vaccinia virus (VACV), cowpox virus (CPXV), and variola virus (VARV). First identified in 1958, MPXV caused its first human infection in 1970 and has since persisted as an endemic pathogen in Central and West Africa, evolving into two distinct clades: West African (Clade II) and Central African (Clade I). Since 2022, MPXV has emerged as a global threat. In two years, MPXV has evolved novel transmission routes and the emergence of a new variant, Clade Ib. The World Health Organization declared MPXV a Public Health Emergency of International Concern (PHEIC) in July 2022 and again in August 2024. With recent Clade Ib outbreaks reported in China, MPXV control poses a significant challenge. Following the global outbreak, the Gao Lab research team led by Academician George F. Gao swiftly initiated studies across three fronts: MPXV basic research, vaccines, and therapeutic antibodies. These works elucidated the molecular mechanism of MPXV polymerase holoenzyme function, developed novel protein subunit and mRNA vaccines, as well as therapeutic antibodies. 

1. Molecular Mechanism of MPXV Polymerase Holoenzyme

MPXV, long overlooked by scientists, required deeper exploration into the high-resolution structure and genome replication mechanisms of its polymerase holoenzyme. Viral DNA replication relies on a holoenzyme complex comprising polymerase F8, processivity factor E4, and the A22 heterodimer. Using cryo-electron microscopy, Gao Lab resolved the high-resolution structure of the MPXV polymerase holoenzyme in its replication-competent state, revealing its unique mechanism for sustained DNA synthesis. The complex includes F8, A22, E4, double-stranded DNA, and dTTP. F8 and E4 have typical DNA polymerase and uracil glycoside hydrolase structure characteristics, respectively. A22 bridges F8 and E4 through its N- and C-terminal domains, with its middle domain showing a degenerate ligase fold. Compared to the structure of the DNA-free vaccinia virus polymerase, the MPXV polymerase undergoes significant conformational changes during replication: a 17° rotation of the finger domain facilitates dNTP interactions, and the thumb domain forms a positively charged groove stabilizing DNA. The polymerase binds DNA primarily via phosphate backbones, minimizing base-specific interactions to enable sequence-independent elongation. Importantly, the A22-E4 heterodimer interacts directly with F8, forming a "forward sliding clamp" via a closed template channel. This mechanism replaces the PCNA trimer-dependent " backward sliding clamp" with other B-family polymerases, preventing template dissociation and enhancing processivity. These findings, published in Science, reveal poxvirus replication mechanisms and provide a structural basis for antiviral drug development targeting the holoenzyme.

2. Novel Mpox Vaccines

Mpox vaccines typically require combinations of multiple antigens for optimal efficacy. Using a structure-based vaccine design strategy previously used for the COVID-19 subunit vaccine ZF2001 (approved in China and four other countries with over 350 million doses administered), Gao Lab developed two novel vaccines: a bivalent chimeric protein subunit vaccine and a trivalent chimeric mRNA-LNP vaccine.

The bivalent protein subunit vaccine preserves key epitopes in MPXV antigens and significantly enhances immunogenicity in mice, eliciting neutralizing antibody titers 28-fold higher than the live-attenuated vaccinia Tian tan strain. It conferred complete protection against lethal vaccinia challenges and accelerated viral clearance in animal organs. Published in Nature Immunology, this vaccine has been licensed to Junshi Biosciences for preclinical development, including toxicity, pharmacology, and formulation studies. Non-human primate trials are ongoing, with clinical trial applications in China expected by Q1 2025. 

The trivalent mRNA-LNP vaccine, encoding three MPXV antigens in a single-mRNA construct, induces robust humoral and cellular immunity in mice, eliciting antibodies that sustain for nearly six months and effectively neutralizing both MPXV and VACV. The vaccine provides complete protection against challenge of lethal dose VACV. In contrast, mice vaccinated with the live-attenuated vaccinia Tian tan strain succumbed to infection. After MPXV challenge, mice vaccinated with chimeric mRNA vaccine exhibited significantly reduced viral loads. Compared to multivalent mRNA vaccines composed of single-antigen mRNA cocktails, the chimeric immunogens design simplifies production, reduces costs, and improves accessibility of mpox vaccines. Published in eBioMedicine, the vaccine is licensed to CSPC Pharmaceutical Group for clinical development, with clinical trial applications in China expected by 2025.

3. MPXV Therapeutic Antibodies

Using a "Normal/Emergent Platform" antibody discovery platform, Gao Lab isolated two monoclonal antibodies (hMB621 and hMB668) targeting distinct epitopes on the MPXV B6R antigen. Both antibodies exhibit broad-spectrum binding to B6R and its homologs in VACV, VARV, and CPXV, with potent neutralizing activity against VACV. In murine models, both antibodies protected against lethal VACV challenges. Structural analysis of the B6R-hMB668 complex revealed B6R structure and the antibody’s binding epitope. Published in Nature Communications, these antibodies are under evaluation for emergency stockpiling, with partnerships sought for further development. 

 

发表论文：

1. Peng, Q., Xie, Y., Kuai, L., Wang, H., Qi, J., Gao*, G. F. and Shi*, Y., 2023, Structure of monkeypox virus DNA polymerase holoenzyme. Science, 379 (6627): 100-105.

2. Wang, H., Yin, P., Zheng, T., Qin, L., Li, S., Han, P., Qu, X., Wen, J., Ding, H., Wu, J., Kong, T., Gao, Z., Hu, S., Zhao, X., Cao, X., Fang, M., Qi, J., Xi, J. J., Duan, K., Yang, X., Zhang, Z., Wang*, Q., Tan*, W. and Gao*, G. F., 2024, Rational design of a 'two-in-one' immunogen DAM drives potent immune response against mpox virus. Nature Immunology, 25 (2): 307-315.

3. Kong, T., Du, P., Ma, R., Wang, H., Ma, X., Lu, J., Gao, Z., Qi, H., Li, R., Zhang, H., Xia, F., Liu, Y., Wang, R., Duan, K., Wang, Z., Wang*, Q., and Gao*, G. F., 2024, Single-chain A35R-M1R-B6R trivalent mRNA vaccines protect mice against both mpox virus and vaccinia virus. EBioMedicine, 109: 105392.

4. Zhao, R., Wu, L., Sun, J., Liu, D., Han, P., Gao, Y., Zhang, Y., Xu, Y., Qu, X., Chai, Y., Chen, Z., Wang*, Q. and Gao*, G. F., 2024, Two noncompeting human neutralizing antibodies targeting MPXV B6 show protective effects against orthopoxvirus infections. Nature Communications, 15 (1): 4660.