Poxvirus Biology

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

(1) Poxviruses are exceptional in having an entry-fusion complex (EFC) consisting of eleven conserved proteins embedded in the membrane of mature virions. With the goal of understanding the function of the EFC, extensive efforts have been made to determine the structures of its components, and to date, have been accomplished for nine of the eleven proteins. During FY2025, we reported the crystal structure of A21, the 10th EFC protein, comprising two α-helices clasping a twisted antiparallel β-sheet stabilized by two conserved disulfide bonds. (2) O-GlcNAcylation, a post-translational modification consisting of O-linked N-acetylglucosamine attached to serine and threonine residues, occurs in thousands of cytoplasmic, nuclear, and mitochondrial proteins but has been reported for relatively few viral proteins. During FY2025, we used click chemistry, specific antibodies, and mass spectrometry to investigate the O-GlcNAcylation of vaccinia virus (VACV) proteins. An O-GlcNAcylated virion protein of ~40 kDa was identified as A4, a highly conserved core component required for virion assembly. The protein was shown to be modified by the cellular O-GlcNAc transferase. Inhbitors of O-GlcNAc transferase reduced O-GlcNAcylation A4 to undetectable levels without diminishing the A4 abundance or infectivity of virus particles. O-GlcNAcylation either has a subtle role in the VACV life cycle, or A4 is an inadvertent substrate of the promiscuous O-GlcNAc transferase.(3) Mpox, the disease caused by monkeypox virus (MPXV) is increasing in Africa and in 2022 spread to more than 100 countries sickening more than 100,000 individuals. Four clades of MPXV have been recognized with differences in severity of disease and extent of human-to-human transmission. Determination of the genetic basis for these differences could help to develop improved therapeutics and vaccines. We previously showed that the Castaneous (CAST) mouse is highly susceptible to MPXV and virulence differences of MPXV clades are statistically significant mimicking their relative severities in humans. In FY2025 we continued our study to evaluate the CAST mouse as a model for investigating genomic differences. The replacement of genes of clade Ia MPXV with homologous gene sequences of the less virulent and less transmissible clade IIa MPXV was expected to reduce virulence. No significant difference was found by replacing more than 40 host-interaction genes. The absence of a significant reduction in virulence can have several explanations that would inform future experiments. (4) Poxvirus replication generates double-stranded RNA (dsRNA), which triggers antiviral responses by activating protein kinase R (PKR) and stimulating 2'5'-oligoadenylate synthetase (OAS) pathways to inhibit translation and degrade RNA, respectively. To counteract these responses, poxviruses express the E3 dsRNA-binding protein and decapping enzymes D9 and D10. The latter prevent dsRNA accumulation by removing 5’ caps from mRNAs allowing their degradation by the cellular XRN1 exonuclease. Mutations in the catalytic sites of D9 and D10 result in an ~15-fold increase in dsRNA compared to wild type virus, resulting in a profound replication defect accompanied by greatly reduced viral late mRNAs and proteins. However, the dsRNA made during poxvirus infection has not been characterized in detail. During FY2025, we purified dsRNA with an anti-dsRNA antibody from cells infected with wild-type and D9/D10 catalytic site mutant VACV using a protocol that avoided annealing of complementary RNAs during isolation. RNAseq analysis showed regions of the genome that account for high levels of dsRNA. (5) Poxviruses express numerous proteins to counteract host defenses. In FY2025, we continued our study of the highly conserved VACV A31 protein, which is present in all Orthopoxviruses and in most Chordopoxviruses. Deletion of A31R from VACV WR, had no discernible effect on replication in many cultured and primary cells. However, A31R gene is essential for the attenuated MVA vaccine strain, which lost numerous genes and became host-range restricted during hundreds of passages in avian cells. We found that replication of an MVA A31 deletion mutant could be significantly restored by replacement of certain genes from VACV WR. (6) The lipoprotein membrane of immature vaccinia virus (VACV) particles is derived from the endoplasmic reticulum (ER) by a poorly understood segmentation process requiring five small proteins (L2, A30.5, A11, A6 and H7) termed viral membrane assembly proteins (VMAPs). Absence of any one of these proteins reduces ER segmentation and results in the formation of empty crescents contiguous with ER membranes. In FY2025, we initiated the use of proximity analysis to determine the intracellular localization of VMAPs and identify interacting host and viral proteins. We showed that the C-terminus of L2 is present in the lumen of the endoplasmic reticulum.