In situ structure of the poxvirus portal complex.

Structure and operating principles of a monkeypox virus replisome.

Poxviruses are double-stranded DNA viruses with large genomes. Among them, monkeypox virus (MPXV) has been responsible for two recent public health emergencies as declared by the World Health Organization1. The MPXV polymerase comprises three subunits-a catalytic subunit (F8) and a heterodimeric processivity factor (A22 and E4). The viral polymerase must coordinate activities with the hexameric helicase-primase (E5) to initiate replication of the viral genome2. Although structures of MPXV E5 (refs. 3,4) and the polymerase5-7 in isolation are available, how they assemble into a functional replisome remains unclear. In isolation, E5 is in an autoinhibited conformation and has very weak helicase activity3,4, and the mechanism for helicase activation is unclear. Here we used cryo-electron microscopy to determine the structures of DNA-bound MPXV replisomes comprising the polymerase holoenzyme (F8, A22 and E4) and the E5 helicase hexamer. We show that, during replisome assembly, E5 undergoes large-scale conformational changes that allow two of its primase domains to interact with the polymerase F8 thumb and A22 subunit. Biochemical assays and single-molecule experiments reveal that this E5 conformational change is coupled to helicase activation and enhances primase activity. Taken together, these findings identify fundamental mechanisms governing coordinated helicase and polymerase activities during DNA replication for an important class of viral pathogens.

High-resolution structure of the vaccinia virus phospholipase D-fold endonuclease K4.

Vaccinia virus (VACV) is an orthopoxvirus closely related to mpox virus, which started global outbreaks in 2022. Poxvirus genomes are flanked by short, inverted complementary hairpin telomeres that feature mismatched bases and insertions essential for viral replication. In this context, a role of the late protein K4 has been proposed. K4 is present in the virion, is apparently non-essential and has a phospholipase D (PLD)-fold as has VACV F13 protein. It also shares fold and nuclease activity with its closest homologue, mammalian PLD3. We established an endonuclease activity against ssDNA and hairpin loops and bubbles in a dsDNA context while RNA is resistant to cleavage. The 2.4 Å cryo-EM structure of K4 shows an unusual octameric assembly, also present in solution. At low concentration, tetramers and dimers similar to the one of hPLD3 are also present. Despite its nuclease activity, in K4 a C-terminal extension blocks the DNA binding pockets. Using an inactive mutant, fortuitously, a DNA 19mer bound simultaneously to 2 sites of the octamer where it displaced the C-termini. DNA binding uses similar residues as the hPLD3 5'-exonuclease, despite different activities and orientations of the DNA. The role of K4 and the control of its activity by the observed auto-inhibition remain enigmatic.

The human papillomavirus L2 protein in the capsid is an ensemble of related structures poised to exit the virus particle

A structural model of L2 within the capsid is developed using cryo-EM single particle analysis of HPV16 pseudovirus capsids with and without the L2 protein combined with AlphaFold3 predictions and molecular dynamics simulations to develop a structural basis for understanding how L2 is organized in the capsid poised to initiate its action during virus entry.

Cryo-electron tomography of Nipah virus structural protein complexes in virus-like particles

This work provides novel insights into paramyxoviral protein complexes, structures, and morphology in NiV by interrogating the protein:protein interactions of the main NiV structural proteins M/N/F/G.

Cryo-EM structure of bacteriophage Nocturne116: insights into the architecture of c2-like Lactococcus viruses

ABSTRACT Nocturne116 is a small bacterial virus with a prolate head and a flexible, non-contractile tail that infects an insect-associated strain of Lactococcus lactis. Nocturne116 has little sequence similarity to other bacteriophages and represents a diverged virus lineage that includes the Lactococcus phage Q54 and phages of the Ceduovirus genus. To better understand the molecular architecture of this group of viruses, we determined the three-dimensional structure of the Nocturne116 virion. The head of the Nocturne116 phage is composed of two types of capsid proteins: the major capsid protein, which assembles into 50 hexamers, and a dedicated vertex protein, which forms 11 pentamers. The particle is stabilized by covalent isopeptide bonds, which result in topologically interlocked capsomer rings in the cylindrical midsection, while in the hemispherical caps, the chainmail-like structure is incomplete. An end vertex of the capsid is occupied by a portal complex, which is connected to the tail via a series of three neck protein rings. The genomic DNA extends through the neck channel up to the second tail tube ring, where it encounters the tail tape measure protein. The tail shaft is built of 26 hexameric rings of the tail tube protein and is capped by an assembly of three minor structural proteins that form a dome-shaped structure at the tail tip. The tail tip lacks characteristic receptor-binding proteins and represents the simplest host-attachment device of any studied Lactococcus phage, suggestive of an unusual mechanism by which the virus recognizes and infects its bacterial host. IMPORTANCE Viruses that infect the widely used food fermenter Lactococcus lactis can cause major problems in the food industry, but despite considerable effort, there are still gaps in the understanding of these viruses. Knowledge of their three-dimensional structure is important for understanding aspects such as particle-stabilizing mechanisms that they employ, or the host recognition mechanisms that these viruses use. The three-dimensional structure of Lactococcus phage Nocturne116 reveals a number of unusual features such as two types of capsid proteins making up the head, covalently crosslinked capsomers which, to our knowledge, are the first such observation in a prolate-shaped virus, or a minimalist tail tip which starkly contrasts with the elaborate machinery of other previously studied Lactococcus phages. Our results also shed light on the general structure of related ceduoviruses which, despite being the third-largest lactococcal virus group, have remained poorly understood from a structural perspective. Viruses that infect the widely used food fermenter Lactococcus lactis can cause major problems in the food industry, but despite considerable effort, there are still gaps in the understanding of these viruses. Knowledge of their three-dimensional structure is important for understanding aspects such as particle-stabilizing mechanisms that they employ, or the host recognition mechanisms that these viruses use. The three-dimensional structure of Lactococcus phage Nocturne116 reveals a number of unusual features such as two types of capsid proteins making up the head, covalently crosslinked capsomers which, to our knowledge, are the first such observation in a prolate-shaped virus, or a minimalist tail tip which starkly contrasts with the elaborate machinery of other previously studied Lactococcus phages. Our results also shed light on the general structure of related ceduoviruses which, despite being the third-largest lactococcal virus group, have remained poorly understood from a structural perspective.

Structural Overview of Herpesvirus Tegument Proteins