Herpes simplex virus type 1 (HSV-1) packages its microns-long double-stranded (ds) DNA genome into a nanometer-scale protein shell, termed the capsid. Upon confinement within the capsid, neighboring DNA strands experience repulsive electrostatic and hydration forces as well as bending stress associated with the tight curvature required of packaged DNA. By osmotically suppressing DNA release from HSV-1 capsids, we provide the first experimental evidence of a high internal pressure of tens of atmospheres within a eukaryotic human virus, resulting from the confined genome. Furthermore, the ejection is progressively suppressed by increasing external osmotic pressures, which reveals that internal pressure is capable of powering ejection of the entire genome from the viral capsid. Despite billions of years of evolution separating eukaryotic viruses and bacteriophages, pressure-driven DNA ejection has been conserved. This suggests it is a key mechanism for viral infection and thus presents a new target for antiviral therapies.
. We previously confirmed that UL25 occupies the vertex-distal region of the CVSC density by visualizing a large UL25-specific tag in reconstructions calculated from cryo-electron microscopy (cryo-EM) images. We have pursued the same strategy to determine the capsid location of the UL17 protein. Recombinant viruses were generated that contained either a small tandem affinity purification (TAP) tag or the green fluorescent protein (GFP) attached to the C terminus of UL17. Purification of the TAP-tagged UL17 or a similarly TAP-tagged UL25 protein clearly demonstrated that the two proteins interact. A cryo-EM reconstruction of capsids containing the UL17-GFP protein reveals that UL17 is the second component of the CVSC and suggests that UL17 interfaces with the other CVSC component, UL25, through its C terminus. The portion of UL17 nearest the vertex appears to be poorly constrained, which may provide flexibility in interacting with tegument proteins or the DNA-packaging machinery at the portal vertex. The exposed locations of the UL17 and UL25 proteins on the HSV-1 capsid exterior suggest that they may be attractive targets for highly specific antivirals.Herpesviruses are incurable human and animal pathogens that generally infect their hosts for life, escaping immune surveillance and producing recurrent infections between periods of latency. They have been shown recently to share functional and structural characteristics with double-stranded DNA (dsDNA) bacteriophages, and the recognition of common features in the capsid assembly pathways has been (and remains) instrumental in identifying roles for herpesvirus proteins that are less easily studied and manipulated than their phage analogs. These common features include (i) initial assembly into an icosahedral procapsid, (ii) maturational proteolysis of structural proteins, (iii) ATP-driven packaging of the dsDNA chromosome through a specialized capsid vertex complex called the "portal," (iv) maturation of the procapsid during packaging, and (v) capsid stabilization effected by binding accessory proteins or by the formation of intersubunit bonds (reviewed in reference 5). While much of the herpesvirus capsid structure has been detailed, particularly by cryo-electron microscopy (cryo-EM), a number of minor proteins that interact with the capsid during assembly are still understood mostly by analogy with identifiable counterparts in phages. Several of these proteins are essential for packaging and retaining the viral DNA and are potentially valuable targets for interfering with herpesvirus replication.The herpesvirus virion, ϳ200 nm in diameter, consists of an icosahedral capsid of 125 nm in diameter enclosing the dsDNA chromosome and an amorphous layer of tegument proteins linking the capsid to an exterior lipid envelope in which different viral glycoproteins are embedded (5,7,12,25). The herpes simplex virus 1 (HSV-1) capsid is composed primarily of the major capsid protein, VP5, organized as hexameric and pentameric capsomers that are termed "hexons" and "pentons,"...
The herpesvirus capsid is a complex protein assembly that includes hundreds of copies of four major subunits and lesser numbers of several minor proteins, all essential for infectivity. Cryo-electron microscopy is uniquely suited for studying interactions that govern the assembly and function of such large and functional complexes. Here we report two high quality capsid structures, from human herpes simplex virus type 1 (HSV-1) and the animal pseudorabies virus (PRV), imaged inside intact virions at ~7 Å resolution. From these we developed a complete model of subunit and domainal organization and identified extensive networks of subunit contacts that underpin capsid stability and form a pathway that may signal the completion of DNA packaging from the capsid interior to outer surface for initiating nuclear egress. Differences in folding and orientation of subunit domains between herpesvirus capsids suggest that common elements have been modified for specific functions.
The herpes simplex virus 1 (HSV-1) UL25 gene product is a minor capsid component that is required for encapsidation, but not cleavage, of replicated viral DNA. UL25 is located on the capsid surface in a proposed heterodimer with UL17, where five copies of the heterodimer are found at each of the capsid vertices. Previously, we demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids. To further define the UL25 capsid binding domain, we generated recombinant viruses with either small truncations or amino acid substitutions in the UL25 N terminus. Studies of these mutants demonstrated that there are two important regions within the capsid binding domain. The first 27 amino acids are essential for capsid binding of UL25, while residues 26 to 39, which are highly conserved in the UL25 homologues of other alphaherpesviruses, were found to be critical for stable capsid binding. Cryo-electron microscopy reconstructions of capsids containing either a small tag on the N terminus of UL25 or the green fluorescent protein (GFP) fused between amino acids 50 and 51 of UL25 demonstrate that residues 1 to 27 of UL25 contact the hexon adjacent to the penton. A second region, most likely centered on amino acids 26 to 39, contacts the triplex that is one removed from the penton. Importantly, both of these UL25 capsid binding regions are essential for the stable packaging of full-length viral genomes.The herpes simplex virus 1 (HSV-1) virion consists of a 152-kbp double-stranded DNA (dsDNA) genome that is enclosed in an icosahedral capsid, which is itself surrounded by an amorphous protein layer called the tegument, and a lipid envelope containing viral glycoproteins. Viral DNA replication generates concatemers of head-to-tail genomes in the nucleus of the infected cell (26,27). DNA replication is concurrent with the expression of HSV late genes, including the capsid structural proteins VP5 (UL19), VP19c (UL38), VP23 (UL18), and VP26 (UL35) (reviewed in reference 24). Using the scaffold proteins encoded by the HSV-1 UL26 and UL26.5 genes, the capsid structural proteins self-assemble into a spherical capsid precursor, or procapsid (19,31,36). Packaging of viral genomes into procapsids requires several tightly coupled events: (i) proteolysis and expulsion of the scaffold proteins, (ii) recognition and cleavage of viral genome termini by the terminase complex, (iii) docking of the terminase-DNA complex on the capsid portal, (iv) insertion of DNA into the procapsid, and (v) sealing of the portal. The product of DNA packaging is an angular capsid that contains tightly packed viral DNA (2, 10). The mature capsid, also known as a C capsid, then exits the nucleus and is incorporated into virions.In the absence of successful packaging, two capsid forms accumulate in the nuclei of infected cells. B capsids contain remnants of the cleaved scaffold proteins VP21, VP22a, and VP24, and A capsids are empty (10). In the current model of HSV DNA packaging, B and A capsids form when DNA packaging is termina...
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