Residues of the UL25 Protein of Herpes Simplex Virus That Are Required for Its Stable Interaction with Capsids

Abstract

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...

The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

“…The conformational changes in capsid structure that accompany genome packaging are believed to expose pUL25 binding sites, resulting in preferential attachment of pUL25 to C capsids and their stabilization (25,64,80). In cells infected with UL25 deletion mutants, DNA-filled C capsids were detected (27,63), whereas upon capsid purification, a larger amount of A capsids, but no C capsids, was seen (24,52). These findings are not necessarily contradictory, since the isolation of nuclear capsids by gradient purification can lead to the loss of capsid-associated proteins and of the packaged DNA (12,13,24).…”

“…This is different from the UL25 protein of alphaherpesviruses, whose abundance increases from B to A to C capsids (24,25,(60)(61)(62)(63)(64). In fact, the HSV-1 UL25/UL17 heterodimer was initially considered to be attached exclusively to C capsids and was thus termed the "C capsid-specific component" (CCSC) (25), yet its subsequent detection in A and B capsids led to its renaming as the "capsid vertex-specific component" (CVSC) (27). The enrichment of CVSC on C capsids described by several groups gave rise to the hypothesis that addition of CVSC concomitant with viral DNA packaging creates a signature indicating that mature capsids are ready for nuclear egress.…”

“…The increased number of alphaherpesvirus A capsids suggested that DNA packaging was initiated, yet the genomes were not stably retained within capsids in the absence of pUL25. In support of this scenario, small amounts of terminal genomic fragments indicative of low-level genome cleavage in the absence of pUL25 were observed (27,78). We searched for genomic termini in RPE-1 cells transfected with the UL77-deleted genome.…”

“…Studies with alphaherpesvirus mutants, for which complementing cells can be more easily established, revealed that two additional viral proteins (pUL17 and pUL25) are involved in encapsidation of genomes and their stable retention within capsids. pUL17 and pUL25 form a heterodimer that is located exclusively around pentons (23)(24)(25)(26) and was therefore named the "capsid vertex-specific component" (CVSC) (27). Loading of the CVSC on DNA-filled capsids is assumed to label mature capsids as ready for nuclear egress, which may be initiated by interaction of the CVSC with the nuclear egress complex (NEC).…”

Differential protein partitioning within the herpesvirus tegument and envelope underlies a complex and variable virion architecture

“…The copy number of GFP-tagged proteins in each H-particle was measured as a proportion of the fluorescence intensity of the capsid protein, pUL25 (24,27,28). Acquisition of pUL25 during assembly is copy-controlled, and estimates of pUL25 copy number were previously obtained by cryo-EM (60 copies; 5 per vertex) and immunoblot (estimated within the range of 75 ± 15 to 82 ± 19 copies) (29)(30)(31). We refined the measurement of pUL25 by comparing the fluorescence of pUL25/GFP to rotavirus (RV) virus-like particles (VLPs), the latter of which invariantly contain 120 copies of GFP (Fig.…”