The Capsid-Associated UL25 Protein of the Alphaherpesvirus Pseudorabies Virus Is Nonessential for Cleavage and Encapsidation of Genomic DNA but Is Required for Nuclear Egress of Capsids

Abstract

To clarify the functional role of UL25 we analyzed the homologous protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL25 was found to be essential for viral replication, as a mutant virus lacking the UL25 protein required UL25-expressing cells for productive propagation. In the absence of the UL25 protein, newly replicated PrV DNA was cleaved and DNA-containing C-type capsids were detected in infected cell nuclei. However, although capsids were frequently found in close association with the inner nuclear membrane, nuclear egress was not observed. Consequently, no capsids were found in the cytoplasm, resulting in an inhibition of virion morphogenesis. In contrast, the formation of capsidless enveloped tegument structures (L particles) in the cytoplasm was readily observed. Thus, our data demonstrate that the PrV UL25 protein is not essential for cleavage and encapsidation of viral genomes, although both processes occur more efficiently in the presence of the protein. However, the presence of the PrV UL25 protein is a prerequisite for nuclear egress. By immunoelectron microscopy, we detected UL25-specific label on DNA-containing C capsids but not on other intranuclear immature or defective capsid forms. Thus, the PrV UL25 protein may represent the hitherto missing trigger that allows primary envelopment preferably of DNA-filled C capsids.Herpesvirus assembly is a multistep process involving many protein-protein, as well as protein-DNA, interactions which occur in at least two distinct subcellular compartments. While capsid assembly and DNA packaging take place in the nucleus of infected cells, the addition of most of the tegument, as well as final envelopment, proceed in the cytoplasm (reviewed in references 42 and 43). In recent years, an increasing amount of data has indicated that the intranuclear stages of herpesvirus assembly, namely, DNA replication, capsid formation, and DNA cleavage/packaging follow pathways similar to those described for the better-studied large double-stranded DNA bacteriophages such as P22, T4, T7, and (26). The similarities even extend to capsid structure, which, together, point to a common ancestry of the eukaryotic Herpesviridae and the prokaryotic Caudovirales (8).In a generalized model of cleavage and packaging of genomic DNA of double-stranded DNA viruses (reviewed in reference 9), the terminase enzyme binds to specialized sites in the concatemeric DNA and subsequently cleaves the duplex DNA to generate one mature genome end. The terminase with the attached concatemeric DNA then binds to the portal complex, a dodecameric ring structure located at one vertex in the capsid which forms a channel. The interaction of the portal with the terminase-genome complex generates the packaging motor, which pumps the DNA against growing mechanical forces into the capsid, fueled by ATP hydrolysis, resulting in removal of the internal scaffold structure. When the full-length genome is packaged, the terminase executes the second cleavage to complete the packaging process. Since the term...

Selection of HSV capsids for envelopment involves interaction between capsid surface components pU L 31, pU L 17, and pU L 25

“…U L 25 null capsids of pseudorabies virus (PRV), a well-studied swine herpesvirus, accumulate against the nuclear membrane of infected cells, suggesting they are able to attach to the INM (33). Thus, components of the capsid other than pU L 25 are sufficient to mediate INM tethering in this system.…”

Isolation and Preliminary Characterization of Herpes Simplex Virus 1 Primary Enveloped Virions from the Perinuclear Space

“…The latter mutant lacks a portion of the large UL36 open reading frame, which results in disruption of secondary envelopment and accumulation of mature packaged C capsids in the cytoplasm (17). The postnuclear supernatant (PNS) of COS-7 cells infected with K⌬UL36 HSV-1 was mixed with a TCE of COS-7 cells infected with ⌬UL15 HSV-1.…”

Vesicle formation from the nuclear membrane is induced by coexpression of two conserved herpesvirus proteins

“…Mutants PrV-⌬UL34 (8), PrV-⌬UL31 (11), and PrV-⌬US3 (25) have been derived from PrV-Ka (30). Mutant PrV-⌬UL25/US3 was isolated after cotransfection of PrV-⌬UL25 genomic DNA (26) and plasmid p⌬US3gfp (25) into RK13-UL25 cells (26). Mutant viruses were propagated on RK13 or RK13-UL31, RK13-UL34, or RK13-UL25 cells (8,11,26).…”

“…Mutant PrV-⌬UL25/US3 was isolated after cotransfection of PrV-⌬UL25 genomic DNA (26) and plasmid p⌬US3gfp (25) into RK13-UL25 cells (26). Mutant viruses were propagated on RK13 or RK13-UL31, RK13-UL34, or RK13-UL25 cells (8,11,26). A cell line complementing simultaneous lack of UL31 and UL34 was generated after cotransfection of a plasmid containing a genomic 5.2-kb DrdI-fragment [nucleotides 28099-33282; GenBank accession no.…”

“…The alphaherpesvirus pUS3 kinase is required for efficient fusion of primary envelopes because, in the absence of pUS3, primary enveloped virions accumulate in the perinuclear space (25). The conserved pUL25 capsid-associated protein apparently plays a role in triggering primary envelopment of DNA-containing mature capsids at the inner nuclear membrane (26).…”