Mutational Analysis of the Herpes Simplex Virus Type 1 UL25 DNA Packaging Protein Reveals Regions That Are Important after the Viral DNA Has Been Packaged

Abstract

The herpes simplex virus type 1 (HSV-1) UL25 gene encodes a minor capsid protein, pUL25, that is essential for packaging the full-length viral genome. Six regions which contain disordered residues have been identified in the high-resolution structure of pUL25. To investigate the significance of these flexible regions, a panel of plasmids was generated encoding mutant proteins, with each member lacking the disordered residues in one of the six regions. In addition, UL25 constructs were produced, which specified proteins that contained missense mutations individually affecting two of the four regions on the surface of pUL25 predicted from evolutionary trace analysis to be important in protein-protein interactions. The impacts of these mutations on viral DNA packaging, virus assembly, and growth were examined. Of the nine mutant proteins analyzed, five failed to complement the growth of a UL25 deletion mutant in Vero cells. These noncomplementing proteins fell into three classes. Proteins in one class did not alter the DNA packaging phenotype of an HSV-1 UL25 deletion mutant, whereas proteins from the other two classes allowed the UL25 null mutant to package full-length viral DNA. Subsequent analysis of the latter classes of mutant proteins demonstrated that one class enabled the null virus to release enveloped virus particles from U2OS cells, whereas the other class prevented egress of mature HSV-1 capsids from the nucleus. These findings reveal a new role for pUL25 in virion assembly, consistent with its flexible structure and location on the capsid.Herpes simplex virus type 1 (HSV-1) has a large, complex virion structure that is composed of four morphologically distinct components. The central core comprises the linear double-stranded DNA genome, which is compactly organized inside an icosahedral capsid containing four different capsid shell proteins. The pUL6 DNA packaging protein forms the portal at the unique vertex. Two additional DNA packaging proteins, pUL25 and pUL17, are found on the capsid, and these proteins are present as heterodimers at multiple sites on the surface, adjacent to the vertices (20,33,35). A protein matrix containing more than 20 different proteins, referred to as the tegument, surrounds the capsid (15). One of the tegument proteins, pUL36, interacts with pUL25 and probably with VP5, the major capsid shell protein (5,19,23), and these associations are likely to be important for anchoring the tegument to the capsid (5). Finally, the outermost layer consists of a lipid membrane embedded with the viral glycoproteins.Viral DNA synthesis and the early stages of virion assembly take place in the nucleus, within replication compartments (8,25). The viral DNA is replicated as head-to-tail concatemers, and cleavage of the viral DNA into unit-length molecules is tightly associated with the packaging of the DNA into a preformed precursor capsid, the procapsid, through the portal. After the viral DNA is encapsidated, the mature C capsid rapidly buds into the inner nuclear membrane and is released i...

Nuclear Egress of Pseudorabies Virus Capsids Is Enhanced by a Subspecies of the Large Tegument Protein That Is Lost upon Cytoplasmic Maturation

“…In the case of viruses deleted for the genes encoding pUL25, pUL31, and pUL34, which are critical for the nuclear egress of capsids, the defect is immediately evident (23,33,35,54,64,66). However, the defect observed in the absence of VP1/2 is not absolute (43).…”

“…Like adenovirus and SV40, herpesviruses encode proteins critical for nuclear egress. These include a capsid protein, pUL25, and a nuclear membrane complex consisting of the viral proteins pUL31 and pUL34 (35,54,63,66). How these proteins coordinate nuclear egress of capsids is also poorly understood.…”

A Physical Link between the Pseudorabies Virus Capsid and the Nuclear Egress Complex

“…With regard to the egress trigger, the finding that pUL25 functions in nuclear egress in addition to its role in C capsid stabilization has placed pUL25 at center stage in current models of egress (54). However, it may be of interest that one of the earliest-described roles of pUL31 is in DNA cleavage and encapsidation of HSV-1, and support for this concept was more recently obtained with MCMV (14,61).…”

“…The interaction between pUL33 and pUL31 was identified in all three subfamilies of the herpesviridae, while the pUL25-pUL31 interaction appeared to be unique to the alphaherpesvirinae (22,86). Both pUL25 and pUL33 are attractive candidates for links between the NEC and capsids, as both are required for capsid nuclear egress and both are capsid-associated proteins (7,35,54). pUL25 is a nonintegral capsid component that contributes to a structure variously referred to as the CCSC for its enrichment on C capsids and, more recently, as the capsid vertex-specific component (CVSC), because the structure was also identified on A and B capsids and is consistently observed around the icosahedral vertices (82,85).…”

“…We therefore instead took a targeted approach and ranked candidate capsid proteins based on their potential roles in recruiting pUL31. The minor capsid protein pUL25 was our top candidate because (i) pUL25 was identified as a pUL31-binding partner in a yeast two-hybrid screen (86); (ii) pUL25 is enriched on C capsids, which are targeted for nuclear egress (18,31,50,85); and (iii) pUL25 is required for nuclear egress independent of its role in genome encapsidation (54).…”