title: A null mutation in the gene encoding the herpes simplex virus type 1 UL37 polypeptide abrogates virus maturation.
abstract: The tegument is an integral and essential structural component of the herpes simplex virus type 1 (HSV-1) virion. The UL37 open reading frame of HSV-1 encodes a 120-kDa virion polypeptide which is a resident of the tegument. To analyze the function of the UL37-encoded polypeptide a null mutation was generated in the gene encoding this protein. In order to propagate this mutant virus, transformed cell lines that express the UL37 gene product in trans were produced. The null mutation was transferred into the virus genome using these complementing cell lines. A mutant virus designated KDeltaUL37 was isolated based on its ability to form plaques on the complementing cell line but not on nonpermissive (noncomplementing) Vero cells. This virus was unable to grow in Vero cells; therefore, UL37 encodes an essential function of the virus. The mutant virus KDeltaUL37 produced capsids containing DNA as judged by sedimentation analysis of extracts derived from infected Vero cells. Therefore, the UL37 gene product is not required for DNA cleavage or packaging. The UL37 mutant capsids were tagged with the smallest capsid protein, VP26, fused to green fluorescent protein. This fusion protein decorates the capsid shell and consequently the location of the capsid and the virus particle can be visualized in living cells. Late in infection, KDeltaUL37 capsids were observed to accumulate at the periphery of the nucleus as judged by the concentration of fluorescence around this organelle. Fluorescence was also observed in the cytoplasm in large puncta. Fluorescence at the plasma membrane, which indicated maturation and egress of virions, was observed in wild-type-infected cells but was absent in KDeltaUL37-infected cells. Ultrastructural analysis of thin sections of infected cells revealed clusters of DNA-containing capsids in the proximity of the inner nuclear membrane. Occasionally enveloped capsids were observed between the inner and outer nuclear membranes. Clusters of unenveloped capsids were also observed in the cytoplasm of KDeltaUL37-infected cells. Enveloped virions, which were observed in the cytoplasm of wild-type-infected cells, were never detected in the cytoplasm of KDeltaUL37-infected cells. Crude cell fractionation of infected cells using detergent lysis demonstrated that two-thirds of the UL37 mutant particles were associated with the nuclear fraction, unlike wild-type particles, which were predominantly in the cytoplasmic fraction. These data suggest that in the absence of UL37, the exit of capsids from the nucleus is slowed. UL37 mutant particles can participate in the initial envelopment at the nuclear membrane, although this process may be impaired in the absence of UL37. Furthermore, the naked capsids deposited in the cytoplasm are unable to progress further in the morphogenesis pathway, which suggests that UL37 is also required for egress and reenvelopment. Therefore, the UL37 gene product plays a key role in the early stages of the maturation pathway that give rise to an infectious virion.
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. pdesai@jhmi.edu
authors.forename: P
authors.initials: P
authors.lastname: Desai
authors.forename: G L
authors.initials: GL
authors.lastname: Sexton
authors.forename: J M
authors.initials: JM
authors.lastname: McCaffery
authors.forename: S
authors.initials: S
authors.lastname: Person
mesh_terms: D000818:Animals; D002213:Capsid / Q000502:physiology; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D018259:Herpesvirus 1, Human / Q000502:physiology*; D008854:Microscopy, Electron; D009154:Mutation; D011817:Rabbits; D014709:Vero Cells; D015678:Viral Structural Proteins / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication
chemical_list: C084763:UL37 protein, Human herpesvirus 1; D015678:Viral Structural Proteins