A Null Mutation in the UL36 Gene of Herpes Simplex Virus Type 1 Results in Accumulation of Unenveloped DNA-Filled Capsids in the Cytoplasm of Infected Cells

Abstract

The UL36 open reading frame (ORF) encodes the largest herpes simplex virus type 1 (HSV-1) protein, a 270-kDa polypeptide designated VP1/2, which is also a component of the virion tegument. A null mutation was generated in the UL36 gene to elucidate its role in the virus life cycle. Since the UL36 gene specifies an essential function, complementing cell lines transformed for sequences encoding the UL36 ORF were made. A mutant virus, designated K⌬UL36, that encodes a null mutation in the UL36 gene was isolated and propagated in these cell lines. When noncomplementing cells infected with K⌬UL36 were analyzed, both terminal genomic DNA fragments and DNA-containing capsids (C capsids) were detected; therefore, UL36 is not required for cleavage or packaging of DNA. Sedimentation analysis of lysates from mutant-infected cells revealed the presence of particles that have the physical characteristics of C capsids. In agreement with this, polypeptide profiles of the mutant particles revealed an absence of the major envelope and tegument components. Ultrastructural analysis revealed the presence of numerous unenveloped DNA containing capsids in the cytoplasm of K⌬UL36-infected cells. The UL36 mutant particles were tagged with the VP26-green fluorescent protein marker, and their movement was monitored in living cells. In K⌬UL36-infected cells, extensive particulate fluorescence corresponding to the capsid particles was observed throughout the cytosol. Accumulation of fluorescence at the plasma membrane which indicated maturation and egress of virions was observed in wild-type-infected cells but was absent in K⌬UL36-infected cells. In the absence of UL36 function, DNA-filled capsids are produced; these capsids enter the cytosol after traversing the nuclear envelope and do not mature into enveloped virus. The maturation and egress of the UL36 mutant particles are abrogated, possibly due to a late function of this complex polypeptide, i.e., to target capsids to the correct maturation pathway.The herpes simplex virus type 1 (HSV-1) virion is comprised of four structural elements: a DNA-containing core; an icosahedral capsid, which encloses the genome; a layer that immediately surrounds the capsid termed the tegument; and an outer membrane or envelope, which encloses the whole structure and in which are embedded the viral glycoproteins (39, 55; reviewed in references 40 and 47). The tegument represents the most diverse structural element of the virus particle in terms of both polypeptide composition and functions.Virus-specified polypeptides that comprise the tegument structure include those that function to activate transcription, shut off host protein synthesis, and uncoat the virus genome, as well as others whose functions are not yet known (reviewed in references 40 and 47). The role of tegument is twofold. First, the tegument can be envisioned as a structure that delivers factors into the cytosol of the infected cell to facilitate the initiation of a successful infection. Components of the tegument that mediate this process...

Determination of Interactions between Tegument Proteins of Herpes Simplex Virus Type 1

“…First, the interaction of UL36 and UL37 appears to have a definite structural role. The absence of either protein in HSV-1 or PRV significantly impairs or blocks further addition of tegument and subsequent viral maturation (2,3,20,21). This fits with their apparent location in the virion, i.e., inner tegument adjacent to the capsid.…”

Functional Characterization of Kaposi's Sarcoma-Associated Herpesvirus ORF45 byBacterial Artificial Chromosome-Based Mutagenesis

“…Tegument proteins are known to be involved in various steps of viral assembly and egress progress. Disruption of the major tegument protein of HSV-1 UL36, which is conserved among all herpesviruses, resulted in accumulation of unenveloped DNA-filled capsids in the cytoplasm (8). Null mutation of HSV-1 UL48 (VP16) interferes with viral egress downstream of the primary envelopment, presumably by impairing virus assembly in the cytoplasm (28).…”

Dynamic ubiquitination drives herpesvirus neuroinvasion

“…Upon virus entry into a cell, pUL36 becomes exposed to the cytosol (4,13,14) and directs delivery of the capsid and its DNA content to the nucleus (2,(15)(16)(17)(18)(19). Following replication, pUL36 is also essential for viral assembly and egress (20)(21)(22).…”