The Herpes Simplex Virus Type 1 UL6 Protein Is Essential for Cleavage and Packaging but Not for Genomic Inversion

Abstract

The HSV-1 genome is composed of two unique regions (UL and US) flanked by inverted repeats. During the course of DNA replication the two unique regions UL and US invert relative to one another. In this report we present evidence that cleavage is not necessary for genomic inversion to occur. We isolated and characterized a UL6::lacZ insertion mutant (hr74) that produces wild-type levels of replicating viral DNA but fails to cleave and package DNA. We demonstrate that this virus is still able to undergo genomic inversion. Furthermore we confirm that replicating DNA from cells infected with wild-type virus contains specific UL terminal but not US termini, whereas cells infected with the mutant hr74 do not contain either US or UL termini. This demonstrates that the specific UL ends found in replicating DNA are the result of the cleavage/packaging process.

DNA Cleavage and Packaging Proteins Encoded by Genes U L 28, U L 15, and U L 33 of Herpes Simplex Virus Type 1 Form a Complex in Infected Cells

“…Six genes of herpes simplex virus type 1 (HSV-1) are known to be essential for this process, namely, U L 6, U L 15, U L 17, U L 28, U L 32, and U L 33. In cells infected with viruses individually lacking these genes, capsids appear normal and are readily detected, but viral DNA is neither cleaved nor packaged (2,3,5,9,12,25,26,30,31,35,37,(40)(41)(42)44).The cleavage and packaging process of herpesviruses is believed to be similar to that employed by double-stranded DNA bacteriophages; consequently, it is useful to consider the mechanisms used by these bacteriophages as a model for the cleavage and packaging events employed by herpesviruses. Such models propose a central role for the viral "terminase," a complex of at least two proteins that (i) binds the viral DNA and links it with the empty viral capsid (HSV-1) or prohead (bacteriophages); (ii) cleaves the viral DNA at precise internal sites, resulting in the separation of unit-length genomes from concatameric DNA; and (iii) hydrolyzes ATP, providing the energy required to drive the DNA into the capsid (reviewed in reference 10).…”

Implication of the product of the bovine herpesvirus type 1 UL25 gene in capsid assembly

“…During the replication process, cleavage and packaging of viral DNA are tightly linked functions at least requiring the following seven genes in HSV-1: UL6, UL15, UL17, UL25, UL28, UL32 and UL33 (Poon & Roizman, 1993;Taus et al, 1998;McNab et al, 1998; Tengelsen et al, 1993;Addison et al, 1990;Lamberti & Weller, 1996al-Kobaisi et al, 1991). Viruses mutated in one of these genes synthesize near-wild-type levels of viral DNA but fail to cleave and package concatemeric DNA into capsids, resulting in an accumulation of B (intermediate) capsids in infected cells, with the exception of the UL25 mutant, which was shown to cleave newly synthesized viral DNA (McNab et al, 1998).…”

Disulfide Bond Formation in the Herpes Simplex Virus 1 UL6 Protein Is Required for Portal Ring Formation and Genome Encapsidation

“…The UL6 protein plays multiple roles during viral assembly and encapsidation, including ring formation, interaction with scaffold and VP5 to nucleate the assembly of the procapsid shell, the docking of the terminase, and acting as a conduit for the uptake of viral DNA into capsids (8,9,20,25,26,28,31,32,40,42,46). It also is likely that the UL6 portal plays a role during subsequent infections.…”

“…The KOS strain of herpes simplex virus 1 (HSV-1) was used as the wild-type (WT) virus and as the parental strain for the generation of recombinant viruses C166A and C254A. The UL6 null virus, hr74, contains an insertion of the E. coli lacZ gene under the control of the HSV-1 ICP6 promoter and was described previously (20). African green monkey kidney fibroblast cells (Vero) were obtained from the ATCC and used to propagate the WT type virus.…”

“…African green monkey kidney fibroblast cells (Vero) were obtained from the ATCC and used to propagate the WT type virus. The UL6 complementing cell line, UL6-31 (20,25), was used to propagate hr74, C166A, and C254A recombinant viruses. Jay C. Brown (University of Virginia Health System) provided anti-UL6 monoclonal antibodies 1C9 and 4G9 (7).…”