Characterization of nuclear structures in cells infected with herpes simplex virus type 1 in the absence of viral DNA replication

Abstract

Herpes simplex virus type 1 DNA replication occurs in nuclear domains termed replication compartments, which are areas of viral single-stranded DNA-binding protein (UL29) localization (M. P. Quinlan, L. B. Chen, and D. M. Knipe, Cell 36:857-868, 1984). In the presence of herpesvirus-specific polymerase inhibitors, UL29 localizes to punctate nuclear foci called prereplicative sites. Using versions of the helicase-primase complex proteins containing short peptide epitopes which can be detected in an immunofluorescence assay, we have found that the helicase-primase complex localizes to prereplicative sites and replication compartments. To determine if prereplicative site formation is dependent upon these and other essential viral replication proteins, we have studied UL29 localization in cells infected with replication-defective viruses. Cells infected with viruses that fail to express one of the three helicase-primase subunits or the origin-binding protein show a diffuse nuclear staining for UL29. However, in the presence of polymerase inhibitors, mutant-infected cells contain UL29 in prereplicative sites. Replication-defective viruses containing subtle mutations in the helicase or origin-binding proteins behaved identically to their null mutant counterparts. In contrast, cells infected with viral mutants which fail to express the polymerase protein contain prereplicative sites in the absence and presence of polymerase inhibitors. We propose that active viral polymerase prevents the formation of prereplicative sites. Models of the requirement of essential viral replication proteins in the assembly of prereplicative sites are presented.on July 10, 2020 by guest http://jvi.asm.org/ Downloaded from FIG. 3. UL29 localization in cells infected with viral mutants that fail to express UL5, UL8, UL52, or UL9. Vero cells were infected with 20 PFU of virus per cell and processed as described in Materials and Methods. (A) Cells were stained with 3-83 and fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody. Cells were infected with the following mutants: panels A, B, and C, hr80; D, E, and F, hr99; G, H, and I, hr114. Infected cells in panels B, E, and H were treated with PAA, and those in panels C, F, and I were treated with ACG. Marker bar, 15 m. (B) hr94-infected cell doubly stained with antibodies (␣) for UL29 and BrdU. Panels A and B show the same group of hr94-infected cells doubly stained, respectively, with 3-83 (fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody) and anti-BrdU (Texas red-conjugated goat anti-mouse secondary antibody). Marker bar, 15 m.

Herpes simplex virus 1 ICP8 mutant lacking annealing activity is deficient for viral DNA replication

“…HSV-1 DNA replication occurs within membraneless subdomains in the nucleus known as RCs (50). RC formation and maturation is believed to be dependent on active DNA synthesis, as the assembly of large mature RCs is blocked by addition of the viral polymerase inhibitor phosphonoacetic acid or by omission of any of the seven essential viral replication proteins during infection (33,(50)(51)(52). Because the QF mutant is unable to complement HD-2 for DNA replication, we asked whether RCs could be detected in Vero cells transfected with plasmids expressing WT or QF mutant protein and superinfected with HD-2.…”

Comparison of the Intranuclear Distributions of Herpes Simplex Virus Proteins Involved in Various Viral Functions

“…Viral DNA was specifically localized to these structures by in situ hybridization (Randall and Dinwoodie, 1986;Knipe, 1990;. In addition to the single-stranded DNA-binding protein, ICP8, the other six essential viral DNA replication proteins, the viral DNA polymerase (Goodrich et al, 1990;Bush et al, 1991), the UL9 origin-binding protein (Olivo et al, 1989), and the helicase-primase complex proteins, UL5, UL8, and UL52 (Olivo et al, 1989;Liptak et al, 1996;Lukonis and Weller, 1996), colocalize within replication compartments. Although their roles in viral replication have not been defined, components of the cell cycle (pRb and p53) and DNA replication (RP-A, PCNA, DNA ligase, and pol␣) machinery are recruited to replication compartments as well (Wilcock and Lane, 1991).…”

Human cytomegalovirus proteins PP65 and IEP72 are targeted to distinct compartments in nuclei and nuclear matrices of infected human embryo fibroblasts

“…Nuclear organization of the host cell plays an important role in the host-virus challenge, particularly for viruses such as herpesviruses, whose replicative cycle takes place in this cellular compartment [Lukonis and Weller, 1996;Burkham et al, 1998Burkham et al, , 2001Monier et al, 2000;Reynolds et al, 2001;Hutchinson et al, 2002;Sourvinos and Everett, 2002]. Since cytomegalovirus can establish different relationships with the host cell, resulting in a lytic cycle or a latent infection, it is likely that this virus has developed different kinds of strategies to exploit the same pathways and nuclear regulatory apparatus, as the cell does in physiological conditions, and/or to modify them to its own benefit.…”