ND10 components relocate to sites associated with herpes simplex virus type 1 nucleoprotein complexes during virus infection

Infections with DNA viruses commonly result in the association of viral genomes and replication compartments with cellular nuclear substructures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. While there is evidence that viral genomes can associate with preexisting ND10, we demonstrate in this study by live-cell microscopy that structures resembling ND10 form de novo and in association with viral genome complexes during the initial stages of herpes simplex virus type 1 (HSV-1) infection. Consistent with previous studies, we found that the major ND10 proteins PML, Sp100, and hDaxx are exchanged very rapidly between ND10 foci and the surrounding nucleoplasm in live cells. The dynamic nature of the individual protein molecule components of ND10 provides a mechanism by which ND10 proteins can be recruited to novel sites during virus infection. These observations explain why the genomes and replication compartments of DNA viruses that replicate in the cell nucleus are so commonly found in association with ND10. These findings are discussed with reference to the nature, location, and potential number of HSV-1 prereplication compartments and to the dynamic aspects of HSV-1 genomes and viral products during the early stages of lytic infection.     

Formation of herpes simplex virus type 1 replication compartments by transfection: requirements and localization to nuclear domain 10.

During infection, the seven essential herpes simplex virus type 1 (HSV-1) replication proteins are found in globular nuclear structures called replication compartments. Replication compartments form adjacent to ND10, nuclear matrix-bound domains which are present in most cell types but whose function is unknown (G. G. Maul, I. M. Ishov, and R. D. Everett, Virology 217:67-75, 1996). We now demonstrate that replication compartments can be formed by cotransfecting Vero cells with constructs expressing the seven essential viral replication proteins and a plasmid containing an HSV-1 origin of DNA replication. Like replication compartments in infected cells, replication compartments formed by cotransfection contain all of the essential viral replication proteins, are sites of DNA synthesis, and are found adjacent to ND10. However, neither the viral origin-binding protein nor a plasmid containing an HSV-1 origin of DNA replication is individually required for the formation of transfection replication compartments, although the presence of each increases the efficiency of replication compartment formation. Further, we provide evidence that UL29 independently localizes adjacent to ND10 and so may play a role in directing replication compartments to these preexisting nuclear structures.     

Interactions of herpes simplex virus type 1 with ND10 and recruitment of PML to replication compartments

Many of the events required for productive herpes simplex virus type 1 (HSV-1) infection occur within globular nuclear domains called replication compartments, whose formation appears to depend on interactions with cellular nuclear domains 10 (ND10). We have previously demonstrated that the formation of HSV-1 replication compartments involves progression through several stages, including the disruption of intact ND10 (stage I to stage II) and the formation of PML-associated prereplicative sites (stage III) and replication compartments (stage IV) (J. Burkham, D. M. Coen, and S. K. Weller, J. Virol. 72:10100-10107, 1998). In this paper, we show that some, but not all, PML isoforms are recruited to stage III foci and replication compartments. Genetic experiments showed that the recruitment of PML isoforms to stage III prereplicative sites and replication compartments requires the localization of the HSV-1 polymerase protein (UL30) to these foci but does not require polymerase catalytic activity. We also examined the stages of viral infection under conditions affecting ND10 integrity. Treatment with factors that increase the stability of ND10, arsenic trioxide and the proteasome inhibitor MG132, inhibited viral disruption of ND10, formation of replication compartments, and production of progeny virus. These results strengthen the previously described correlation between ND10 disruption and productive viral infection.     

Oligomerization of ICP4 and rearrangement of heat shock proteins may be important for herpes simplex virus type 1 prereplicative site formation

Herpes simplex virus type 1 (HSV-1) DNA replication occurs in replication compartments that form in the nucleus by an ordered process involving a series of protein scaffold intermediates. Following entry of viral genomes into the nucleus, nucleoprotein complexes containing ICP4 can be detected at a position adjacent to nuclear domain 10 (ND10)-like bodies. ND10s are then disrupted by the viral E3 ubiquitin ligase ICP0. We have previously reported that after the dissociation of ND10-like bodies, ICP8 could be observed in a diffuse staining pattern; however, using more sensitive staining methods, we now report that in addition to diffuse staining, ICP8 can be detected in tiny foci adjacent to ICP4 foci. ICP8 microfoci contain UL9 and components of the helicase-primase complex. HSV infection also results in the reorganization of the heat shock cognate protein 70 (Hsc70) and the 20S proteasome into virus-induced chaperone-enriched (VICE) domains. In this report we show that VICE domains are distinct but adjacent to the ICP4 nucleoprotein complexes and the ICP8 microfoci. In cells infected with an ICP4 mutant virus encoding a mutant protein that cannot oligomerize on DNA, ICP8 microfoci are not detected; however, VICE domains could still be formed. These results suggest that oligomerization of ICP4 on viral DNA may be essential for the formation of ICP8 microfoci but not for the reorganization of host cell chaperones into VICE domains.     

Spatial and temporal organization of adeno-associated virus DNA replication in live cells

Upon cell entry, the genomes of herpes simplex virus type 1 (HSV-1) and adenovirus (Ad) associate with distinct nuclear structures termed ND10 or promyelocytic leukemia (PML) nuclear bodies (NBs). PML NB morphology is altered or disrupted by specific viral proteins as replication proceeds. We examined whether adeno-associated virus (AAV) replication compartments also associate with PML NBs, and whether modification or disruption of these by HSV-1 or Ad, both of which are helper viruses for AAV, is necessary at all. Furthermore, to add a fourth dimension to our present view of AAV replication, we established an assay that allows visualization of AAV replication in live cells. A recombinant AAV containing 40 lac repressor binding sites between the AAV inverted terminal repeats was constructed. AAV Rep protein and helper virus-mediated replication of this recombinant AAV genome was visualized by binding of enhanced yellow fluorescent protein-lac repressor fusion protein to double-stranded AAV replication intermediates. We demonstrate in live cells that AAV DNA replication occurs in compartments which colocalize with AAV Rep. Early after infection, the replication compartments were small and varied in numbers from 2 to more than 40 per cell nucleus. Within 4 to 8 h, individual small replication compartments expanded and fused to larger structures which filled out much of the cell nucleus. We also show that AAV replication compartments can associate with modified PML NBs in Ad-infected cells. In wild-type HSV-1-infected cells, AAV replication compartments and PML NBs did not coexist, presumably because PML was completely disrupted by the HSV-1 ICP0 protein. However, alteration or disruption of PML appears not to be a prerequisite for AAV replication, as the formation of replication compartments was normal when the ICP0 mutants HSV-1 dl1403 and HSV-1 FXE, which do not affect PML NBs, were used as the helper viruses; under these conditions, AAV replication compartments did not associate with PML NBs.     

Interaction of Herpes Simplex Virus ICP0 with ND10 Bodies: a Sequential Process of Adhesion,Fusion, and Retention

On entry into the nucleus, herpes simplex virus 1 (HSV-1) DNA localizes to nuclear bodies known as ND10. Gene repression imposed by ND10 is released by a viral protein, ICP0, via degradation of the ND10 constituents promyelocytic leukemia protein (PML) and Sp100 and the subsequent dispersal of ND10 bodies. In order to understand the dynamic interaction between ICP0 and ND10, we carried out deletion mapping to identify the domains of ICP0 responsible for its association with ND10. Here, we report the following. (i) An ND10 entry signal (ND10-ES), located between residues 245 and 474, is required for ICP0 to penetrate and fuse with ND10. ICP0 lacking ND10-ES adheres to the surface of ND10 but fails to enter. (ii) In the absence of ND10-ES, the E3 ubiquitin ligase of ICP0 facilitates the transient adhesion of the truncated ICP0 to the ND10 surface, whereas the presence of ND10-ES in ICP0 renders ND10 fusion regardless of the E3 ligase activity. (iii) The C terminus of ICP0 is required for retention of ICP0 in ND10 but plays no role in the recruitment process. (iv) The adverse effects of an inactive RING domain on viral replication are partially reversed by deleting either ND10-ES or the C-terminal retention domain, suggesting that additional ICP0 functions require the release of ICP0 from ND10. Based on these results, we conclude that association of ICP0 and ND10 is a dynamic process, in which three sequential steps—adhesion, fusion, and retention—are adopted to stabilize the interaction. A faithful execution of these steps defines the ultimate productivity of the virus.     

SUMO pathway dependent recruitment of cellular repressors to herpes simplex virus type 1 genomes

Components of promyelocytic leukaemia (PML) nuclear bodies (ND10) are recruited to sites associated with herpes simplex virus type 1 (HSV-1) genomes soon after they enter the nucleus. This cellular response is linked to intrinsic antiviral resistance and is counteracted by viral regulatory protein ICP0. We report that the SUMO interaction motifs of PML, Sp100 and hDaxx are required for recruitment of these repressive proteins to HSV-1 induced foci, which also contain SUMO conjugates and PIAS2β, a SUMO E3 ligase. SUMO modification of PML and elements of its tripartite motif (TRIM) are also required for recruitment in cells lacking endogenous PML. Mutants of PML isoform I and hDaxx that are not recruited to virus induced foci are unable to reproduce the repression of ICP0 null mutant HSV-1 infection mediated by their wild type counterparts. We conclude that recruitment of ND10 components to sites associated with HSV-1 genomes reflects a cellular defence against invading pathogen DNA that is regulated through the SUMO modification pathway.     

H-1 parvovirus-associated replication bodies: a distinct virus-induced nuclear structure

We have identified a nuclear structure that is induced after infection with the autonomous parvovirus H-1. Using fluorescence microscopy, we observed that the major nonstructural protein (NS1) of H-1 virus which is essential for viral DNA amplification colocalized with virus-specific DNA sequences and sites of ongoing viral DNA replication in distinct nuclear bodies which we designated H-1 parvovirus-associated replication bodies (H-1 PAR-bodies). In addition, two cellular proteins were shown to accumulate in H1 PAR-bodies: (i) the proliferating cell nuclear antigen (PCNA) which is essential for chromosomal and parvoviral replication and (ii) the NS1-interacting small glutamine-rich TPR-containing protein (SGT), suggesting a role for the latter in parvoviral replication and/or gene expression. Since many DNA viruses target preexisting nuclear structures, known as PML-bodies, for viral replication and gene expression, we have determined the localization of H-1 PAR- and PML-bodies by double-fluorescence labeling and confocal microscopy and found them to be spatially unrelated. Furthermore, H-1 PAR-bodies did not colocalize with other prominent nuclear structures such as nucleoli, coiled bodies, and speckled domains. Electron microscopy analysis revealed that NS1, as detected by indirect immunogold labeling, was localized in ring-shaped electron-dense nuclear structures corresponding in size and frequency to H-1 PAR-bodies. These structures were also clearly visible without immunogold labeling and could be detected only in infected cells. Our results suggest that H-1 virus does not target known nuclear bodies for DNA replication but rather induces the formation of a novel structure in the nucleus of infected cells.     

Recruitment of cellular recombination and repair proteins to sites of herpes simplex virus type 1 DNA replication is dependent on the composition of viral proteins within prereplicative sites and correlates with the induction of the DNA damage response

Herpes simplex virus type 1 (HSV-1) DNA replication is associated with nuclear domains called ND10, which contain host recombination proteins such as RPA, RAD51, and NBS1 and participate in the cell's response to DNA damage. The stages of HSV-1 infection have been described previously. Infected cells at stage IIIa are observed after the initial disruption of ND10 and display nuclear foci, or prereplicative sites, containing the viral single-stranded-DNA-binding protein (UL29), the origin-binding protein (UL9), and the heterotrimeric helicase-primase. At stage IIIb, the viral polymerase, its processivity factor, and the ND10, protein PML, are also recruited to these sites. In this work, RPA, RAD51, and NBS1 were observed predominantly in stage IIIb but not stage IIIa prereplicative sites, suggesting that the efficient recruitment of these recombination proteins is dependent on the presence of the viral polymerase and other replication proteins within these sites. On the other hand, Ku86 was not found in any of the precursors to replication compartments, suggesting that it is excluded from the early stages of HSV-1 replication. Western blot analysis showed that RPA and NBS1 were (hyper)phosphorylated during infection, indicating that infection induces the host response to DNA damage. Finally, RPA, RAD51, and NBS1 were found to be associated with UL29 foci observed in transfected cells expressing UL29 and the helicase-primase heterotrimer and containing intact ND10. The ability to recruit recombination and repair proteins to various subassemblies of viral replication proteins thus appears to depend on several factors, including the presence of the viral polymerase and/or UL9 within prereplicative sites and the integrity of ND10.     

Herpes simplex virus type 1 prereplicative sites are a heterogeneous population: only a subset are likely to be precursors to replication compartments.

When herpes simplex virus type 1 (HSV-1) DNA replication is blocked by viral polymerase inhibitors, such as phosphonoacetic acid (PAA) or acyclovir (ACV), UL29 (ICP8) localizes to numerous punctate nuclear foci which are called prereplicative sites. Since this pattern can form in cells infected with mutants which are defective in UL5, UL8, UL9, or UL52 in the presence of polymerase inhibitors (C. J. Lukonis and S. K. Weller, J. Virol. 70:1751-1758, 1996; L. M. Liptak, S. L. Uprichard, and D. M. Knipe, J. Virol. 70:1759-1767, 1996), we previously proposed that it is unlikely that these numerous UL29 foci actually represent a functional subassembly of viral replication proteins that could lead to the formation of replication compartments (C. J. Lukonis and S. K. Weller, J. Virol. 70:1751-1758, 1996). In this paper, we have investigated the requirement for formation of the prereplicative site pattern by using double mutants of HSV. From the analysis of mutants lacking both UL5 and UL9, we conclude that neither viral helicase is required for the prereplicative site pattern to form as long as a polymerase inhibitor is present. From the analysis of mutants defective in both UL30 and UL5, we suggest that the prereplicative site pattern can form under conditions in which viral and/or cellular polymerases are inhibited. Furthermore, reexamination of the UL29 staining pattern in cells infected with wild-type virus in the presence of PAA reveals that at least two different UL29 staining patterns can be detected in these cells. One population of cells contains numerous (greater than 20) punctate UL29 foci which are sites of cellular DNA synthesis. In another population of cells, fewer punctate foci (less than 15) are detected, and these structures do not colocalize with sites of cellular DNA synthesis. Instead, they colocalize with PML, a component of nuclear matrix structures known as ND10. We propose that ND10-associated UL29 sites represent domains at which replication compartments form.     

PML contributes to a cellular mechanism of repression of herpes simplex virus type 1 infection that is inactivated by ICP0

Promyelocytic leukemia (PML) nuclear bodies (also known as ND10) are nuclear substructures that contain several proteins, including PML itself, Sp100, and hDaxx. PML has been implicated in many cellular processes, and ND10 are frequently associated with the replicating genomes of DNA viruses. During herpes simplex virus type 1 (HSV-1) infection, the viral regulatory protein ICP0 localizes to ND10 and induces the degradation of PML, thereby disrupting ND10 and dispersing their constituent proteins. ICP0-null mutant viruses are defective in PML degradation and ND10 disruption, and concomitantly they initiate productive infection very inefficiently. Although these data are consistent with a repressive role for PML and/or ND10 during HSV-1 infection, evidence in support of this hypothesis has been inconclusive. By use of short interfering RNA technology, we demonstrate that depletion of PML increases both gene expression and plaque formation by an ICP0-negative HSV-1 mutant, while having no effect on wild-type HSV-1. We conclude that PML contributes to a cellular antiviral repression mechanism that is countered by the activity of ICP0.     

Mutations that decrease DNA binding of the processivity factor of the herpes simplex virus DNA polymerase reduce viral yield, alter the kinetics of viral DNA replication, and decrease the fidelity of DNA replication

The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is essential for viral replication and possesses both Pol- and DNA-binding activities. Previous studies demonstrated that the substitution of alanine for each of four arginine residues, which reside on the positively charged surface of UL42, resulted in decreased DNA binding affinity and a decreased ability to synthesize long-chain DNA by the polymerase. In this study, the effects of each substitution on the production of viral progeny, viral DNA replication, and DNA replication fidelity were examined. Each substitution mutant was able to complement the replication of a UL42 null mutant in transient complementation assays and to support the replication of plasmid DNA containing herpes simplex virus type 1 (HSV-1) origin sequences in transient DNA replication assays. Mutant viruses containing each substitution and a lacZ insertion in a nonessential region of the genome were constructed and characterized. In single-cycle growth assays, the mutants produced significantly less progeny virus than the control virus containing wild-type UL42. Real-time PCR assays revealed that these UL42 mutants synthesized less viral DNA during the early phase of infection. Interestingly, during the late phase of infection, the mutant viruses synthesized larger amounts of viral DNA than the control virus. The frequencies of mutations of the virus-borne lacZ gene increased significantly in the substitution mutants compared to those observed for the control virus. These results demonstrate that the reduced DNA binding of UL42 is associated with significant effects on virus yields, viral DNA replication, and replication fidelity. Thus, a processivity factor can influence replication fidelity in mammalian cells.