Herpesviral replication compartments move and PNAS PLUS coalesce at nuclear speckles to enhance export of viral late mRNA Lynne Changa, William J. Godinezb, Il-Han Kimb, Marco Tektonidisb, Primal de Lanerollec, Roland Eilsb, Karl Rohrb, and David M. Knipea,1 aDepartment of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115; bDepartment of Bioinformatics and Functional Genomics, Biomedical Computer Vision Group, BIOQUANT, Institute of Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg and German Cancer Research Center (DKFZ) Heidelberg, 69120 Heidelberg, Germany; and cDepartment of Physiology and Biophysics, College of Medicine, University of Illinois, Chicago, IL 60612 Edited* by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved April 11, 2011 (received for review March 1, 2011) The role of the intranuclear movement of chromatin in gene Viruses are simple genetic entities and have often provided expression is not well-understood. Herpes simplex virus forms precise probes of cellular molecular mechanisms. Viral chro- replication compartments (RCs) in infected cell nuclei as sites of mosomes of herpes simplex virus (HSV) are replicated and un- viral DNA replication and late gene transcription. These structures dergo late transcription in intranuclear structures called replica- develop from small compartments that grow in size, move, and tion compartments (RCs) (15), which are known to move and coalesce. Quantitative analysis of RC trajectories, derived from 4D coalesce in nuclei of infected cells during the course of infection images, shows that most RCs move by directed motion. Directed (16, 17). The function and mechanism of RC movement are movement is impaired in the presence of actin and myosin inhibitors unknown. However, lepidopteran nucleopolyhedroviruses, large as well as a transcription inhibitor. In addition, RCs coalesce at and double-stranded DNA viruses that also replicate inside the host reorganize nuclear speckles. Lastly, distinct effects of actin and nucleus, have been shown to manipulate nuclear G- and F-actin myosin inhibitors on viral gene expression suggest that RC move- for viral gene expression and progeny production (reviewed in ment is not required for transcription, but rather, movement results ref. 18). Pseudorabies virus and HSV have also been shown MICROBIOLOGY in the bridging of transcriptionally active RCs with nuclear speckles to induce F-actin formation in nuclei of certain cells, and viral to form structures that enhance export of viral late mRNAs. capsids seem to be associated with these structures (19, 20). Other studies have shown that HSV capsids undergo directed gene movement | nuclear export movement inside the nucleus (21), which is inhibited by the actin polymerization inhibitor latrunculin A (lat A) and the putative ovement of chromatin and its interactions and location in myosin inhibitor 2,3-butanedione monoxime (BDM) (22, 23). Mthe nucleus are believed to be important for regulation of Based on the increasing evidence for the role of intranuclear gene expression (1). A number of studies have shown an asso- movement in gene regulation and its involvement during viral ciation between the movement of chromosomal domains and infection, we were interested in the possible roles for movement changes in transcriptional activity. In yeast, activation of genes is in HSV-1 gene expression. associated with targeting of the gene to the nuclear periphery (2, HSV-1 transcription, DNA synthesis, and capsid assembly all 3), and this is believed to be because of the linkage of actively occur in the nucleus and are temporally regulated by a cascade of transcribed genes to the nuclear pore complex for RNA export immediate-early (IE), early (E), and late (L) gene expression. As (4). Lymphoid cell genes move to positions near centromeric mentioned above, these processes are also spatially regulated in the form of intranuclear compartmentalization (24). RCs, which heterochromatin coincident with transcriptional silencing (5). fi fi Transcriptional activation of the β-globin locus involves move- were rst identi ed by the presence of infected cell protein (ICP) ment of the gene away from centromeric heterochromatin (6). 8 (an HSV single-stranded DNA binding protein) (15), develop During B-cell development, the IgH and Igκ loci are localized at from small structures that grow in size, move, and coalesce, ul- timately filling the entire nucleus and marginalizing host chro- the nuclear periphery in hematopoietic progenitor cells, but they – move internally coincident with transcriptional activation in pro- matin to the nuclear periphery (17, 25 27). RCs have also been B cells (7). Similarly, the β-globin gene relocalizes to the nuclear shown to be transcriptionally active by the presence of a viral interior as it is activated during murine erythroid differentiation transactivator protein ICP4 (28, 29) and RNA pol II (30) that (8). Actively transcribed genes are also thought to be brought into colocalize with ICP8. To better understand the mechanism and function of intranuclear movement of RCs, we carried out 4D close contact with nuclear speckles (9, 10), sites often regarded imaging of cells infected with a recombinant HSV-1 strain as hubs of RNA metabolism. Although these results link gene expressing ICP8 fused to GFP to visualize RCs. Our results movement with changes in transcriptional activity, it is unclear showed that the majority of RCs move by directed motion and whether intranuclear movement of chromatin is the cause or require nuclear actin, myosin, and ongoing transcription. RC result of changes in transcriptional activity (1). Therefore, it is important to define systems in which the function of chromatin movement can be experimentally assessed. Nuclear forms of actin and myosin have been implicated – Author contributions: L.C., W.J.G., R.E., K.R., and D.M.K. designed research; L.C., W.J.G., in mediating long-range directed movement (11 13), but the I.-H.K., and M.T. performed research; L.C., W.J.G., P.d.L., R.E., K.R., and D.M.K. contributed mechanism by which this putative nucleoskeletal system operates new reagents/analytic tools; L.C., W.J.G., K.R., and D.M.K. analyzed data; and L.C., W.J.G., is unknown. In addition to their possible roles in mediating in- P.d.L., K.R., and D.M.K. wrote the paper. tranuclear movement, there is increasing evidence for roles of The authors declare no conflict of interest. nuclear actin and myosin in cellular transcription, chromatin *This Direct Submission article had a prearranged editor. remodeling, and mRNA export (reviewed in ref. 14). How and 1To whom correspondence should be addressed. E-mail: [email protected]. whether their roles in movement are connected to regulation of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. gene activity are unclear. 1073/pnas.1103411108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1103411108 PNAS Early Edition | 1of9 Downloaded by guest on October 2, 2021 movement resulted in coalescence at nuclear speckles. In- for α often did not characterize this type of motion accurately. terestingly, inhibition of directed movement by lat A led to Because the positions representing a directed motion trajectory decreased accumulation of viral gene transcripts, whereas in- exhibit a highly anisotropic scatter, a more effective way to detect hibition by BDM resulted in a defect in export of a late viral this type of motion was to characterize the shape of the trajectory mRNA. Combined, our data suggest that HSV RC movement by its 3D relative shape anisotropy κ2 (32) (Fig. 2A shows sample results in bridging transcriptional domains within RCs to RNA RC trajectories, their ellipsoid of gyration, and κ2 values, which processing structures in the nucleus to enhance export of a subset are defined as a function of the squared lengths of the semiaxes of late viral mRNAs. of the ellipsoid). Our hierarchical approach for determining the motion type of the RCs, therefore, first used κ2 to distinguish Results trajectories exhibiting directed motion from those displaying RCs Move by an Active Process. To better understand the process random motion. Trajectories with isotropic shapes were then of RC movement in the context of the 3D nucleus, we carried additionally classified into confined diffusion, obstructed diffu- out 4D (3D space and time) confocal imaging of cells infected sion, or simple diffusion based on their α-values and the classi- with a recombinant HSV-1 strain expressing ICP8 fused to GFP fication scheme by Bacher et al. (34) (Fig. 2B shows sample MSD (8GFP virus) (17). Cells were imaged from 4 to 7 h postinfection curves that correspond to trajectories shown in Fig. 2A). Based (hpi). ICP8-GFP localized to bright foci clustered within RCs. on this hierarchical analysis approach, 74% of RCs appeared to Smaller and dimmer individual foci, most likely representing undergo directed motion, 13% displayed simple diffusion, and viral prereplicative sites (15), were also present along the nuclear 13% displayed obstructed diffusion (control; n = 5 nuclei, 23 periphery and in the nucleoplasmic space between RCs (Fig. 1). RCs analyzed) (Fig. 2C). None of the RCs displayed confined These smaller individual foci exhibited highly constrained move- diffusion. These results showed that the majority of the RCs ment, whereas whole RCs displayed long-range movement, often to another RC (Fig. 1 and Movie S1). When the movement of moved by an active process. RCs was tracked relative to the position of the nucleus in the 4D Active Movement of RCs Depends on Nuclear Actin, Myosin I, and datasets, these structures moved at an average speed of 0.24 μm/ Transcription. Active movement of transgenes has been reported min (up to 0.35 μm/min; SD = 0.056 μm/min). RCs traveled to involve nuclear actin and nuclear myosins such as nuclear distances of up to 5.65 μm during the 3-h imaging period (as myosin I (NMI) (11–13).