Molecular characterization of baculovirus-induced chromatin marginalization and architectural alteration

To facilitate rapid replication and assembly of progeny, baculovirus is known to manipulate the host nuclear microenvironment by inducing chromatin changes in localization and architecture. However, the molecular mechanisms underlying these changes remain unknown. Here, we revealed that the nuclear lamina (NL) protein Lamin A/C interacts with the heterochromatin protein 1 alpha (HP1a) and identified the middle region of HP1a as critical for this interaction. Suppression of Lamin A/C and HP1a expression resulted in a significant inhibition of chromatin marginalization mediated by baculovirus infection. Moreover, the heterochromatin modification H3K9me3, which is recognized and bound by HP1a, also participated in the process of chromatin marginalization. Our live-cell imaging and quantitative analysis unveiled a passive function of marginal chromatin, which involves the formation of a physical barrier that impedes the nuclear egress of the nucleocapsids. Furthermore, baculovirus-induced nuclear F-actin altered the steady-state of intranuclear actin pool, thus regulating the nucleosome disassembly. Overall, our findings illustrate the molecular mechanisms dictating chromatin marginalization and structural alterations during baculovirus infection, shedding new light on the potential function of marginalized chromatin in the origin of its biphasic life cycle. Author Summary In our previous study, we illustrated the organization and accessibility of chromatin marginalized by baculovirus infection through a combination of ATAC-seq and biochemical assays. Here, we further dissect the molecular mechanism underlying the baculovirus infection induced chromatin marginalization and disassembly. Specifically, baculovirus utilizes the Lamin A/C-HP1a-H3K9me3 axis to mediate chromatin marginalization at the nuclear periphery. When the interaction between Lamin A/C and HP1a is disrupted, the marginalization of chromatin is also affected. Furthermore, our single-virion tracking results indicate that the marginalized chromatin forms a physical barrier, impeding the nuclear export of nucleocapsids at the very late stage of infection. For the changes in chromatin architecture, we propose a model in which baculovirus infection induced nuclear F-actin compromises the dynamics of nuclear actin pool, which in turn promotes chromatin disassembly. Taken together, we provide a comprehensive understanding of molecular mechanism of baculovirus infection induced changes in chromatin localization and organization, which lay the foundation for studies on how DNA viruses manipulate the nuclear microenvironment.


Abbreviations
The following abbreviations are used in this manuscript:  Recently, baculovirus infection induced changes in the accessibility and organization of marginalized chromatin have been characterized 4 . The data suggested that the marginalized chromatin genome-widely gains accessibility with disassembly of multi-nucleosomes. The potential roles of host nuclear lamina and heterochromatin protein 1 alpha (HP1a, also known as CBX5) in mediating the tethering of host chromatin to the nuclear periphery were discussed. NL is a dense filamentous meshwork underlying the nuclear membrane principally composed by the Lamin proteins 9 . In addition to functioning as the nucleoskeleton, Lamin proteins can also serve as a scaffold for orderly organizing the heterochromatin at the nuclear periphery through interacting with Lamin B receptor (LBR) and HP1a 10,11 . In mammals, the Lamin proteins are grouped into two classes: A-type (Lamin A/C) and B-type (Lamin B1, B2 and B3) 12 . Within the host of baculovirus, most invertebrates only possess a single Lamin gene in their genomes. Such as, the domestic Bombyx mori genome contains a Lamin A/C gene but lacks the B-type Lamin proteins 13

Characterization of the localization and interaction patterns between Lamin A/C and HP1a during viral infection
To better identify the baculovirus infection and track the single virion movement, we constructed a recombinant virus, named VP39-3mC, with vp39 gene under its native promoter and fused to 3×mCherry at its 3' terminus, which is based on a vp39 knockout bacmid preserved by our laboratory (Supplementary Fig. 1A). As a major capsid protein, VP39 mainly localizes at the VS and fused with 3×mCherry red fluorescent protein can be used to observe the motility of single virion in living cells 22 1A). As previous research reported, VP39-3mC infection also induced nuclear lamina disruption 23 (Fig. 1D). HP1a was fused with a tagBFP fluorescent protein to indicate the localization of heterochromatin and Histone H2B was fused with a GFP fluorescent protein to indicate the localization of host chromatin. During VP39-3mC infection, HP1a gradually marginalized to the nuclear periphery and exhibited same location dynamics as the host chromatin (Fig. 1A). We observed a co-localization pattern between HP1a and Lamin A/C at 48 h p.i. (Fig. 1C), whereas this pattern was absent at 0 h p.i. (Fig. 1B). Additionally, they were not co-localized at the site where the nuclear lamina was disrupted (Fig. 1D Fig. 2B), which is found to be important for the protein-protein interaction and phase separation 24,25 . To identify the region responsible for the interaction between HP1a and Lamin A/C, we constructed three HP1a truncated mutants as shown in Fig. 1G. The Co-IP results indicated that truncating the Hinge region of HP1a eliminates the interaction between Lamin A/C (Fig.   1H).
Taken together, our findings suggested that in Bombyx mori HP1a interacts with Lamin A/C by its Hinge region and this interaction may participate in the baculovirus infection induced chromatin marginalization.

Lamin A/C-HP1a interaction mediates the baculovirus infection induced chromatin marginalization
To elucidate the function of Lamin A/C-HP1a interaction, we knocked down (

Marginalized chromatin impedes the nuclear export of progeny virions
Generally, it is widely believed that viruses remodel the nuclear microenvironment to enhance their replication and proliferation. Recently, however, several researches suggested that HSV-1 infection induced marginalized chromatin physically restricted the diffusion of its capsids in the nucleus 5,28 . While HSV-1 capsids move through diffusion 29 , baculovirus capsids transport within the nucleus by utilizing nuclear Factin 22 . Given the distinct nuclear motion types and different virion morphologies between these two viruses 6, 30 , we want to reveal whether the nuclear export of baculovirus is also hindered by the marginalized chromatin. VP39-3mC was used to within the nucleus and these actin filaments disappeared at 48 h p.i. (Fig. 5A).
Subsequently, fluorescence recovery after photobleaching (FRAP) was employed to identify the dynamics of nuclear actin pool. We transiently transfected a plasmid to express wild-type actin fused with EGFP and NLS. The time of recovery for wild-type actin at 0 h p.i. was greater than at 24 h p.i. (Fig. 5B), CD treatment (Fig. 6C and 6D). Notwithstanding, we could not conclude that the different observation in chromatin compaction is mediated by nuclear F-actin, because CD could induce actin depolymerization both in the cytoplasm and the nucleus. Hence, mCherry-NLS-R62D was transiently expressed to rescue the dynamics of nuclear actin pool and inhibit the formation of nuclear actin filaments. As expected, mCherry-NLS-R62D also could prevent the chromatin decompaction during viral infection ( Fig. 6E and 6F).
To more directly confirm the role of nuclear actin in chromatin organization, MNase digestion was performed to measure the nucleosome assembly 31 . Consistent with us previous findings, baculovirus infection resulted in multi-nucleosome disassembly ( Fig. 6G and 6H). Moreover, CD treatment or expressing mCherry-NLS-R62D appeared more resistance to MNase digestion compared with DMSO or mCherry-NLS-Actin (Fig. 6I-L Lamin B-LBR-HP1a 11,34 . We found that this conserved mechanism is also exploited by baculovirus to mediate the whole genome marginalized to the nuclear lamina. The crucial step in this process is the G9a-mediated repositioning of H3K9me3, which leads to H3K9me3 localized in the nucleoplasm providing more targets for HP1a recognition.
Recently, a study has attempted to explore the significance of Lamin A/C and LBR on HSV-1 infection 3 . They found that Lamin A/C and LBR knockout impaired HSV-1 genes expression, as well as the formation of VRC and the marginalization of chromatin.
Owing to the compromised HSV-1 genes expression and VRC formation, it is challenging to straightforwardly confirm how Lamin A/C and LBR prevent the

Cell culture, virus strain, antibodies and plasmids
BmN cells were maintained at 27 ℃ as a monolayer in the Sf-900 II SFM medium

RNA interference
The control siRNA and siRNAs targeted the host Lamin A/C and HP1a genes were