Baculoviruses and nucleosome management

Negatively-supercoiled-ds DNA molecules, including the genomes of baculoviruses, spontaneously wrap around cores of histones to form nucleosomes when present within eukaryotic nuclei. Hence, nucleosome management should be essential for baculovirus genome replication and temporal regulation of transcrip- tion, but this has not been documented. Nucleosome mobilization is the dominion of ATP-dependent chromatin-remodeling complexes. SWI/SNF and INO80, two of the best-studied complexes, as well as chromatin modi ﬁ er TIP60, all contain actin as a subunit. Retrospective analysis of results of AcMNPV time course experiments wherein actin polymerization was blocked by cytochalasin D drug treatment implicate actin-containing chromatin modifying complexes in decatenating baculovirus genomes, shutting down host transcription, and regulating late and very late phases of viral transcription. Moreover, virus-mediated nuclear localization of actin early during infection may contribute to nucleosome management.


Introduction
The study of baculoviruses, pathogens of larval lepidopteran insects, has been justified mainly by aims of improving their abilities to serve humanity-as pesticides, expression vectors, vaccine platforms, drug therapy vectors and more (Moscardi, 1999;Chuang et al., 2007;Airenne et al., 2013;Fernandes et al., 2013;Assenberg et al., 2013;Contreras-Goḿez et al., 2013;Grabherr and Ernst, 2013;Van Oers et al., 2014). Investment in the utility of baculoviruses as laboratory tools and industrial workhorses has overshadowed investment in basic studies on how these viruses actually function. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the type species of the Alphabaculovirus genus of Baculoviridae and is the most-studied of the baculoviruses (Herniou et al., 2011). AcMNPV arguably has received more attention than any other insect virus that is not pathogenic for humans. Even so, large gaps remain in understanding the molecular strategies that AcMNPV and other baculoviruses use to reproduce and to make the cells they infect such powerful protein expression machines.
The current paradigm holds that baculovirus gene expression occurs in four temporal phases and is regulated primarily at the transcriptional level; that baculoviruses are the only nuclear replicating DNA viruses that use a combination of host and viral polymerases for viral gene transcription; that TATA boxes and CAGT motifs promote transcription during the early phases, and that ATAAG, GTAAG or TTAAG motifs promote transcription during the late phases (Rohrmann, 2013). Genome sequence has provided the basis for most baculovirus studies conducted during the last several decades and a great deal has been learned. But baculoviruses, like papillomaviruses and polyomaviruses of vertebrates, have circular, negatively-supercoiled, doublestranded (ds) DNA genomes, and any effect genome structure might have on regulation of transcription and replication of baculoviruses has yet to be explored. This omission in baculovirus lore may be attributable to the influence of the long-standing view that DNA is a passive polymer bullied by proteins that dictate their interactions (Cozzarelli et al., 2006). This view has changed, however. DNA topologists have unequivocally demonstrated that DNA structure has an active role in regulating biological processes (Liu et al., 2009;Fogg et al., 2012).
Two lessons from DNA topology have immediate relevance for baculoviruses: (1) covalently-closed circular (ccc) supercoiled DNA replication results in intertwined rings of DNA that are decatenated in eukaryotes by topoisomerase 2 (Topo 2) (Liu et al., 2009); and (2) negatively-supercoiled DNA immediately forms nucleosomes on entering host nuclei making nucleosome manipulation essential both for baculovirus replication and temporal regulation of transcription (Patterton and von Holt, 1993;Baranello et al., 2012). To date, these principles have not been integrated into the baculovirus paradigm Models of negatively supercoiled ds DNA behavior abound (Witz and Stasiak, 2010). Chromosomal and plasmid DNA molecules are negatively supercoiled in all bacteria, except for in extreme thermophiles, giving DNA topologists and microbiologists ample material with which to study the properties of negatively supercoiled DNA in biological systems (Schvartzman et al., 2013). Current evidence suggests that because torsional tension diminishes the DNA helicity and facilitates strand separation, negative supercoiling assists in DNA replication, transcription and aids DNA topoisomerases in decatenating DNA. In short, negative supercoiling has emerged as a major factor in the governance of biological activities (Witz and Stasiak, 2010;Schvartzman et al., 2013).
The AcMNPV genome is negatively supercoiled and forms nucleosomes Summers and Anderson (1973) used cesium chloride density gradient centrifugation to show that baculovirus genomes consist of ccc, supercoiled ds DNA. That the supercoiling is negative was demonstrated by the rapid inactivation of baculoviruses by exposure to psoralens in 365 nm light (Hartig et al., 1992;Weightman and Banks, 1999). Psoralens are bi-functional photoreactive molecules that preferentially intercalate into negatively supercoiled ds DNA and photoreact at 365 nm, forming cross-links between the apposing strands (Naughton et al., 2013;Kouzine et al., 2014). Wilson and Miller (1986) noted that parental AcMNPV DNA formed chromatin-like structures within 2 h of uncoating within the nucleus, consistent with the properties of a negatively supercoiled ds DNA molecule (Baranello et al., 2012). Additionally, treatment of DNA from infected cells with micrococcal nuclease resulted in monosome-sized fragments that contained both cellular and viral DNA. Extending these findings, Wilson and Price (1988) observed that infection resulted in increased association of cellular histones with the nuclear matrix, and that progeny AcMNPV DNA incorporated histones prior to 24 hpi, all supporting the idea that AcMNPV DNA forms nucleosomes in the nucleus.
Nucleosomes consist of 147 bp of DNA wrapped around a core histone octamer, and these structures are spaced every 10 to 50 bp in chromatin (Moshkin et al., 2012). Not surprisingly, they can impede access of DNA binding proteins to their target sequences, thereby preventing both transcription and replication. There is no basal transcription on cellular chromatin templates in vitro without factors mediating nucleosome repositioning or ejection (Moshkin et al., 2012). Transcription takes place when AcMNPV chromatin templates are transfected into susceptible host cells, however, showing the immediate early genes are open for business (Burand et al., 1980). Chromatin-modifiers present within the transfected cells may have a role in immediate early gene transcription, but negative supercoiling allows access to binding sites not available in positively supercoiled or even neutral DNA (Baranello et al., 2012). Accordingly, transfection studies have shown that baculovirus genome replication is 4 times more efficient when the template is supercoiled than when it is nicked, and 15-150 times more efficient than when it is linearized (Burand et al., 1980;Kitts et al., 1990).
If negative supercoiling enables transcription of AcMNPV immediate early genes and thereby offers a way out of nucleosomal constraints, then subsequent viral transcriptional programs (delayed early, late and very late) would be expected to depend on virus-encoded factors capable of modulating chromosome-remodeling systems.

Moving nucleosomes
Nucleosome mobilization is the specialty of ATP-dependent chromatin remodeling complexes (Ho and Crabtree, 2010). Each complex is named after the primary ATPase that does the work. INO80 and SWI/SNF comprise two of the four main families of these complexes. They are evolutionarily conserved from yeast through vertebrates. These complexes are thought to have played a major role in the evolution of multicellularity and the demand for tissue-specific and developmental-stage-specific expression of genes (Ho and Crabtree, 2010;Moshkin et al., 2012).
The ATP-dependent chromatin remodeling complexes consist of 8-15 combinatorially assembled components with a core group of essential subunits surrounded by others that, in vertebrates, at least, switch in and out during specific stages of development and differentiation. All of the complexes are thought to have specialized, non-redundant roles in development. Some of the complexes are celltype and developmental-stage specific (Ho and Crabtree, 2010). Use of a combinatorial complex for chromatin remodeling during development and differentiation enables the formation of hundreds of complexes for diverse gene expression patterns (Moshkin et al., 2012).
This wide selection of complexes could be vulnerable to interception and repurposing by negatively supercoiled viruses with nucleosome management issues of their own. Subunit modification or faux subunit synthesis could be used to help recruit the ATP-dependent complexes to viral chromatin for specific tasks. In this regard, it is worth noting that all group I NPVs have orthologs of SNF2 (Katsuma et al., 2008). SNF2 ATPases are the major ATPases in many SWI/SNF and INO80 chromatin-remodeling complexes. These ATPases have active roles in both substrate recognition and catalytic activities (Morrison and Shen, 2009). Moreover, during BmNPV infection of Bm5 cells, genes related to organism development are up-regulated differentially throughout early, late and very late viral gene expression (Xue et al., 2012).

Actin-containing chromatin remodeling complexes
Actin is an abundant, conserved, 42 kDa ATPase that is regulated by a plethora of actin-binding proteins for many different cytoplasmic and nuclear functions (Krauss et al., 2003;Cisterna et al., 2009;Visa and Percipalle, 2010;Simon and Wilson, 2011;Rajakylä and Vartiainen, 2014). Cytoplasmic functions of actin inevitably depend on its ability to polymerize. Interestingly, this activity may be dispensable for some nuclear functions (Posern et al., 2002;Johnson et al., 2013;Kapoor and Shen, 2014).
Histone acetyltransferase TIP60 (dTip60 in Drosophila) is part of a multimeric protein complex that unites HAT with chromatin remodeling activities that include both gene repression and activation. dTip60 is involved in widespread gene regulation in Drosophila, especially genes involved in chromatin-related functions (Schirling et al., 2010).
INO80 and SWI/SNF are ATP-dependent chromatin remodeling complexes that depend on their actin subunits to bind chromatin, and thereby, to function (Zhao et al., 1998;Kapoor et al., 2013;Kapoor and Shen, 2014). INO80 figures prominently in transcriptional regulation, DNA replication and DNA repair (Morrison and Shen, 2009). The actin subunit in INO80 forms a module with actin-related proteins 4 and 8 that interacts with chromatin during INO80-chromatin interaction (Kapoor and Shen, 2014). Interestingly, actin may not have to retain the ability to polymerize in order perform this function (Kapoor et al., 2013).
The two SWI/SNF complexes in Drosophila are called Bhramaassociated proteins (BAP) and polybromo-containing BAP (PBAP), and in vertebrates, BAF and PBAF (F for factors) (Fig. 1). BAP and PBAP are both powered by Brahma (Brm), and each contains 11 subunits, 10 of which are identical. BAP and PBAP can serve as transcriptional activators or suppressors, and are known to interact with various transcription factors in different cell types. For example, BAP is essential for intestinal stem cell proliferation and damage-induced midgut regeneration in Drosophila (Jin et al., 2013). Notably, alphabaculoviruses establish initial infection in differentiating larval lepidopteran midgut cells (Keddie et al., 1989;Washburn et al., 1995). Some studies have indicated that actin in SWI/SNF complexes needs to be polymerizable to retain full function, but this point is still being debated (Rando et al., 2002;Zhao, et al., 1998).

Evidence for BAP and Topo 2 activity in baculovirus genome processing
The formation of baculovirus chromatin would seem to necessitate that both DNA replication and temporal regulation of transcription would involve the mobilization of nucleosomes, yet no studies have been conducted to test this possibility directly. Nonetheless, evidence from experiments conducted prior to the recognition of legitimate nuclear actin might shed some light on this topic.
Actin was first shown to be essential for AcMNPV progeny production in 1987 when it was noted that cytochalasin D (CD), a drug that prevents actin polymerization, blocked nucleocapsid assembly in AcMNPV-infected cells (Volkman et al., 1987). These results were puzzling at the time because baculovirus nucleocapsid assembly takes place in the nucleus, and in 1987, actin was thought to function only in the cytoplasm. Nonetheless, the requirement for actin for progeny virus production was shown to be uniformly true for a wide variety of alphabaculoviruses infecting several different genera and species of cell lines (Kasman and Volkman, 2000). Viral DNA synthesis was not affected by CD, suggesting nucleosome mobilization for early gene expression and DNA replication do not depend on actin polymerization (Volkman, 1988). DNA processing was affected, however, as shown in time course experiments wherein viral DNA was analyzed by field inversion gel electrophoresis of viral DNA (Fig. 2, Oppenheimer, 1995). Most of the viral DNA produced in the presence of CD remained stranded at the top of the gel, apparently too large to penetrate, with very few individual supercoiled ccc forms apparent at 16 through 24 hpi. In the absence of CD, supercoils of control viral DNA first appeared at 12 hpi and were clearly pronounced by 16 through 24 hpi. In 1995, these results were difficult to explain, but a recent study provides a plausible hypothesis. DNA topology rules dictate that baculovirus DNA replication results in intertwined rings of DNA that are decatenated by Topo 2. Topo 2 needs BAF to mediate its binding to the chromatin matrix (Dykhuizen et al., 2013). If BAP,  figure (for example, BAF60A, B, C indicates that one of these three subunits is present). Some assemblies of the vertebrate brahma-associated factor (BAF) complexes are tissue-specific and have unique developmental roles. Other assemblies might coexist in a specific cell type and perhaps target specific genes or function together with specific transcription factors. As in D. melanogaster, a second set of BAF complexes, the PBAF complexes, exists containing polybromo (also known as BAF 180). The fundamental activity of promoting nucleosome mobility has been highly conserved. BRD, bromodomain-containing protein; BRG1, brahmarelated gene 1; MOR, Moira; PHD, plant homeodomain; PtdIns (4,5)P 2 , phosphatidylinositol-4,5-bisphosphate; SAYP, supporter of activation of yellow protein; SNR1, Snf5related protein 1.  Oppenheimer (1995). Time course of viral DNA isolated from AcMNPV-infected Sf-21 cells grown in the absence (left) and presence (right) of cytochalasin D, resolved using field inversion gel electrophoresis, then probed with [ 32 P]-dATP labeled total viral DNA as outlined in Oppenheimer and Volkman (1997). Times (hours) post infection when the samples were taken are indicated. DNA isolated from AcMNPV budded virus (BV) is shown on the left as a control for supercoiled ccc DNA (runs at 1500 kb) and linear DNA at 134 kbp. similarly, is needed to facilitate Topo 2 binding to viral chromatin for genome processing, then CD might prevent processing by blocking BAP binding and thereby mislocating Topo 2.

Baculovirus-mediated nuclear localization of actin
During AcMNPV early gene expression, a considerable amount of cytoplasmic monomeric actin is translocated to the nucleus and retained there (Ohkawa et al., 2002). Viral DNA transfection experiments revealed that five early genes of AcMNPV (in addition to the ie0-ie1gene complex) appear to be involved in nuclear actin accumulation (Ohkawa et al., 2002). Ac004, Ac152, and pe38 transfected 1 day prior to he65 and Ac102, leads to the nuclear localization of actin (NLA) (Ohkawa et al., 2002). In viral infection experiments, however, only one of the five viral genes, Ac102, is essential for NLA. That AC102 is needed for some other function(s) as well can be inferred from the failure of actin directed to the nucleus by an NLS motif to rescue an Ac102 deletion mutant (Gandhi et al., 2012).
The nuclear localization of actin may be due, in part, to host heat shock genes that are rapidly upregulated by 6 hpi, and are important for DNA replication and baculovirus production (Salem et al., 2011;Lyupina et al., 2010). Heat shock is well known to promote nuclear translocation of actin (Iida et al. 1986;Johnson et al., 2013).
Actin transcript levels do not increase during AcMNPV infection; rather, the actin transcripts disappear at around 12 hpi along with transcripts of most other host genes (Ooi and Miller, 1988). Transcript levels of other cytoskeletal elements, however, such as profilin, subunit 4 of the Arp2/3 complex, and actin depolymerizing factor 1, do increase prior to 12 hpi in BmNPV-infected cells (Xue et al., 2012).
Why would a virus cause such a massive shift in cytoplasmic versus nuclear actin? The answer could be that the virus is reprogramming the host nucleus to a more embryonic state with increasing concentrations of nuclear actin (Ho and Crabtree, 2010) (Fig. 3). Nuclear actin is known to play a major role in nuclear dedifferentiation (Miyamoto and Gurdon, 2013). If the virus-induced influx of monomeric actin triggers nuclear reprogramming, then viral late gene and very late gene expression could be regulated in a manner similar to that of a differentiation pathway using virusmodified chromatin remodeling complexes. Consistent with this hypothesis, during BmNPV infection of Bm5 cells, genes related to organism development are up-regulated differentially throughout early, late and very late viral gene expression (Xue et al., 2012).

Evidence for actin-containing chromatin modifier-involvement in shut down of host transcription, and late and very late baculovirus transcription
Late gene expression occurs coincident with viral DNA synthesis and the massive halt of most host transcription (Ooi and Miller,1988). According to DNA topology rules, all three of these processes should require nucleosome rearrangement (Fogg et al., 2012). Indeed, RNA silencing experiments targeting various AcMNPV-encoded late expression factors (lef-1, lef-2, lef-3 and lef-11), p143 or dnapol, showed each of them was essential for all three processes (Schultz and Friesen, 2009). Moreover, Nagamine et al. (2008) showed that lef3 and p143 also were important for host chromatin relocation to the nuclear margins, a cytopathic effect that occurs during late gene expression in baculovirusinfected cells. Schultz and Friesen (2009) additionally showed that when another group of AcMNPV-encoded factors (lef-8, lef-9, p47 and pp31) were silenced, only late gene expression was affected. All these factors are candidates for subverting ATP-dependent chromatin remodeling complexes.
The presence of CD in AcMNPV-infected cells delays virusinduced shut-down of host protein synthesis (Talhouk and Volkman, 1991). It also leads to delays in the onset and shutdown of late protein synthesis even though there is no change in timing or amount of viral DNA synthesized (Talhouk and Volkman, 1991;Volkman, 1988). Further, when CD is added to AcMNPVinfected cells after host shut-down has occurred already, host mRNA and protein synthesis are revived, showing there is a window of reversibility (Talhouk and Volkman, 1991). All these observations could be explained if CD-sensitive chromatin modifiers were involved in shutting down host protein synthesis and in transitioning between early and late phase of viral transcription. Again, candidates for recruiting these activities include lef-8, lef-9, p47 and pp31 (Schultz and Friesen, 2009). Embryonic stem cells (ESCs) are characterized by hyperdynamic chromatin, which is compacted when these cells exit from their pluripotent state and differentiate into cells of multiple lineages. In the self-renewing state, chromatin remodellers are required to prevent this chromatin compaction (CHD1) and to repress and refine the inappropriate expression of genes (esBAF and the TIP-p400 complex) that would otherwise be allowed by the permissive chromatin landscape. Exit from this self-renewing state into a state that allows multilineage commitment involves global changes in chromatin configuration, such as the formation of heterochromatin and the silencing of pluripotency genes (BAF complexes and NURD complexes). Evidence is emerging that chromatin remodellers such as BAF complexes are crucial for the reversal of development and the reactivation of pluripotency genes such as Oct 4, which occurs during the nuclear reprogramming of a committed cell type back into an ESC-like state. The proteins known to be involved are listed, together with the chromatinremodelling complex they are found in (CHD, orange; SWI/SNF, green; ISWI, yellow; and INO80, pink).
CD similarly delays the switch to the very late phase of transcription (hyperexpression of polyhedrin and p10 mRNAs) but only when present during the transitional period, 15-23 hpi (Wei and Volkman, 1992). Once the transition is passed and hyper-transcription has begun, addition of CD has no effect on p10 or polyhedrin mRNA expression levels. If CD is added before the transition, recovery by removing CD is possible but only if protein synthesis ensues (Wei and Volkman, 1992). These results suggest that viral-encoded protein(s), such as VLF-1 (a known very late expression factor) and CD-sensitive chromatin modifiers are needed for the transition to hyperexpression to take place.

Supercoiling and hyperexpression
The transition of oncogene c-myc from basal level to full level of transcription is both BAF-regulated and dependent upon negative supercoiling (Brooks and Hurley, 2009;Baranello et al., 2012). The SWI/SNF complexes are able to generate superhelical torsion in nucleosome DNA (Olave et al., 2002). Upstream of the main promoter of c-myc is an AT-rich, supercoil-sensitive sequence called FUSE. In response to increasing levels of negative supercoiling, FUSE melts, enabling binding of transcription activator FUSE-binding protein (FBP) that increases the promoter activity and transcriptional rate to full output ( Fig. 4A) (Baranello et al., 2012;Kouzine et al., 2008). Similarly, polyhedrin and p10 genes have AT-rich BURST sequences close to the transcription start sites that bind transcription activator VLF-1 (Kumar et al., 2009;Rohrmann, 2013). VLF-1 and the BURST sequences are both known to promote hyperexpression (Rohrmann, 2013). VLF-1 preferably binds cruciform structures, structures that are stabilized by negative supercoiling (Mikhailov and Rohrmann, 2002). It is possible, therefore, that c-myc, polyhedrin and p10 are similarly regulated ( Fig. 4A and B). For polyhedrin and P10 production, plenty of template is available. Only a small fraction of the replicated viral DNA is encapsidated for virus assembly (2-4% for budded virus) and that may account for the massive output in infected cells (Rohrmann, 2013). If, indeed, supercoiling is found to be involved in hyperexpression of polyhedrin and p10, then baculoviruses could become valuable tools for elucidating the role(s) of host factors involved in regulating supercoiling and gene expression, important goals for cancer research (Baranello et al., 2012).

Vertebrate supercoiled ds DNA viruses and nucleosome management
Papillomaviridae and Polyomaviridae are the only families among nuclear-replicating, ds DNA vertebrate viruses known to have negatively supercoiled, ccc genomes, assuring the formation of viral nucleosomes during infection. The genomes are small, 8 and 5 kb, respectively, and have only two phases of transcription: early and late. Both polyomaviruses and papillomaviruses have been studied extensively because of their cancer-causing capabilities (de Villiers et al., 2005;Hou et al., 2005). Their link with cancer likely involves their subversive effects on chromatin modifiers (Dhamne et al., 2007).
Papillomaviruses are more difficult to study than polyomaviruses because of the lack of convenient cell culture systems that support their replication. Papillomaviruses infect basal epithelial cells but replicate only when the cells differentiate. This dependence on differentiation could be mediated by the availability of appropriate ATP-dependent chromatin-modifying complexes. U2OS cells, a cell line established from bone tissue of a 15 yr old female osteosarcoma victim, will support genome replication when transfected with papilloma virus genomes (Reinson et al., 2013).
Polyomaviruses, in comparison, are less demanding than papillomaviruses. Even when susceptible host cells are quiescent, polyomavirus can induce the synthesis of cellular DNA and cellular enzymes related to DNA synthesis (Dulbecco, 1970). This capability is remarkable because quiescent cells are devoid of nuclear actin, which, in turn, reduces DNA synthesis and destabilizes the binding of RNA polymerases II and III to their binding sites (Spencer et al., 2011). The ease of establishing infection and replication in culture helped to make SV40, type species of Polyomaviridae, the beststudied negatively-supercoiled ds DNA virus of all.
ATP-dependent chromatin modifying complexes play pivotal roles in SV40 replication and genome processing. Nucleosome positioning is critical for cleavage of SV40 chromatin by Topo 2 (Capranico et al., 1990;Ishimi et al., 1991;Halmer and Gruss, 1997). Replication efficiency of SV40 is linked directly to the accessibility of topoisomerases I and II to the viral chromatin, which is, in turn, dependent on the overall chromatin structure (Halmer and Gruss, 1997). Treatment of SV40-infected CV-1 cells with a Topo 2 catalytic inhibitor results in catenated dimers of genomic DNA that are resolved when the inhibitor is removed (Ishimi et al., 1995). In another example, SV40 DNA chromatin in Drosophila embryo extracts uses the chromatin-modifying complex CHRAC to alter the nucleosomal structure at the origin to enable access of the large T-antigen and efficient initiation of replication in the presence of ATP (Alexiadis et al., 1998).
Infections by polyomaviruses and papillomaviruses induce double strand breaks and the DNA damage response (DDR) during genome replication and processing (Reinson et al., 2013;Dahl et al., 2005). Baculoviruses also induce the DDR during genome replication and processing; and, as it does for the polyomaviruses and papillomaviruses, this response contributes to baculovirus production (Huang et al., 2011;Mitchell and Friesen, 2012;Mitchell et al., 2013). It should be noted that the DDR includes recruitment of chromatin-modifying complexes, which is favorable for viral DNA replication and processing (Morrison and Shen, 2009).

Baculovirus ancestry
There is no doubt that the era of DNA sequencing has led to a vast trove of information that never would have been discovered any other way. For insect virologists, the pay off included the discovery that baculoviruses, nudiviruses and bracovirusesthree families of insect viruses with different strategies for tracking and infecting  Baranello, L., et. al.. The importance of being supercoiled: How DNA mechanics regulate dynamic processes, pg 636, 2012, with permission from Elsevier. Regulation of c-myc compared to the potential regulation of AcMNPV polyhedrin and p10 genes. A. During transcription of c-myc, the melting of the supercoil-sensitive sequence FUSE promotes the recruitment of factors that enhance (fuse-binding protein; FBP) or repress (FBP-interacting repressor; FIR). B. Postulated mechanism of hyperexpression of AcMNPV polyhedrin and p10 genes. The AT-rich BURST sequence melts enabling cruciform structures to form and bind VLG-1, leading to enhanced expression. their hostsare all genetically related (Wang et al., 2007;Cheng et al., 2002;Bézier et al., 2009;Burand et al., 2012;Burke et al., 2013).
Because bracovirus genomes are fully integrated into the DNA of their mutualist partners, braconid parasitoid wasps, the threevirus-family phylogeny could be calibrated using the age of fossil amber-embedded wasps of the bracovirus-bearing microgastroid complex as the standard (Strand, 2010;Thézé et al., 2013). The phylogenetic analysis showed that bracoviruses and nudiviruses shared a common ancestor 190 million years ago (Mya) while baculoviruses and nudiviruses shared a common ancestor 310 Mya, during the Paleozoic era.
Besides sharing at least 13 orthologous genes, viruses representing these seemingly disparate families all replicate within the nucleus, all have large ds DNA supercoiled genomes (Huang et al., 1982;Strand, 2010), and all infect metabolically active, differentiating cells (for example, see Table 7.2 in Jehle, 2010), assuring the presence of ATP-dependent chromatin remodeling complexes.
Bracoviruses are produced only in ovarian calyx cells in pupal and adult female braconid wasps (Strand, 2010;Bitra et al., 2011). This celltype specific, developmental-linked trigger likely involves development-and cell-specific ATP-dependent chromatin-modifying factors. Similarly, nudivirus HzNV-2 is reproductive-tissue specific, but in males and females, both. Nevertheless, there is a need for tissue specific nucleosome management, so one might expect that a selection of ATP-dependent chromatin modifying complexes could serve as important infection factors for HzNV-2, as well.
Finally, in view of the shared orthologous genes and the common aspect of negatively supercoiled genomes, one might expect that at least a portion of the shared genes be devoted to viral chromatin remodeling. In support of this idea, AcMNPV lef 8 and p47, both required for late gene expression, are among the shared orthologues (Schultz and Friesen, 2009;Thézé et al., 2013).

Summary
The rules of DNA topology state that negatively-supercoiled-ds DNA molecules wrap around cores of histones to form nucleosomes when present within eukaryotic nuclei. These rules apply to negatively supercoiled viral genomes, such as those of baculoviruses, and imply that nucleosome management must occur for viral regulation of transcription and replication to take place. Nucleosome mobilization is the dominion of ATP-dependent chromatin-remodeling complexes. The SWI/SNF and INO80 families of ATP-dependent chromatin-remodeling complexes are highly conserved and contain actin as a subunit active in binding to chromatin. Histone acetyltransferase TIP60 complexes also contain an actin subunit that may contribute to chromatin binding. Retrospective analysis of results of AcMNPV time course experiments wherein actin dynamics were disrupted by cytochalasin D drug treatment were consistent with the involvement of the SWI/ SNF family in decatenating baculovirus genomes. Shutting down host transcription and regulating late and very late phases of viral transcription likely involves actin-containing chromatin modifying complexes as well. Nucleosome management may be the purpose of virus-mediated nuclear localization of actin early during infection. Full understanding of baculovirus-based nucleosome management strategies could lead to valuable, new baculovirus-based technologies.