Baculoviridae is a diverse family of insect viruses with large, double-stranded, circular DNA genomes packaged in rod-shaped, enveloped nucleocapsids. A characteristic feature of baculoviruses is the production of paracrystalline occlusion bodies (OBs) which surround the assembled virions at late times of infection. Baculoviruses produce lethal infections in their hosts and OBs protect the virions in the environment after death of the insect until uptake by another susceptible host. Other insect viruses also produce OBs such as entomopoxviruses (EPV) and cytoplasmic polyhedrosis viruses (CPV). EPV and CPV replicate in the cytoplasm of infected cells; in contrast, baculoviruses replicate within the nucleus. According to the size and shape of OBs, baculoviruses were traditionally classified into two genera: nucleopolyhedrovirus (NPV), which produce large OBs known as polyhedra, and granulovirus (GV), which produce small ovoid OBs or granules. The major proteins that form each class of OB are known as polyhedrin and granulin, respectively. Recently, after several baculoviral genomes have been sequenced, a new classification based on the phylogenetic relationships between species within the family was accepted [1-2]. Four genera were defined:
2. Two types of virus progeny serve at different steps of host invasion
Alphabaculoviruses and betabaculoviruses produce virions of two phenotypic classes: occlusion-derived virus (ODV) and budded-virus (BV). Nucleocapsids of both types of virus particles are assembled in the nucleus of the infected cell. During the late phase of infection, when proteins of the nucleocapsid are expressed, BVs are produced as the newly formed nucleocapsids exit the cell, acquiring their envelopes from the cell membrane during the budding process. BVs disseminate the infection within the host by entering other cells via a mechanism of receptor-mediated endocytosis. ODVs arise at very late times, when nucleocapsids are enveloped in membrane units derived from the nuclear envelope to finally be embedded in the OBs. OBs persist in the environment after liquefaction of the insect cadaver and are responsible for the horizontal transmission of the virus between hosts. Upon ingestion by a susceptible insect larva, OBs are dissolved in the alkaline environment of the midgut and ODVs are released. The ODVs move through the peritrophic membrane and nucleocapsids are delivered into midgut epithelial cells through a mechanism of membrane fusion mediated by specific viral proteins known as
3. Nuclear events associated with infection
Baculovirus infection causes cells to enlarge and stop dividing; the nucleus swells and forms the virogenic stroma (VS), which is the nuclear compartment where the viral DNA is replicated and the nucleocapsids of virus progeny are assembled. The host chromatin adopts a marginal distribution at 24 hours post infection (hpi), when the VS becomes evident. At this time, it was shown that histone H4 fused to fluorescent protein markers colocalizes with the chromatin in the periphery but not with the VS . The new distribution of the chromatin is determined by the replication of the virus and may have effects in changes operated in the expression of host genes and the progression of the cell cycle. At late times, ODVs become occluded into OBs in the periphery of the VS.
Apparently, the nucleocapsids of NPVs enter the nucleus through the nuclear pores, whereas the genome of GVs is probably injected . Virions of baculoviruses are devoid of histones, in turn the DNA in the nucleocapsid is packed in association with viral protein P6.9, a basic DNA-binding protein. This small polypeptide is rich in arginine, serine and threonine residues, a feature similar to proteins called protamines present in the nuclei of spermatids in many animals and plants. The positive arginine residues in protamines neutralize the negative charges in the DNA backbone while serine and threonine mediate interaction between protamine molecules, resulting in a high condensation of genomic DNA. Once it is uncoated into the nucleus, the DNA dissociates from P6.9 through the phosphorylation of the protein. During infection the viral DNA appears to be organized in the form of nucleosomal-like structures in association with P6.9, as suggested by experiments of micrococcal nuclease digestion of isolated nuclei .
4. Transcription program of baculovirus genes
In general, genes of DNA viruses are transcribed in a temporal sequence and the process is highly regulated by infection-derived mechanisms and proteins from both host and viral origin. This stepwise mode of gene expression ensures the availability of gene products required for the progression into the next phase of the infection. Baculoviruses express their genes in three successive phases designated as early, late and very late (figure 1). Early genes are transcribed by the host RNA polymerase II before virus DNA replication, while late and very late genes are transcribed by a virus-encoded RNA polymerase, after starting of viral DNA replication. Products of a number of early genes are required for virus DNA synthesis and for expression of late/very late genes, and at least one late gene product is also needed for expression of very late genes. The progression of the infection into the late phase correlates with the transcriptional shutoff of cellular and early viral genes. Genes belonging to the different temporal classes are encoded in both DNA strands without any associative distribution in the genome. A number of baculovirus genes contains promoters with sequence elements characteristic of both early and late classes. Their transcription is regulated independently at each temporal phase which ensures their expression throughout the infection . Most of our knowledge of baculovirus gene regulation comes from studies in
5. Expression of early genes
At the time the virus reaches the nucleus, the template DNA for transcription as well as proteins of the virion that are carried over and may activate transcription are at their lowest levels. Therefore, the success of the infection depends on the ability of the virus to efficiently redirect the cellular system to express early genes encoded in its genome. Baculovirus DNA is infectious without any accompany protein, as proved by the infection that follows after transfection of permissive cells with viral DNA. This indicates that baculovirus early promoters are responsive to the RNA pol II and accordingly, their structural organization resembles that of the host genes which are transcribed into mRNAs.
Promoters of baculovirus early genes consist in a core region and regulatory proximal sequences that may be recognized for specific binding of transcription factors from either the host or the virus. The core promoter includes two characteristic elements: a TATA-box-like sequence and a transcription initiator (INR), although one or both are absent in some early promoters . The TATA box is the site for assembly of the preinitiation transcription complex by first binding of TATA binding protein (TBP). After the RNA pol II is recruited to the complex, transcription starts about 30 nucleotides downstream of the position of the TATA-box. The INR determines the starting site for transcription (nucleotide +1 of the primary transcript), and ensures proper initiation when there is no TATA-box present. CAGT is the most conserved INR sequence motif in baculovirus early promoters. Other activating sequences can be found either upstream of the core promoter or downstream, in the 5´UTR of the regulated gene.
Besides the sequences within the promoter region that modulate expression of a gene through the binding of regulatory proteins, there are sequences that enhance transcription from promoters even if they are located at a long distance. In baculoviruses there are non-coding regions known as homologous regions (
Baculovirus early genes can be subdivided into two categories: immediate-early (
5.1. Immediate early transactivator IE1
AcMNPV IE1 is a 582 aminoacids long protein exhibiting general characteristics of transcription factors (figure 2). It has a modular organization with domains associated to different functions: dimerization, nuclear import, DNA-binding, transactivation and replication [16-17]. Dimer formation is required for nuclear localization since the protein mutated in the dimerization domain cannot be imported into the nucleus . IE1 transactivates early promoters including its own. Two mechanisms are postulated by which IE1 is capable of activating an early promoter: one independent and one dependent on DNA-binding. In the first one, IE1 activates transcription by interaction with cell factors recruited to the promoter regulatory regions. In the DNA-binding-dependent mechanism, the activation depends on binding of IE1 to
It has been reported that AcMNPV IE1 down-regulates the expression of certain genes. Promoters of these genes contain a sequence motif similar to one half of a typical
IE1 is the only known baculoviral gene that is expressed as part of a product of alternative splicing designated IE0. Compared to IE1, AcMNPV IE0 contains 54 additional aminoacids at its N-terminus. Both protein species are required for an efficient infection, although each one is dispensable given the other is present .
6. Expression of late and very late genes
Experiments using α-amanitin, an inhibitor of RNA pol II, showed that the synthesis of virus-specific mRNA becomes resistant to the drug with the progression of the infection, indicating that a novel RNA polymerase is induced in infected cells [24-25]. In AcMNPV-infected cells this viral encoded enzyme transcribes late genes mostly from 6 to 24 hpi and very late genes between 18 and 72 hpi. These times correlate with the production of BVs and ODVs, respectively. Expression of late genes depends on viral DNA replication. When replication is blocked with the DNA synthesis inhibitor aphidicolin, transcription of late genes is also inhibited . In accordance to this dependency, whose nature is not known, genes involved in DNA replication are also required for transcription of late genes, and therefore, they are considered as a subset of the factors regulating late gene expression, collectively known as late expression factors (
|6||210||core||Replication||primase accessory protein|
|37||112||α-I, α-II, β, γ||Replication|
|67||385||α-I, α-II, β||Replication||ssDNA binding protein|
|147||582||α-I, α-II, β||Replication||transactivator of early genes, |
|125||226||α-I, α-II*, β*||Replication||possible ssDNA binding protein|
|151||408||α-I||Replication||transactivator of early genes,|
|135||299||α-I*, α-II*, β*||Replication||apoptosis inhibitor|
|90||464||core||Transcription||RNA polymerase subunit, capping enzyme,|
|50||876||core||Transcription||RNA polymerase subunit|
|62||490||core||Transcription||RNA polymerase subunit|
|40||401||core||Transcription||RNA polymerase subunit|
|99||265||core||Transcription||transcription initiation factor|
|28||173||α-I, α-II, β||Transcription|
|36||275||α-I, α-II*, β||Transcription||DNA binding protein|
|53A||78||α-I, α-II*, β*||Transcription|
6.1. Replication lefs
The actual function of IE1 in DNA replication is poorly understood, nonetheless it appears to depend on the
Other genes have a stimulatory effect in transient replication/late expression assays. These are
6.2. Transcription lefs
The viral RNA polymerase is a complex of the products of four baculovirus core genes:
6.3. Late and very late mRNAs synthesis and regulation
Promoters of late genes contain a TAAG sequence motif from which transcription is initiated. There are less TAAG motifs in baculovirus genomes than expected by random occurrence, suggesting that the activity of this sequence as a late promoter selects negatively its random distribution. The integrity of this motif is strictly necessary for transcription, while adjacent sequences up to eighteen nucleotides may affect the level of expression . There may be more than one functional TAAG over a variable distance upstream the translational start codon of the regulated gene .
Late transcripts usually span more than one ORF; likewise, one specific ORF may be represented in transcripts with different 5' or 3' ends. The significance of these polycistronic messages is not known and it is generally assumed that only the leading gene in the message is translated into protein. Late genes are encoded in both DNA strands, distributed over the genome, therefore there may be opposite late transcripts with complementary stretches. It is not known if this may play any regulatory role considering that baculovirus genes are susceptible to silencing by double-stranded RNA . Late transcripts are capped and polyadenylated at their 5' and 3' ends, respectively. At least two enzymatic activities required for capping reside in the LEF4 subunit of the RNA polymerase. This protein functions as RNA triphosphatase and guanylyltransferase but lacks activity of N7-methyltransferase, which is required for methylation of the cap structure in position N7 of guanine [49-50]. A gene responsible for this activity has not been identified in baculovirus. The structure of cap 1 mRNAs includes methylation of the 2'hydroxyl group of the ribose of the first transcribed nucleotide by an RNA cap 2'O-methyltransferase (MTase-I). Several alphabaculoviruses have a MTase-I gene. The gene of AcMNPV has been found to stimulate late gene expression in transient assays . Late transcripts are terminated by the polymerase at U-rich sequences present in their 3'UTR, and subsequently the enzyme adds adenosine residues independently of template .
The most expressed very late genes in AcMNPV are those encoding polyhedrin and P10. Their transcription depends on a TAAG initiation promoter but their high level of expression depends on the presence of an AT-rich sequence known as the “burst” sequence, located between the TAAG and the translational start codon [53-54]. The burst sequence binds very late expression factor-1 (VLF1; ) originally identified in a temperature sensitive AcMNPV mutant defective in occluded virus production . VLF1 is a baculovirus core gene that is essential for the packaging of DNA into normal nucleocapsids.
7. Cellular responses to infection and changes in host gene expression
Early in infection, baculoviruses produce cell cycle arrest at G2/M or S phase, prior to viral DNA replication . The AcMNPV early transcription coactivator IE2 is considered to be involved in regulation of cell-cycle . The progression of infection is accompanied by profound changes in the expression of cellular genes. The host protein synthesis is shutoff starting at around 12-18 hpi . This was found to be mostly the result of a reduction in the levels of transcripts rather than in translation of mRNAs , though the actual mechanism of the decrease in the steady-state level of host messages is not known.
Despite host genes are eventually down-regulated at late times, Nobiron and co-workers  found that the transcript of a cognate heat shock protein (hsc) 70 gene was transiently up-regulated early in AcMNPV-infected Sf9 cells. In a comprehensive study of gene expression profile of Sf21 cells using microarrays designed from an EST database of
The actual level of cellular proteins during infection may vary with a different kinetics of that of the steady-state level of their mRNAs. For example, in a study by Rasmussen and Rohrmann , the level of TBP in AcMNPV-infected Sf9 cells, revealed constant until 72 hpi. In other study, TBP was actually found to increase between 16 and 72 hpi in Sf21 and TN368 cells, and to co-localize with viral DNA replication centers within the nucleus . Therefore, TBP appears not be targeted for degradation as it is in other viral systems. However, the functional significance of its increment is unclear, given that it coincides with decreasing levels of transcripts synthesized by RNA pol II.
Currently, due to the relevance of AcMNPV as vector for the expression of proteins in cultured insect cells, it is of special interest to understand the global shutoff of host protein synthesis. In this system, the expression of foreign proteins is driven by the promoter of polyhedrin gene, which is most active at very late times of infection. By this time many processes and pathways appear highly compromised, and the expression of certain classes of proteins may be severely affected, especially those involved in traffiking through the ER and Golgi.
Baculoviruses induce apoptosis of infected cells . Programmed cell death functions as an antiviral defense response to prevent production of virus progeny and spreading of the infection. To counteract the apoptotic response, baculoviruses encode antiapoptotic genes. P35 is a potent antiapoptotic protein of AcMNPV that inhibits the activity of effector caspases. The results of experiments using an AcMNPV
8. Baculoviral microRNAs
MicroRNAs (miRNAs) are small RNA molecules of ~20-22 nt that regulate gene expression posttranscriptionally in a sequence dependent way. miRNAs have been widely described in animals and plants and regulate expression of protein coding genes involved in numerous processes. Genes coding for miRNAs are transcribed by the RNA pol II. The primary transcript (pri-miRNA) containing a hairpin loop is processed by the RNase III-like enzyme Drosha releasing the precursor miRNA (pre-miRNA). The pre-miRNA is a ~80 nt molecule that contains an imperfect hairpin loop and is exported to the cytoplasm by Ran-GTP dependent Exportin 5. Once in the cytosol, the pre-miRNA loop is cleaved by another RNAse III enzyme, Dicer, leaving the RNA duplex consisting of the mature miRNA and its complement (miRNA*). One of these strands (the mature miRNA) is then incorporated in the RNA-Induced Silencing Complex (RISC), which is then ready to target the specific mRNA and either represses its translation or degrades it .
Viruses were also found to encode miRNAs. Strikingly, nearly all the virus encoded miRNAs were reported from DNA viruses, especially those that have a nuclear cycle, with access to the microRNA processing proteins. The majority of the viral miRNAs described belong to herpesviruses. Interestingly, studies of virus-host interactions revealed a complex miRNA regulation with both viral and host microRNAs regulating both viral and host mRNA targets [68-69]. Regarding insect viruses miRNAs, little is known yet. Two viruses, belonging to
A further study on
9. Persistent infections
It is known that some viruses are capable of persisting in their hosts without causing disease. This can be accomplished by producing either a latent or a persistent infection. The main difference between both is that during latent infection the virus is not replicating and keeping a minimal gene expression while in persistent infections all the genes are expressing, at low levels, without causing any symptom. Herpesviruses can establish latent infections in specific cell types . This state is characterized by a unique transcriptional program that involves the expression of latency-associated transcripts (LATs) as the only viral products synthesized in large quantities. The virus is maintained as an independent quiescent genetic material within the host cell nucleus. An alternative mechanism is observed in measles virus by which the virus remains at low levels with the production of viral proteins. This is usually referred to as persistent infection .
The White Spot Syndrome Virus (WSSV) is a non-occluded virus pathogenic to shrimp, phylogenetically related to baculoviruses. It was found at very low levels in asymptomatic shrimps. The virus may reside within cells in a quiescent state as in a latent infection or causing a persistent infection . Similarly, a nudivirus was found infecting persistently the cell line IMC-Hz1, derived from the corn earworm
Baculoviruses are highly lytic, causing a lethal disease in infected larvae. Epizootics caused by these viruses can reduce dramatically their host population . Persistence of baculoviruses in the environment is mainly thought to be due to the OBs that protect virions from UV light and allow horizontal transmission. But there seems to exist another way for baculoviruses to persist in the environment at low host densities. Baculoviruses can cause sublethal infections, and so be vertically transmitted from adult to offspring [77-79] or may as well become persistent or latent . A laboratory colony of
There is accumulating evidence of persistent baculoviral infections. Kemp et al  detected baculoviral presence (CfMNPV, CfDEFMNPV and a GV) in laboratory and field populations of
On the whole, baculoviruses seem to use different strategies to persist in nature: on one hand OBs permit their subsistence outside the host for horizontal transmission while, on the other hand, they can persist as covert infections in the host, allowing vertical transmission too. Moreover, these covert infections can be triggered to overt infections producing the typical lethal disease in the host. Nevertheless, the mechanisms of reactivation of these sublethal infections remain to be elucidated.
10. Concluding remarks
Among nuclear DNA viruses, baculoviruses have developed a unique strategy to synthesize late mRNAs which consists in having their own DNA-directed RNA polymerase. This enzyme recognizes viral late promoters that are different to promoters responsive to the cellular RNA polymerase. By this means, the infected cell produces high levels of viral proteins at times of the infectious cycle at which the cellular protein synthesis is mostly shutdown. A late viral progeny with a distinct phenotype is embedded in proteinaceous occlusion bodies (OBs) that assemble after overexpression of the major OB protein. In order to exploit their high protein synthesis capacity, baculoviruses have been developed as vectors for expression of heterologous proteins in insect cells. This system is continuously evolving to new biotechnological applications. However, there is still a lack of knowledge about the molecular mechanisms governing the complex baculovirus infectious cycle. A better understanding of these mechanisms would also benefit the development of baculovirus as biopesticides. To this regard, the array of viral factors involved in regulation of gene expression is an important component of the specific virus-host interactions that determinate the susceptibility to the virus of different cell types within a host and of different hosts within a range of insect species.