**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 [57]. The AcMNPV early transcription coactivator IE2 is considered to be involved in regulation of cell-cycle [38]. 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 [58]. This was found to be mostly the result of a reduction in the levels of transcripts rather than in translation of mRNAs [59], though the actual mechanism of the decrease in the steady-state level of host messages is not known.

**8. Baculoviral microRNAs**

its translation or degrades it [67].

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 (miR‐ NA\*). 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

Baculovirus Gene Expression http://dx.doi.org/10.5772/56955 69

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 *Ascoviridae* and *Baculoviridae*, were reported to code for miRNAs. The first report of a miRNA encoded by an insect virus was from the *Heliotis virescens* ascovirus (HzAV). This virus codes for a miRNA that targets viral DNA polymerase and regulates viral replication [70]. More recently, Singh and colleges [71] presented a study in which they found and validated four miRNAs encoded by *Bombyx mori* nucleopolyhedrovirus (BmNPV): *bmnpv-miR-1*, *-2*, *-3* and *-4*. This was achieved by sequencing small RNAs obtained from infected tissues of *B. mori* larvae followed by *in silico* analysis and validation using northern blot hybridization, stemloop RT-PCR and poly(A)-tailed RT-PCR. Interestingly, closely related baculoviruses were found to contain these miRNA in their genomes in conserved positions. All four BmNPV miRNAs are present with 100% identity in AcMNPV, BomaNPV and PlxyMNPV whereas three miRNAs were conserved in RoMNPV and only one in MaviNPV. In contrast of what occurs in animals and plants (miRNAs coded in intergenic regions or introns), these micro‐ RNAs were found in genomic locations completely overlapping viral ORFs, either in the coding or the complementary strand. *In silico* predictions revealed putative targets, either viral or from the host. Viral predicted targets include *dna binding protein*, *chitinase*, *bro-I*, *bro-III*, *lef8*, *fusolin*, *DNA polymerase*, *p25* and *ORF 3* of BmNPV. Cellular predicted target genes encode proteins related to antiviral defense mechanisms, such as prophenoloxidase and hemolin, or proteins that play an important role in small RNA-mediated gene regulation like GTP binding nuclear protein Ran, DEAD box polypeptides and eukaryotic translation initiation factors [72].

A further study on *bmnpv-miR-1* revealed the sequence dependent interaction of this miRNA with cellular Ran mRNA. The GTP-binding nuclear protein Ran is an essential component of the Exportin-5-mediated nucleocytoplasmic transport machinery involved in the transport of

Despite host genes are eventually down-regulated at late times, Nobiron and co-workers [60] found that the transcript of a cognate heat shock protein (hsc) 70 gene was transiently upregulated 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 *S. frugiperda*, Salem and co-workers [61] confirmed the general shutoff of host transcription over time of AcMNPV infection, but interestingly, they found that about 25% of host genes were slightly up-regulated at 6 hpi. The expression of heat shock proteins (HSPs) of the 70-kDa family in infected cells was followed by western analysis [62]. The results of this study showed changes in the cellular pattern of HSP/HSC70s. Moreover, the infection potentiated the response to heat shock, boosting the HSP/HSC70s content of cells several-fold in comparison with uninfected cells.

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 [63], 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 [64]. 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 [39]. 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 *p35* mutant that causes apoptosis upon infection in Sf21 cells showed that apoptosis is triggered by replication of the viral DNA [65]. Apoptosis induced by this mutant was inhibited when each one of the AcMNPV genes required for replication was independently silenced by RNAi. Silencing of these genes also inhibited shutoff of host proteins synthesis, suggesting that both processes are linked. These cellular responses resemble that of vertebrates which arises as consequence of cell cycle arrest or DNA damage. In a recent report Huang and co-workers [66] presented evidence indicating that infection of Sf9 cells with AcMNPV induces a DNA damage response which is required for efficient replication of the virus.
