Cellular transcriptional machinery

Once the cell has been targeted, the vector particle has to be internahzed by the cellular uptake machinery via endocytotic or phagocytotic uptake processes. This step can be taken rather easily by polyplexes, as appropriate receptor-binding ligands and/or cationic charges may enhance intracellular uptake of particles into endosomal vesicles. Several intracellular barriers then have to be overcome for successful transgene expression. Endosomal release was found to be a major bottleneck for many non-viral vectors [151,167]. The vector particle needs to survive and escape from the endosomal vesicular compartment, traffick the cytoplasmic environment, target the nucleus, enter the nucleus, and expose the carried nucleic acid to the cellular transcription machinery. 

Comment It is critical to remove excess cap analogue from in vitro transcribed transcripts prior to transfection, because the cap analogue will compete with transcripts for the cellular translational machinery. Also, even after DNase treatment, the RNA sample may still contain traces of functional pDNA, which may interfere with subsequent detection by RT-PCR. Furthermore, plasmids containing a mammalian promoter may even give rise to de novo transcription in transfected cells. 

Alper H, Stephanopoulos G (2007) Global transcription machinery engineering a new approach for improving cellular phenotype. Metab Eng 9 258-267... 

The lytic cycle Is somewhat more complicated for DNA viruses that Infect eukaryotic cells. In most such viruses, the DNA genome Is transported (with some associated proteins) Into the cell nucleus. Once Inside the nucleus, the viral DNA Is transcribed Into RNA by the host s transcription machinery. Processing of the viral RNA primary transcript by hostcell enzymes 3delds viral mRNA, which Is transported to the cytoplasm and translated Into viral proteins by host-cell ribosomes, tRNA, and translation factors. The viral proteins are then transported back Into the nucleus, where some of them either replicate the viral DNA directly or direct cellular proteins to replicate the viral DNA, as In the case of SV40 discussed In the last section. Assembly of the capsid proteins with the newly replicated viral DNA occurs in the nucleus, 3deldlng hundreds to thousands of progeny virions. 

As described above, the El A-induced entry into the cell cycle, accompanied by the many changes of cellular transcription and the beginning of viral DNA replication, triggers the cellular machinery for programmed cell death. Therefore, Ads have evolved multiple mechanisms to interrupt this intrinsic cellular death program, primarily involving two proteins encoded in the ElB transcription unit, ElB/ 19K and E1B/55K. Both proteins are expressed early, shortly after ElA. 

Intracellular protein delivery is considered to be the most direct, fastest and safest approach for curing gene-deficiency diseases or disorders affecting primarily cellular processes, such as cystic fibrosis, coagulation disorders, otl-antitrypsin deficiency, immunoglobulin deficiencies, endocrine disorders and lysosomal storage diseases, where the expression of required proteins in the host cells is limited due to the defects in the required cellular (endogenous transcriptional) machinery/ ... 

Herpes simplex virus (HSV) has been the most extensively studied of the human herpesviruses owing to its ability to easily infect cells in vitro to produce infectious virus. As with all herpesviruses, HSV encodes a number of proteins for efficient viral gene expression, viral DN A replication, and the shutoff of cellular gene transcription and translation [53], These virally expressed proteins do not function in isolation but associate with a variety of cellular and viral proteins. Furthermore, many have exhibited multiple different functions. In an effort to understand the biology of HSV and the function of its proteins, a proteomics approach has been used to study a critical viral transactivator (ICP27), the alteration of the cellular translation machinery, and components of the viral replication complex, which will be reviewed here. 

The human HS cycle can be considered broadly as a period which leads to the dramatic shift in activities of the transcriptional and translational machinery followed by eventual recovery and resumption of original activities preceding stress. Figure 1 depicts many of the key events in the HS cycle for a typical human cell line such as cervical carcinoma-derived HeLa cells. Most cells respond in an identical fashion, but some cell types that have distinctive HS responses. These differences are manifested by shifts in the relative concentrations of accumulated HS proteins and possibly in the pattern of posttranslational modifications. In all cases, however, the cellular stress response is heralded by induction of a specific transcription factor whose DNA binding activity facilitates increased expression of one or more of the stress-inducible genes. 

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