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The first vaccinia genome sequenced was from the vaccinia Copenhagen strain. The open reading frame names were determined based on its Hind III restriction map. VACV is a large (about 191,636 kbp encoding for 2063 proteins) double stranded DNA containing virus with a complex enveloped virion . The virus replicates entirely in the cytoplasm of infected cells; thus, it encodes all of the enzymes and proteins necessary to transcribe the DNA genome.
1.3.2 Vaccinia virus molecular biology
Fig. 1.2 The overview of vaccinia virus structure and replication cycle. (A) Virion structure includes several membranes and a core containing a double stranded DNA, enzymes, factors, v.v. for the initial replication of virus in host cells. (B) life cycle happens entirely in the cytoplasm and occurs in three phases; early, intermediate and late, accompanying with different forms of virion . Mature vaccinia virions exist in four forms which differ in the number of surrounding membranes and the location of viral particles. Intracellular mature virus (IMV) is a first assembled and simplest infection form of virus with one membrane, which is remained inside of infected cells following virus maturation. Intracellular enveloped virus (IEV) is essentially IMV with two additional surrounding layers which are derived from Golgi apparatus or endosomal cisternae . Cell-associated enveloped virus (CEV) is derived from IEV after outer IEV membrane is fused with plasma membrane. CEV retains attached to the outer surface of cells. And extracellular enveloped virus (EEV) is CEV which has been released from the cell surface. Therefore, EEV is mainly responsible for the spread of virus during infection .
The initial step for virus entry is attachment of virus to the cell plasma membrane. The detailed mechanism of action is still not well understood. It can occur via direct fusion at plasma membranes or low-pH dependent endosomal pathways . Three viral membrane proteins: H3L  D8  and A27 , were showed to be involved in the attachment of virus to glycoaminoglycans (GAGs) at the cell surface. But virus can also use GAG independent pathway to enter the cell . Soon after the entry, the core is released from virions and virus uses packaged materials to start an early phase transcription, followed by intermediate and late phases as illustrated in fig. 1.2. The life cycle of virus happens entirely in the cytoplasm of host cells, hence will not interfere or integrate to the host structural genome. Viral proteins which are necessary for functions of certain phases will be synthesized at different stages of infectious cycle. Gene expression during VACV infection occurs in a tightly regulated temporal cascade featuring sequential synthesis of early, intermediate and late gene classes which are distinguished by special transcriptional promoters and its enclosed factors.
About half of VACV genes belong to the early class . The VACV early class mRNA appears in minutes (20 min) and reaches a peak at about 100 min after virusâ€™ entry into cells . Viral mRNAs are synthesized inside the core and are extruded outside its surface by packaged initial materials along with DNA genome which is necessary for early transcription and translation . They include early transcription factors such as 80 kDa and 72 kDa proteins encoded by A7L and B6L correspondingly  which bind to promoters with assistance of RNA polymerase to activate the transcription processes [24-26]. Subsequently, the synthesis of early proteins is made. They include proteins needed for core wall uncoating reactions, viral DNA replicational and transcriptional factors to activate intermediate genes.
Early viral DNA releasings are those templates for intermediate-gene transcriptions which then are used to translate into transcription factors that at the end will be used for the transcription of late- phase genes. The intermediate genes are believed to be few in number . The late gene expression becomes detectable about 140 min after infection , mainly encodingfor viral structures, virion enzymes as well as essential proteins such as early initial proteins which must be incorporated into virus particles during assembly. Once all necessary materials are synthesized, the assembled process is initiated to first form immature virions. These viral particles mature with brick shape called IMVs. IMVs acquire a second, double membrane from Golgi apparatus to form IEVs. Next step, IEVs release to outside of the infected cell and may contain one more layer of cell plasma membrane named EEV or CEV (if it is still retained on cell plasma membrane). While there is no evidence for the participant of host proteins in early phase of virus infection, the situation is different in intermediate and late phases. Virus appears to borrowhost cell proteins for mRNA synthesis which have been reported elsewhere [27-28]. Soon after infection, VACVs develop multiple mechanisms to interfere the host gene expression such as inhibition of host mRNA synthesis , induction of actin and tubulin mRNA degradation. Eventually, in about 2-4 hours, host translation is drastically impaired in order to temporally maximize the expression of viral genetic information [30-31].
1.3.3 Vaccinia virus as an oncolytic agent
There are numerous inherited biological properties that make VACV suitable for development as an oncolytic agent. As a member of poxvirus family, VACV has been reported with broad spectrum of host range in which virus can use multiple host entrance mechanisms [16, 19, 32]. VACV is one of the safest viruses which have been intensively studied about molecular biology and pathogenesis, as a result of being used as a vaccine for eradiation of smallpox disease in human. Besides, it owns natural tumor tropism which can selectively infect, replicate and destroy tumor cells while leave the harmless to normal cells [7, 33]. VACV owns a large double DNA genome (~200 kbp) encoding almost all needed enzymes and factors for virus replication in the cytoplasm, and therefore physically independence on host-genome modification. In addition, this virus is able to carry multiple large transgenes, up to 25 kbp which allows a variety of genes to be added and engineered without affecting viral replication . Recently, there are many genetically engineered VACVs generated based on wide type VACV backbones [10, 35-36]. These new generations showed significantly improved tumor selectivity and efficacy in both cell culture and animal studies of various cancer cell types as well as some potential candidates are being tested in different tumor models in clinical trials (reviewed in ).