Viruses cloning

Watanabe S, Temin HM. 1983. Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors. Mol Cell Biol. 3 2241-2249. 

Frenkel N, Spaete RR, Vlazny DA, Deiss LP, Locker H (1982) The herpes simplex virus amplicon—a novel animal-virus cloning vector. hr Eucaryodc Viral Vectors (Gluzman Y, ed), pp 205—209. New York Cold Spring Harbor Laboratory. 

Screening of Identified Mutations in Various Plaque-Purified Virus Clones... 

Spaete RR, Frenkel N. 1982. The herpes simplex virus amplicon a new eucaryotic defective-virus cloning-amplifying vector. Cell 30 295-304... 

It has long been known that cells vary greatly in their susceptibility to viral infection, even when no differences can be demonstrated in their surface receptors for virus adsorption. Moreover, the same virus clone can inhibit macromolecular synthesis in one cell type to a greater extent than another, even though virus yields may not differ significantly. For example, Baxt and Bablanian (1976 ) showed that VSV inhibits nucleic acid synthesis in BHK-21 celts more readily than it does in LLC-MK2 cells. Week and Wagner (1978) also reported that MFC-11 mouse myeloma celts were more susceptible to VSV shut-off of cellular RNA synthesis than were BHK-21 or mouse L cells. 

Abstract In 2007, the world celebrated the 50th anniversary of the discovery of interferon (IFN) by Isaacs and Lindemnann. Subsequently, the IFN-a gene was cloned, fully sequenced and IFN-a was produced in recombinant form. Recombinant IFN-a is now used as the basis for treatment of chronic hepatitis C virus infection and can also be used to treat certain forms of chronic hepatitis B virus infections. IFNs have also been used in other viral infections, although with less success. The antiviral mechanisms of IFNs are reviewed in this chapter as well as the utility of IFNs in the treatment of persistent viral infections. 

Neural progenitor cells Lawrence et al. (2004) When nestin+ multi-potential cells are differentiated towards a neuronal lineage post-infection, there is a negligible increase in virus rephcation. When these cells were transfected with a pNL4-3 molecular clone and stimulated with TNP-a, there is an upregulation in virus production, suggestive of reactivation from latency... 

The genes of the inducible and the constitutively expressed forms of NOS have been cloned and expressed. The expression of inducible NOS in the brain tissue of animals with experimentally induced neurological disorders (boma disease virus and rabies virus in rats), herpes simplex virus (mice) and experimental allergic encephalitis (in rats) suggests that NO produced by induced NOS may be a toxic fector in the pathogenesis of neurological diseases (Koprowski et /., 1993). 

Nagasawa T, Nakajima T, Tachibana K, et al. Molecular cloning and characterization of a murine pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin. Proc Natl Acad Sci U S A 1996 93 14726-14729. 

Linnen, J., era/. (1996). Molecular cloning and disease association of hepatitis G virus A transfusion-transmissible agent. Science 271,505-508. 

As discussed above, alternative recombinant DNA techniques are necessary to efficiently generate genome-scale clone sets. One alternative exploits the ability of the Vaccinia virus DNA topoisomerase I to both cleave and rejoin DNA strands with high sequence specificity (Shuman, 1992a Shuman, 1992b). In the reaction, the enzyme recognizes the sequence 5 -CCCTT and cleaves at the final T whereby a covalent adduct is formed between the 3 phosphate of the cleaved strand and a tyrosine residue in the enzyme (Fig. 4.1). The covalent complex can combine with a heterologous acceptor DNA that has a 5 hydroxyl tail complementary to the sequence on the covalent adduct to create a recombinant molecule (Shuman, 1994). 

Figure 4.4. Schematic illustration of directional topoisomerase cloning of PCR products into the pUNI vector. The PCR product to be cloned has the sequence 5 -CACC appended at the 5 end to direct the orientation of cloning. The Vaccinia virus topoisomerase I enzyme forms a covalent adduct with the cloning vector to create a cloning competent plasmid construct. The loxP site is 5 to the insertion site. The vector and PCR product are designed to fuse the ORF in-frame with loxP.

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