1050: Forgot Algebra

What is retrovirus? Retrovirus belongs to the group of RNA viruses. Their genetic information is not stored in DNA, but in RNA, so they are the only known viruses whose genomes are not haploid. The retroviral genome is diploid with two identical single-stranded positive-stranded RNA with long-terminal repeat (LTR) at both ends. It contains strong promoters and enhancers, which play an important role in the transcriptional regulation of viral DNA. The virus core contains reverse transcriptase and integrase. Unlike other RNA viruses, retroviral RNA does not replicate itself. After entering the host cell, the RNA synthesizes double-stranded DNA by reverse transcriptase, and then integrates the double-stranded DNA into the chromosomal DNA of host cell by integrase, thereby forming proviruses and establishing lifelong infection, and transmitting it to offspring cells along with the division of host cells. Classification of retroviruses According to the current international classification criteria of viruses, retroviruses are divided into the following seven genera: 1. Alpharetrovirus 2. Betaretrovirus 3. Gammaretrovirus 4. Deltaretrovirus 5. Epsilonretrovirus 6. Lentivirus 7. Spumavirus Common retroviruses are monkey leukemia virus, murine leukemia virus, hpv retrovirus, HIV and so on. Basic features of retroviruses The most basic feature of retroviruses is that the existence of a reverse transcription process in its life processes in which genetic information is transmitted from RNA to DNA, and during reverse transcription, DNA is integrated into the chromosomal DNA of host cells. The process of retroviral transcription is carried out in the nucleocapsid of the virus. When the virus enters the cell, it uses tRNA as a primer, uses positive-stranded RNA as a template for the virus genetic material, synthesizes complementary negative-stranded DNA, and forms a hybrid double strand of RNA-DNA. The hybrid double-stranded RNA is hydrolyzed by RNA enzyme H activity in reverse transcriptase, and then duplicated into double-stranded DNA by negative-stranded DNA under reverse transcriptase and DNA polymerase activity. The double-stranded DNA is then cyclized and stabilized in the cytoplasm under the protection of the virus nucleocapsid. When the host cell divides, the nuclear membrane disintegrates and chromosomes are exposed. At this time, the nucleocapsid of the virus disintegrates, the viral DNA is released to bind to the integrase, and the combined integrase integrates into the DNA of the host cell chromosome to form a provirus, which establishes a lifelong infection through cell division and transmits it to the offspring cells. Retroviral RNA and retroviral DNA contains long terminal repeats (LTR) , strong promoters and enhancers, which play an important role in the transcription of proviruses. When intracellular conditions are met, the proviruses activate and transcribe a large number of RNA, some of which are used to synthesize viral proteins, some are capped and tailed as genetic material of progeny viruses. Finally, the synthesized progeny viruses are released to the host cells by germination. Construction of retroviral vector Retrovirus packaging requires the following steps. 1. Recombinant DNA technology Recombinant DNA technology can be used to remove viral transformed genes, and to replace viral coding genes with target genes and selective marker genes. The LTRs of viruses can effectively transcribe the inserted foreign genes. These vectors often contain marker genes, such as Neo gene. Therefore, retroviral pre-DNA vectors can be constructed in vitro, including two-terminal LTRs, intermediate insertion of marker genes, bacterial replicons and single-site insertion of exogenous genes. 2. Establishment of packaging cell lines These cells contain integrated defective reverse transcription DNA (the packaging signals has been removed), and no viral particles are assembled despite the presence of a large number of viral proteins. 3. Transfection of packaging cells with constructed retroviral vector The vector inserted into the target gene can be amplified in the cultured cells and then transfected into mouse cells. Because the retroviral vector is defective, it can only be replicated in the presence of auxiliary virus or packaging cells. Therefore, the obtained virus contains both vector virus and auxiliary virus, and the protein provided by the latter can be used to package the carrier RNA instead of its own RNA. Using this helper virus cell, there is no need to label genes. The resulting viruses contain vector RNA, but no helper virus RNA. 4. Infecting target cells with the acquired defective heavy helper virus By interacting with the host cell receptor, it enters the cell and integrates into the host genome through reverse transcription. This viral transcript does not encode any protein needed to produce a viral shell. It usually encodes the target gene. In addition, many non-replicative vectors encode another promoter (pro), which synthesizes the RNA of another gene in the viral genome. Because such vectors do not encode capsid proteins, cells infected by non-replicating vectors cannot produce additional viral particles. Thus, the viral genome is transmitted from one infected cell to another, whereas the viral genome can be transmitted to all offspring through the normal reproductive genetic process of the host gene. Application prospect of retroviral vector Because the advantages of the retroviral vector method are very prominent, it has broad application prospects in both theory and practice. The advantages of this method include: a. retrovirus have high transfection efficiency b. Transcriptional viruses have DNA stages in their life cycle, and their genome structure is simple, with a length of only 8-10Kb, which is convenient for gene manipulation. c. The capacity of the retrovirus is large. The deletion of structural genes in retrovirus genome does not affect the function of other parts. All of them can be replaced by exogenous genes, which can meet the needs of most exogenous genes. d. recombinant retrovirus does not cause cytopathy after infecting cells, and the resulting virus particles are released from the cell membrane by budding and are present in the cell culture supernatant. It is easy to isolate from the host cell and has a low probability of contaminating the DNA and other components of the host cell. e. Recombinant retroviruses lacking structural genes are pseudotyped. They can only integrate exogenous genes into the chromosomes of target cells and cannot replicate to produce progeny viruses, thus improving the safety of retroviruses. Retroviral vector for gene therapy Retroviral vectors for gene therapy have attracted increasing attention because of their high transfer efficiency, stable expression and wide host range. Retroviral vector-based method has become an important strategy in gene therapy. In addition, the retroviral vector method can be used to determine the structure and function of genes, and to carry out developmental biology research, carcinogenesis model research and so on. High concentrations of retroviral vector particles are artificially infected with pre-implantation or post-implantation embryos, and embryos can be directly co-cultured with single-layer cultured cells that can release retroviruses to achieve the purpose of infection.