Retroviral Vectors

Basics of the retrovirus virion and infection. Retrovirus virions contain a protein capsid that is lipid encapsulated. Virions range in diameter from 80 to 130 nm. The viral genome is encased within the capsid along with the proteins integrase and reverse transcriptase. The genome consists of two identical positive (sense) single-stranded RNA molecules ranging in size from 3.5 to 10 kilobases. Following cellular entry, the reverse transcriptase synthesizes viral DNA using the viral RNA as its template. The cellular machinery then synthesizes the complementary DNA which is then circularized and inserted into the host genome. Following insertion, the viral genome is transcribed and viral replication is completed. The majority of retroviruses are oncogenic although the degree to which they cause tumors varies from class to class.　

Retroviral receptors. Retroviruses of cats and mice are typically classified by host range. This has led to the use of the following termonology. Ecotropic viruses are viruses which use receptors unique to mice and are only able to replicate within the murine species. Xenotropic viruses uses receptors found on all cells in most species except those of mice. Polytropic and amphotropic viruses use different receptors found in both murine and nonmurine species.　

The retroviral genome. The retroviral genome consists of little more than the genes essential for viral replication. The prototype and simplest genome to describe is that of the Moloney murine leukemia virus (MMLV) in contrast to the highly complex genomes of the HTLV and HIV retroviruses. The genome can be divided into three transcriptional units: gag, pol and env. The gag region encodes genes which comprise the capsid proteins; the pol region encodes the reverse transcriptase and integrase proteins; and the env region encodes the proteins needed for receptor recognition and envelope anchoring. An important feature of the retroviral genome is the long terminal repeat (LTR) regions found at each of the gene. The LTR plays an important role in initiating viral DNA synthesis and its integration as well as regulating transcription of the viral genes.　

MMLV genome:

Retroviral Vectors for Gene Transfer : MMLV　

MMLV vectors. To date, these vectors have been used more than any other gene transfer vehicle. They are produced simply by replacing the viral genes required for replication with the desired genes to be transferred. Thus, the genome in retroviral vectors will contain an LTR at each end with the desired gene or genes in between. The most commonly used system for generating retroviral vectors consists of two parts, the retroviral DNA vector and the packaging cell line.　

Retroviral DNA vectors are plasmid DNAs which contain two retroviral LTRs in the region internal to these LTRs for insertion of the desired gene. A portion of a retroviral plasmid DNA vector, LNSX, is shown below. (Methods in Enzymology. 1993; 217:584.)

The gene of interest is cloned into the multicloning site following the simian virus SV40 promoter (SV). As one can imagine, these plasmid DNAs can be manipulated to meet a variety of needs allowing for multiple applications and the design of very elegant vectors.　

Packaging cell lines provide all the viral proteins required for capsid production and the virion maturation of the vector. These packaging cell lines have been made so that they contain the gag, pol and env genes. Early packaging cell lines contained replication competent retroviral genomes and a single recombination event between this genome and the retroviral DNA vector could result in the production of a wild type virus. This led to the term "helper virus contamination" and to this date, with all viral vectors systems, it is important to insure that the vector be free of helper virus. Current packaging systems require that three homologous recombination events occur for any wild type virus production. Since this is an extremely rare event, current cell lines are considered to be helper free, although it is still wise to test for contamination of any wild type virus. There have been a wide variety of packaging cell lines produced as well as retroviral DNA vectors, and most are commercially available. For the MMLV vectors it is the packaging cell line which determines if the vector is ecotropic, xenotropic or amphotropic. Dependent upon the target cells it is important to ensure that the correct packaging cell line be chosen.　

Vector preparartion. Following insertion of the desired gene into in the retroviral DNA vector, and maintainance of the proper packaging cell line, it is now a simple matter to prepare retroviral vectors. The retroviral DNA vector is transferred into the packaging cell line using calcium phosphate mediated transfection, a procedure which we will describe in later lectures. After approximately two days for virion production, the virus is harvested, and this virus is then used to infect a second packaging cell line. Doing this will allow you to produce a virus with a variety of host ranges. The flow chart below is a quick schematic of vector production. (Methods in Enzymology 1993; 217:589).

Retroviral Vectors for Gene Transfer: HIV-Based Vectors　

Human immunodeficiency viruses (HIV) are the most recently discovered members of the retrovirus family and have led to the new classification of lentivirus. Like other members of the retroviral family, the HIV genome contains the gag, pol and env genes. In addition, several other nonstructural proteins which serve regulatory functions are contained within its genome. Below is the diagram of the HIV genome considered to be one of the most complex among the retroviruses. Notice that the basic genome units (gag, pol and env) and LTRs predominate the genome.

Potential applications. Rather than an in depth focus on HIV biology, we will discuss the current applications of the HIV virus in gene therapy. HIV-based vectors are recent developments in the field of gene therapy and focus on treatment of AIDS. Vector production is similar to that of MMLV vectors, and HIV vectors possess all the same advantages and disadvantages as their MMLV counterparts. An exciting application of HIV vectors is to use HIV vectors to target genes selectively into HIV-containing cells. The goal is that genes delivered by the HIV vector would allow selective killing of any cell previously or subsequently infected with HIV. An example of how this might be accomplished focuses on the tat and rev genes found in the HIV genome. These genes are also found only in cells infected with HIV. By using a tat and rev induced promoter to drive the expression of a toxin gene, any cell infected with HIV should be selectively killed upon expression of the toxin gene. Of course this system does require that a cell become infected with HIV but once infected the cell would terminate as would the HIV virus. A major concern in using HIV vectors is the fact that there is a strong possibility for genetic recombination between infectious HIV and the HIV vector itself. Such an event would result in the HIV vector acting as an infectious HIV particle. One possible method to overcome this risk would be the inclusion of a suicide gene into the HIV vector genome. The suicide gene would make cells infected with the HIV vector genome sensitive to a drug that would poison only those cells containing the HIV vector genome. An example of a suicide gene is the Herpes simplex virus thymidine kinase gene, and ganciclovir is the drug which would yield selective toxicity.

The table below overviews the advantages and disadvantages of retroviral vectors.

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