Team:Freiburg/Project/The viral vector


The AcCELLerator

The Viral Vector

Retroviral Introduction

Retroviruses are enveloped viruses that are found in fish, avian and mammals. The diameter of a retroviral virion is approximately 80 – 100 nm. The outmost layer of the virus is a lipid bilayer derived from the host cell membrane. It surrounds the spherical capsid in which two identical single stranded RNA strands are located (Fig. 2). They are positive-sense and approximately 7 – 11 kb long [5]. The retroviral life cycle can be divided in two distinct phases: infection and replication. In the following sections we will focus on the Moloney Murine Leukemia Virus (MuLV), which is the origin of the viral vector we are using for our project.

Fig.1: Steps in viral transduction.

Retroviral Tropism And Infection Mechanism

Viruses are classified as ecotropic, polytropic or amphotropic depending on the number of different host cells they are able to infect [1]. The Moloney Murine Leukemia Virus (MuLV) is an ecotropic gamma-retrovirus; it only infects rodent cells (mice and rat). This tropism of the virus is granted by its high specificity towards the mCAT-1 receptor [7]. Once attached to the receptor the MuLV enters the target cell and the single stranded viral RNA is transferred into the cytoplasm.

Within the cell a complementary strand of DNA is created by the viral reverse transcriptase, which is then replicated to form a double strand DNA (dsDNA). During mitosis the dsDNA can enter the nucleus due to the breakdown of the nuclear membrane. Once in the nucleus the viral DNA is further processed by the viral Integrase that recesses the the 3’ ends of the DNA by 2 bp. Double stranded viral DNA is then integrated at random into the host genome and, termed as provirus, it starts to express viral enzymes and proteins.

The Retoviral Genome

The MuLV is a simple retrovirus naturally encoding only three genes called gag, pol and env. These so called trans-gene elements are flanked by long terminal repeats (LTR). The 5 ’ LTR harbors a promoter region, which initiates transcription of the provirus, whereas the 3’ LTR is needed for polyadenylation of the proviral mRNA. The gag (group specific antigen) genes are precursor polyproteins that form the major components building up the core particle, RNA binding proteins and the nucleoprotein core particle. The pol genes encode for the reverse transcriptase, RNase H and the Integrase. It is mandatory for proper processing of the viral RNA genome. The env gene codes for the envelope protein. This protein is responsible for the viral tropism and is located in the viral lipid bilayer which is derived from the host cell membrane. The retroviral psi-packaging sequence is a cis-acting RNA element which allows the transcribed viral RNA to be incorporated into the assembly of the new virus [3]. After proper packaging the virus is leaving the cell and can start the next round of infection.

Fig.2: Schematic retrovirus.

Retroviral Vectors

Retroviral vectors are used to deliver variable gene cargos into cells, a process termed transduction (Fig. 1). The viral vector recognizes a specific receptor on the surface of the cells allowing its internalization and integration into the host genome. To ensure safe handling in the production of viral particles the cis- and trans-acting gene elements are separated from each other. All the structural proteins (gag, pol and env) needed in order to package a new virus is encoded by a helper plasmid within the packaging cell. The genes are under the control of non-MuLV promoters in order to minimize the probability of a recombination event that would lead to the production of self-replicating viruses.

The gene of interest is located, together with the psi packaging sequence, on the viral vector plasmid. Within the packaging cell line structural genes required for assembly of a new virus are transcribed and translated by the helper plasmid. The viral vector plasmid gets also transcribed but lacks any of the structural genes. The end result is a replicative incompetent recombinant retrovirus that includes the transgene, but lacks any of the structural genes to form a new virus in the host target cell [8]. The advantages of retroviral vectors are especially the stable integration of genes and their high specificity but also their flexible genome.


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