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Retroviral Guide


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Retroviral Guide

A retrovirus is an RNA virus which is duplicated in a host cell by the reverse transcriptase enzyme to produce DNA from its RNA genome. Then, the DNA is incorporated into the host’s genome by an integrase enzyme. The virus thereafter replicates as part of the host cell’s DNA. Recently, retroviral vectors have become increasingly vital tools for the delivery of nucleic acids to a number of cell types in a variety of experimental systems. Apart from being important in laboratory settings for use in both in tissue culture and animal models, they have also been applied in clinical trials to treat genetic diseases.

Retroviridae

Retroviridae is subdivided into Orthoretrovirinae and Spumaretrovirinae. Under Orthoretrovirinae, the various genuses are Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, and Lentivirus, whereas under Spumaretrovirinae only one genus is present spumavirus. The host range of retrovirus includes human, murine, feline (cat), bovine (pig), and avian (birds), and it is dependent upon the viral envelope, structural proteins and glycoproteins, involved in integration. Infections with a number of retroviruses can lead to serious conditions, such as AIDS, neurological diseases, a range of malignancies, and added clinical conditions. Moreover, some retroviruses can even become integrated as DNA in the germ line and passed as endogenous viruses from generation to generation. Using retrovirus in research has built up the need to advance the investigation in detail regarding the viral particles and genomes, their modes of replication, integration, and host immune evasion. The basic replication of retroviruses includes that the ssRNA become double-stranded DNA (dsDNA) and gets into the host genetic material and employs host machinery for the synthesis of new virions (Figure 1).

Figure 1. The reverse of Cricks’ central dogma that occurs in retroviruses.

Retrovirus structure, genome, and proteins

The typical retrovirus structure is enveloped, spherical to pleomorphic in shape, and they have a diameter of 80–100 nm. The different genuses of retrovirus virions have diverse morphology. However, they have their same virion component, which includes the two copies of the genetic material, outer envelope coat, and the viral proteins. Envelope consists of lipids which are obtained from the host plasma membrane during the budding process and the glycoprotein. The retroviral envelope serves three separate functions, including the outer lipid bilayer protects from the extracellular environment, aiding in the entry and way out of host cells by endosomal membrane trafficking, and the facility to straightforwardly enter cells by fusing with their membranes.

Simple and complex retroviruses both contain two copies of linear, nonsegmented, single-stranded RNA of 7-12 kb in length encoding the gag, env, and pol genes. Gag encodes a polyprotein that is translated from an unspliced mRNA which is then cleaved by the viral protease (PR) into the CA, MA, and NC proteins. The Env gene also encodes a polyprotein precursor that is cleaved by a cellular protease into the surface (SU) envelope glycoprotein gp120 and the transmembrane (TM) glycoprotein gp41. Pol is expressed as a Gag-Pol polyprotein because of ribosomal frameshifting during Gag mRNA translation, and encodes the enzymatic proteins PR, RT, and IN. These three proteins are associated with the viral genome within the virion. The RT protein possesses three distinct activities: 1) RNA-dependent DNA polymerase activity, responsible for transcribing the two RNA genomes into a single cDNA; 2) DNA-dependent DNA polymerase activity; and 2) RNase H activity.

Figure 2. Simple and Complex Retrovirus Virion Structure.

Replication of retroviruses

Replication is a multistep process; each step is important for the virus entry and multiplies itself in the host cell. There are seven steps in the replication cycle of the retrovirus (Figure 3). The first step is attachment, in which the retrovirus uses one of its glycoproteins to bind to one or more specific cell-surface receptors on the host cell. Some retroviruses also employ a secondary receptor, referred to as the co-receptor. Some retroviral receptors and coreceptors have been identified. For instance, CD4 and various members of the chemokine receptor family on human T cells act as the HIV receptors and coreceptors.

Figure 3. The seven steps in the replication cycle of the retrovirus

The second and third steps are penetration and uncoating, individually. Retroviruses infiltrate the host cell through direct fusion of the virion envelope with the plasma membrane of the host. The fourth step is replication, which happens after the retrovirus undergoes partial uncoating thus releasing its genome and three essential enzymes, including RT, integrase, and pol gene coding enzymes. In this step, the RNA genome is converted by RT into double-stranded DNA, followed by integration into the host genome, transcription and translation of viral proteins along with the host.

The fifth step is assembly, in which retrovirus capsids are assembled in an immature form. The sixth step is budding, in which the immature viral particle acquires the host plasma membrane. Finally, step seven is "maturation." At this stage, the Pol and Gag proteins of the retrovirus are cleaved by the retroviral protease, thereby forming the mature and infectious form of the virus.

Application of retroviruses

Nowadays, the central goals of retrovirology are the treatment and the prevention of human and non-human diseases and to use this virus in research. Recent studies have suggested that retroviruses can be used in many ways such as model for biological research, for understanding of molecular and cell biology studies. The use of retroviral vectors is one of the most common methods to develop novel gene therapy approaches. Recombinant retroviruses such as the murine leukemia virus (MLV) have the capacity to integrate into the host genome in a stable manner. The stable integration of the engineered vector provides a potential long term therapeutic gene expression to treat patients. Because of its powerful efficiency to alter the genome, it is necessary to be sure that retroviral vectors integration sites are not random in the mammalian genome.

The complete understanding of retrovirus can help the researchers and clinicians to use them in various fields of biology and medicine for the development of new methodologies and techniques. Ongoing investigation on application of retroviruses in gene therapy and anti-cancer agents makes these type a widely studying group. The way retroviruses enter and target the specific cells and integrate itself into the host genome was very fascinating to the scientists globally, and these can be used as models to develop new vectors which could be employed in research.

References:

  1. Saxena, Shailendra K., and Sai V. Chitti. "Molecular Biology and Pathogenesis of Retroviruses." Advances in Molecular Retrovirology. InTech, 2016.
  2. O’KEEFE, E. P. Nucleic acid delivery: lentiviral and retroviral vectors. Labome com, 2013.
  3. Gonçalves A. Use of Retroviral and Lentiviral Vectors to Deliver New Gene Therapies. J Hum Virol Retrovirol, 2017

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