Many microorganisms afflict humans and other organisms. These include bacteria, viruses, fungi, and others. Viruses, in particular, are much smaller than bacteria (25 nm to 400 nm in size). (Encylopædia Britannica, 2009). They are essentially a compact core of nucleic acid surrounded by a capsid (outer protein coat). Nucleic acids are a type of macromolecule that store genetic information and consist of a nitrogenous base, a 5-carbon sugar (ribose or dexoxyribose), and a phosphate group. While the human genome is exclusively sequestered in DNA (deoxyribose nucleic acid), viruses can utilize both DNA and RNA (ribose nucleic acids). 

---

 

Retrovirus Background

Viruses are mainly classified based on their genetic content; DNA viruses & RNA viruses. They can have single stranded or double stranded DNA or RNA in their genome. A group of RNA viruses are known as retroviruses. Their genome consists of solely single stranded RNA. Following infection of a host cell, the viral genome is replicated via a process known as reverse transcription. This is essentially a reverse of the first step of the Central Dogma of genetics (DNA undergoes transcription to form -> RNA which undergoes translation to form -> protein). In the cytosol (cytoplasm) of a retrovirus-infected cell, however, the viral RNA gets converted to DNA by an enzyme called reverse transcriptase (an RNA-dependent DNA polymerase enzyme). This "provirus" is then randomly integrated into the host genome which undergoes transcription/translation, propagating the viral genome. Because of this random integration, retroviruses display a high degree of mutation (which greatly hampers the research and production of a vaccine). As much as 5-8% of the human genome has been found to have endogenous retroviral gene sequences (endogenous referring to retrovirus genomes that have been passed down from generation to generation vs. exogenous where a retroviral particle infects the host cell). (Betshaw et. al. 2003). Retroviruses may not be lethal to the host cell; however they can continue to produce new viral particles to infect more host cells. Retroviruses also play a role in cancer. If its genome integrates in a host cell near special gene sequences that have been linked to cancer (called proto-oncogenes), it can cause excessive transcription of said proto-oncogene.

 

 

Figure 1: Retrovirus Life Cycle

(Courtesy of Clontech, 2009)

---

 

 

Retrovirus Structure

Retroviruses are enveloped viruses--containing an outer lipid bilayer membrane. This envelope contains special proteins that recognize receptors on the external surface of a host cell, thereby causing infection. Retroviruses are diploid viruses, with 2 single strands of RNA. The RNA is packaged into a protein capsid and consists of 3 primary genes: gag, pol, and env. Gag (group specific antigen) codes for proteins coating the capsid, pol (polymerase) codes for reverse transcriptase and other enzymes (protease and integrase), and env (envelope) codes for proteins making up the envelope. The viral genome is approximately 7 to 12 kilobases in size, wich each end containing special regions known as "long terminal repeats" (LTRs). (NIH, 2009). The LTR on the 5' end of the virus has functions similar to a promoter, while the LTR on the 3' end functions as a terminator sequence.  LTRs essentially are the control center for gene expression of the retrovirus.

Figure 2, (Courtesy of Access Excellence, 2009)

 

 

As seen in Figure 3, retroviruses can enter the host cell via different mechanisms. In each case, the viral envelope is important as the appropriate surface receptors need to be recognized by both the virus and the host cell for fusion to occur.

Figure 3 (Courtesy of Stanford, 2009).

 

Click here to see a short video of a retrovirus life cycle. (Flash Player required). (Courtesy of 1lec, 2009). 

---

 

 

 

Retrovirus History (Key Points)

• 1st retrovirus discovered = Rous sarcoma virus (RSV): sarcomas in chickens
• 1st oncogenes of mammals discovered = mouse mammary tumor virus (MMTV) and the Gross mouse leukemia virus
• 1970s: first use of gene delivery by a virus by Paul Berg (modified SV40 virus from a lambda bacteriophage to infect monkey kidney cells in vitro)
• 1st pathogenic human retrovirus discovered = Human T-Cell Leukemia virus (1981)
• 1983: HIV (Human Immunodeficiency Virus) discovered 

---

 

Retrovirus Classification

 

---

 

Retroviral Vectors

Retroviruses are of importance in the field of genetic engineering. Its genome can be modified and used to insert a genetic sequence of our choice. Because it integrates into the host cell genome, retroviruses can be thought of as a "permanent" gene delivery vehicle, or vector. This process where genetic information is inserted into another cell is known as transduction. Most research on gene therapy focuses on diseases due to mutations and/or abnormalities from a single chromosome or segment thereof (i.e., cystic fibrosis-chromsome 7 and hemophilia A-X chromosome) since it is far simpler to change a single gene than multiple genes in a disorder such as breast cancer. Transduction can take place either in the lab (in vitro) or in a living body (in vivo).

 

Most retroviral gene vectors available commercially today are based on the Moloney murine leukemia viruses (Mo-MLV). It has evolved over millions of years to achieve efficient entry into the host genome. It remains relatively stable following integration and has a wide host range. It is non-pathogenic in humans, can be readily modified using existing techniques (restriction enzymes), and has been shown to have potential in treating genetic disorders. In a study done in 2000 by a French laboratory, a retroviral vector derived from Mo-MLV was shown to show positive effect at treating a disease known as Human Severe Combined Immunodeficiency Disease (SCID-X1). (Calvo, 2000). There are two main types of these vectors. The virus can be rendered incapable of replication by having the gene sequences that code for this function replaced or deleted. Basically the retroviral vector particle contains a "packaging construct" that incorporates all of the viral particles (gag/pol/env) in trans conformation. The vector genome retains its cis conformation elements, while the packaging signal (ψ) is deleted, preventing incorporation of the packaging information into viral particles. The virus is still able to penetrate the host cell, but is unable to cause cell death from excessive replication. In the second type, the virus can still replicate. However, since its genome is now longer, the amount of new genetic information that can be inserted is limited to a small size (less than 8 kb). Unfortunately, Mo-MLV-derived vectors necessitate the cells to be mitotically active. This means that these vectors cannot adequately target tissue that does not normally divide such as neural tissue. Furthermore, there is a risk of mutation from the insertion into the host genome.

 

Figure 4: Mechanism of Retroviral Vector Infection of Host Genome

(Courtesy of Clontech, 2009)

 

 

What about cells that don't have the receptor that responds to the specific retrovirus? This is relatively simple, simply replacing the env gene (which codes for the viral envelope) on the retrovirus with a gene from another virus (i.e., vesicular stomatitis virus, rabies). This is known as pseudotyping the vector.

 

Recently, researchers have been investigating vectors based on lentiviruses (a subclass of retroviruses that include the HIV virus). Lentiviruses can integrate into non-mitotically active cells, which is a clear benefit over that of simple retroviruses. (Uchida et al, 2009). They are more complicated than simple retroviruses, containing 6 additional proteins: tat, rev, vpr, vpu, nef, and vif. Research into lentiviral vectors has currently focused on modifying HIV-1. Studies have shown promising results, showing a positive response from neuronal, muscular, and hepatic tissue, with a limited inflammatory reaction from the injection. (Blmer et al, 1997). This vector certainly offers a wealth of future study possibilities.

 

 

 

Figure 5: Comparison of MLV/HIV-1 Activity in Host Cell Integration

(Courtesy of Wu et al, 2003)

 

---

 

 Limitations

The most important factors in selecting a gene vector are obviously safety (low toxicity is important), cost (it should be cheap for maximal commercial usage), efficacy (it should be stable), and specific (it should only affect the targeted cells). Gene therapy is still in its infant stages and bears with it many risks. For instance, in 1999, a patient died during an experimental gene therapy procedure because the vector affected his immune system instead of the target cells. (PLOS, 2004).

 

Risks associated with current human gene therapy trials include:

 

• viruses can infect more than one type of cell
• when a gene is added to DNA, there is a risk that the new gene can be inserted in the wrong place, possibly causing cancer or other damage.
• when DNA is directly injected into a tumor, there is a risk that the gene can be unintentionally inserted into germ cells (sperm or eggs), producing heritable changes
• overexpression of transferred genes (too much gene product/protein is produced, potentially leading to harm)
• virus can be transmitted to the environment or other patients
• viral vector can cause inflammation or an immune reaction
• ability to ensure precise regulation of the transplated genes by the body's normal physiological signals.
• capacity for therapeutic gene insertion is small (less than 8 kb)

---

 

 Conclusions & Notes

Researchers need to overcome a few problems before gene therapy becomes a common method of treating diseases. There is a need to find effective and efficient ways of delivering genes into the body to safely treat cancers, AIDS and other diseases. These vectors must identify the target cells (i.e., tumor cells) throughout the body and be able to integrate the desired gene sucessfully into the cells. New vectors are also currently being tested (such as adenoviruses). It is impossible to predict when these various obstacles will be overcome although there is a growing increase in the amount of funds being poured and attention being paid into genetic research. Perhaps in a decade or two, physicians and veterinarians will be using genetic science to treat patients suffering from cystic fibrosis, hereditary anemias, and immune system disorders. It may even be possible to treat nongenetic disorders such as hypertension.

---

Glossary (Klug et al., 2009).

• central dogma = genetic information flows from DNA to RNA to proteins.
• long terminal repeat (LTR) = sequence of several hundred base pairs found at the ends of retroviral DNAs.
• nucleoside = a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar molecule.
• nucleotide = a nucleoside covalently linked to a phosphate group.
• promoter = region having a regulatory function and to which RNA polymerase binds prior to the initiation of transcription.
• restriction enzyme = an enzyme (usually a nuclease) that recognizes specific nucleotide sequences in a DNA molecule and cleaves or nicks the DNA at those sites.
• reverse transcriptase = a polymerase that uses RNA as a template to transcribe a single stranded DNA molecule into a product.
• transcription = transfer of genetic information from DNA by the syntehsis of an RNA molecule copied from a DNA template.
• transduction = transfer of virally mediated genes from one bacterium to another or the transfer of eukaryotic genes mediated by a retrovirus.
• translation = the derivation of the amino acid sequence of a polypeptide from the base sequence of an mRNA molecule in association with a ribosome.
• vector = in recombinant DNA, an agent such as a phage or plasmid into whcih a foreign DNA segment will be inserted.

---

 

 References

 

1Lec. (2009). Replication Cycle of a Retrovirus. Flash file retrieved from <http://www.1lec.com/Microbiology/Replication%20Cycle%20of%20a%20Retrovirus/index.html>

 

Access Excellence. (2009). Diagram of a Retrovirus. Image retrieved from <http://www.accessexcellence.org/RC/VL/GG/diagram.php>

 

Belshaw, R.; Pereira, V.; Katzourakis, A.; Talbot, G.; Paces, J.; Burt, A.; Tristem, M. (2003). Long-term reinfection of the human genome by endogenous retroviruses. Proceedings of the National Academy of Sciences USA 101 (14): 4894-99

 

Blmer, U., Naldini, L., Kafri, T., Trono, D., Verma, I. M. and Gage, F. H. (1997). Highly efficient and sustained gene transfer in adult neurones with a lentivirus vector. Journal of Virology 71: 6641-6649.

 

Calvo, M.C. (2000). Gene therapy of human severe combined immunodeficiency (SCID)-X1 Disease. Science. 288(5466): 669-672. 

 

Clontech. (2009). Wild Type Retrovirus Life Cycle Image retrieved from <http://www.clontech.com/support/tools.asp?product_tool_id=54271&tool_id=154902>.

 

Encyclopædia Britannica. (2009). virus (biology) -- Britannica Online Enclopedia." Retrieved from <http://www.britannica.com/EBchecked/topic/630244/virus>

 

Horton, H.R.; Moran, L.A.; Scrimgeour, K.G.; Perry, M.D.; Rawn, J.D. (2006). Principles of Biochemistry 4th edition. Pearson Education Inc. New Jersey. p.583-585

 

Klug, W.S.; Cummings, M.R.; Spencer, C.A. (2007). Essentials of Genetics 6th edition. Pearson Education Inc. New Jersey. p.G2-G15

 

National Center for Biotechnology Information (NCBI). (2009) Retroviruses. National Institutes of Health. Retrieved from <http://www.ncbi.nlm.nih.gov/retroviruses/>.

 

Public Library of Science (PLOS). (2004) Retroviruses integrated into human genome. PLoS Biol, 2, (8), 281. Retrieved from <http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020281>

 

Stanford University's Nolan Lab. (2009). Viral Infection I: Entry. Image retrieved from <http://www.stanford.edu/group/nolan/tutorials/ret_7_readthrough.html>

 

Uchida, N.; Washington, K.N.; Hayakawa, J.; Hsieh, M.M.; Bonifacino, A.C.; Krouse, A.E.; Metzger, M.E.; Donahue, R.E.; Tisdale, J.F.; (2009). Development of a human immunodeficiency virus type 1-based lentiviral vector that allows efficient transduction of both human and rhesus blood cells. Journal of Virology. 83(19): 9854-9862

 

Wu, X.; Li, Y.; Crise, B.; Burgess, S.M. (2003). Transcription start regions in the human genome are favored targets for MLV integration. Science. 300 (5626):1749-51

---

 

External Links

http://www.genetherapynet.com/

http://www.ncbi.nlm.nih.gov/