The history of retroviruses has been shaped by five distinct parameters.
The history of retroviruses has been shaped by five distinct parameters. Since the discovery that sarcoma could be transmitted among chickens in the early part of the 20th century, the scientific world has also seen the development of the Focus assay and the discoveries of reverse transcriptase, retroviral oncogenes, and the first human retrovirus. It all began in 1910 with Peyton Rous’ observation that a spindle-cell sarcoma tumor isolated from a chicken could cause more sarcoma tumors when put into other chickens. In 1911, he also showed that filtering the cells out and injecting the fluid from the tumors could also induce sarcoma in the chickens. This was the first hint that an infectious agent could be a cause of cancer. The virus causing this sarcoma was eventually called the Rous sarcoma virus, and was shown to contain an RNA genome in 1961.
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One of the characteristics of cells that have been transformed by a virus is that they have loss of control over cell division, loss of contact inhibition, and are anchorage independent. Normal cells will divide until they touch each other or a surrounding barrier and then stop, forming a monolayer. They also stick to the surface they are on and are considered anchorage-dependent. Transformed cells however, keep dividing because all the stop signals have been inhibited. They grow to extremely high densities often building up on top of each other. When transformed cells form clumps they are called foci. Howard Temin and Harry Rubin were the first scientists to develop an assay for transformed cells. Temin and Rubin’s research focused on the Rous sarcoma virus and its transformation of chicken embryo cells. The assay known as the Focus assay is based on the formation of foci by transformed cells. When they infected cells with a known quantity of RSV, the cells formed foci which were then counted. The number of foci formed was shown to be proportional to the concentration of virus used and the measurement of the infectivity of the virus was given as focus-forming units per milliliter. They also demonstrated it only takes one RSV virus particle to transform a cell. Development of the Focus assay allowed for scientists to study virus-host interactions and opened up the door for many more retrovirus discoveries including the role of oncogenes in human cancer.
Retroviruses have genomes composed of RNA, yet they integrate themselves into host genomes which are composed of DNA. In order for the viruses to do this, they must first use an enzyme to convert the RNA genome to DNA. The enzyme used by retroviruses for this task is known as reverse transcriptase and it was discovered by Howard Temin, Mitzutani, and David Baltimore in 1970. Temin and Mizutani were interested in how retroviruses, then called RNA tumor viruses, caused oncogenic transformation and Baltimore was interested in the negative strand RNA genome of the vesicular stomatitis virus. Both discovered the enzymatic activity of reverse transcriptase. This caused a complete overhaul of the idea that the orientation of gene expression is DNA to RNA to proteins. It also allowed for more investigation into how retroviruses insert into host genomes to cause latent infections, and reverse transcriptase is often used today convert mRNAs to cDNAs.
The next great discovery in retroviruses was the identification of viral oncogenes, in particular src. Temperature sensitive mutants of the Rous sarcoma virus were shown to replicate, but not transform cells at certain temperatures. In addition, cells transformed by the virus at favorable temperatures would revert back to their untransformed status when put into temperatures unfavorable to the virus. Peter Duesberg was able to show that transforming ability of the virus was due to one gene and Joan Brugge and Ray Eriksonwere were able to isolate the src protein product. Mark Collett and Art Levinson showed the Src protein is a protein kinase. Dominique Stehelin and Peter Vogt were able to use reverse transcriptase to convert the RNA genome to cDNA and created a probe. They found the probe hybridized to various avian DNAs suggesting the gene was originally from a cellular host. This was proven when the src gene was cloned and it was seen that it had intron structures like that of a cellular gene. These viral oncogenes were termed proto-oncogenes. The importance of the discovery of proto-oncogenes is that it showed cellular oncogenes did not cause cancer unless mutated or overexpressed.
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Finally, the last of the five parameters of retroviral history was the identification of the first human retrovirus. Peter Gallo discovered the human T-cell leukemia virus, HTLV-1. He approached the task by first assaying human lymphocytic leukemias for reverse transcriptase which would only be produced by a virus and not by the host cell. He developed an assay that could distinguish reverse transcriptase from host polymerases. Then he developed a method for growing human leukemic T-cells as a cell line using interleukin-2. Electron microscopy showed virion particles in a T-cell leukemia cell line produced from a patient. Finally, he was able to show that he could isolate the virus from a patient, that it could infect T-cells in vitro, that there were antibodies to the virus in the patient’s blood, and that the virus’ DNA was integrated into the infected T-cells’ genomes. Peter Gallo’s work allowed for the discovery of many more human retroviruses including HIV.
Type C viruses that cause cancer in mammals are grouped in leukemias and sarcomas. Three type C viruses in mammals are the Harvey murine sarcoma virus, the Moloney murine sarcoma virus, and the simian sarcoma virus. The Harvey murine sarcoma viruses are known to contain the ras proto-oncogene. The cellular Ras protein (c-Ras) is a GTPase that activates expression of Type-D cyclins involved in moving a cell from the G1 phase to S phase. c-Ras is active when bound with GTP and inactive when bound with GDP. Certain proteins, in particular GAP, control the activity of Ras by hydrolyzing the GTP to GDP so it becomes inactivated. However, the v-Ras can’t be hydrolyzed and therefore is active at high levels. This means that v-Ras activates expression of the Type-D cyclins constituitively and the cells keep replicating and dividing. Thus, v-Ras transforms cells to promote proliferation. It has been found that the Harvey sarcoma ras is transcribed the most in cells of the brain, muscle, and skin.
The Moloney murine sarcoma virus contains the viral proto-oncogene v-mos that can cause sarcoma even in cells that do not normally express cellular mos (c-mos). c-Mos is a mitogen activated protein kinase (MAPK) that phosphorylates serine and threonine residues on proteins. c-Mos is involved in inducing Meiosis I and Meiosis II and is needed for the maturation of germ cells. It is expressed in germ cells, but not normally in somatic cells. v-Mos can directly activate the MAP kinase pathway in somatic cells despite the fact that they don’t normally express c-Mos. Protein kinase C is known to activate v-Mos through phosphorylation. When v-Mos is activated, it activates the MAP kinase pathway leading to proliferation of cells and its characteristic sarcoma.
The Simian sarcoma virus has a gene v-sis that encodes a homolog of the B chain of platelet derived growth factor (PDGF). PDGF binds to platelet derived growth factor receptor (PDGFR) on cells to stimulate cell growth and differentiation. Altered expression of growth factors is associated with malignancy in cells. The v-sis protein, though produced intracellularly, can localize to the cell membrane surface and bind to the PDGFRs to activate transformation of the cell. It is also interesting that the helper-virus derived env gene which is encoded directly before v-sis needs to be intact for v-sis translation. The env gene contains three methionine codons that have been shown to be required for v-sis translation to be initiated.
Retroviruses have replication schemes that contain some steps similar to other viruse and other steps that are completely unique. Like other viruses they have glycoproteins that recognize receptors for specific molecules on cell surfaces. These glycoproteins determine the host range for the virus to infect. When the virion glycoprotein binds to a cell receptor it recognizes, the virion membrane fuses with the cell membrane. The virus enters the cell during which the virus is uncoated and the viral core is released into the cytoplasm. The RNA genome is reverse transcribed and undergoes strand displacement synthesis to produce double stranded DNA. This is completed by the reverse transcriptase carried in the virion. The double stranded DNA and proteins for integration are brought into the nucleus, usually during mitosis when the nuclear membrane is no longer intact. Integration is accomplished by the viral enzyme integrase, also carried by the virion, and the virus genome is often inserted in genes that are highly transcribed. The integrase creates nicks on both ends of the viral DNA and in the host DNA so they can bind to each other. The virus is now considered to be a provirus. The earlier process of reverse transcription provided the double stranded DNA with long terminal repeats containing a promoter region that can be recognized by host RNA Polymerase II. Transcription and translation of the gag, pol, and env genes produces the capsomeres, reverse transcriptase, and a structural polyprotein containing glycoproteins respectively. Transcription also creates single stranded RNA viral genomes. The Gag protein assembles the virus particles containing the genome and enzymes near the membrane and the particles begin to bud from the membrane. Cellular ESCRT (endosomal sorting complex required for transport) complexes aid in clipping off the bud so the virion is released with an envelope coating.
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