Retroviruses are a family of enveloped RNA viruses that are defined by their common characteristics in structure, replication properties and composition. These viruses are important in research in many different areas of science, such as biology, genetics, medicine, cancer, and biotechnology. The process of pseudotyping for retroviruses, in particular, allows researchers to investigate the entry of these viruses into cells. One particular retrovirus, the Ebola virus, was investigated to illustrate how the virus glycoproteins are integrated into retroviruses that are defective and still enter the virus into other cells. Previous investigations into the role of glycoprotein have found that the glycoprotein plays a key role in the infection of the Ebola virus.
The effects of covalent modifications in the glycoprotein, GP1 and GP2, were analyzed. It was hypothesized that the elimination of one of the cysteines in the cysteine pairs would cause elimination of the other cysteine, resulting in effects of the glycoprotein processing. The results of the study suggest a model for the cysteine bridge for the Ebola virus glycoprotein, which helps determine the specific domain for each cysteines. Overall, the study provided information in the understanding of the structure and function of the glycoprotein in the Ebola virus.
Retroviruses are a family of enveloped RNA viruses that are defined by their common characteristics in structure, replication properties and composition. The viruses range from 80-100 nm in diameter and have an outer lipid envelope composed of viral glycoproteins. The structure and location of their inner protein core is a specific characteristic to the members in this family of viruses. In addition, the replication strategy, known as reverse transcription, of the RNA into a linear double stranded DNA is the hallmark of this family of viruses. Furthermore, retroviruses are broken into two types of categories: simple and complex. The type of category depends of their genetic structure. In a retrovirus there are three coding domains that contain the information for the virion proteins, which supports the synthesis of the internal virions that form the nucleoprotein structures.
This nucleoprotein structures are responsible for the reverse transcriptase. Retroviruses are also broken down into seven additional groups based on evolutionary relationships. Five of the seven groups are composed of oncogenic viruses (cancer causing viruses) and the other groups are referred to as lentiviruses and spumaviruses. (Hughes and Varmus, 1997) Retroviruses are important in research in many different areas of science, such as biology, genetics, medicine, cancer, and biotechnology. For instance current research is geared towards retroviruses that undergo the process of pseudotyping. The process of pseudotyping allows the retrovirus to obtain other types of enveloped viruses to use them for entry into other cells. One particular retrovirus, the Ebola virus, is currently undergoing research for gene therapy at the David Sanders lab at Purdue University.
The researchers at this laboratory have been able to demonstrate pseudotyping in the Ebola virus. For instance, the researchers have illustrated how the Ebola virus glycoproteins are integrated into retroviruses that are defective, but can still facilitate entry of the virus into other cells. In addition, the research allows scientists to study how the Ebola virus is able to enter cells independent of other steps required in the virus type of life cycle. Furthermore, the research has focused on the Ebola virus application in gene transfer and gene-therapy experiments. (2007, Dave Sanders Lab) The current paper discusses research conducted on the modification of the Ebola virus glycoprotein, beginning with a background on the method of retrovirus replication and pseudotyping.
The retrovirus replication method occurs through entry from host cell through a method of attachment with the surface glycoproteins on plasma membrane receptors. This attachment fuses the virus to the host cell membrane. In addition, the virus is very specific to animal species and target cells in the animal. Once the virus attaches to the cell and enters the RNA genome, the RNA is transcribed into a double-stranded DNA molecule, further resulting in sequences of the viral DNA. The transcription of DNA includes two occurrences of the reverse transcriptase from the 5 terminal end and the 3 terminal end on the original template cell. (Hughes and Varmus, 1997)
Pseudotyping and Ebola Virus
Pseudotyping refers to the lack of the structural protein being encoded by a nucleic acid. Basically, pseudotype viruses use recombinant viral gene transduction. Pseudotype viruses are important in research due to the outer shell. Ebola virus is a type of pseudotype virus that is from the Order Mononegavirales, Family Filoviridae. Viruses from the Family Filoviridae cause a hemorrhagic disease in both humans and nonhuman primates. In addition, this virus is classified as a biosafety level 4. (2007, Dave Sanders Lab) Ebola virus has also been classified as an aggressive pathogen and was first recognized near the Ebola River valley in Zaire in the year 1976. Since then, outbreaks of this virus have been observed in central Africa.
In fact there was on outbreak in the Republic of the Congo with125 deaths in 2003. The natural host for the Ebola virus is unknown and therefore, it has been impossible to implement a control system to destroy the virus and prevent transmission to humans. In addition, the virus has a quick progression when infected in a human, which further complicates the control of the virus and decreases the ability to acquire immunity of the disease. Furthermore, there are currently no antiviral drugs or vaccine that works against the virus in the human population. (Sullivan et al., 2003)
The progression of the Ebola virus has been known to take course within 14-21 days after infection. The symptoms of the disease include fever, malaise, and myalgia. As the infection runs its course, the patient starts to undergo severe bleeding to include gastrointestinal bleeding and rashes, as well as lymphopenia and neutrophilia, which are hematological problems. Through the studies that have been conducted, it has been found that when the virus enters the host, it releases cytokines which cause the inflammatory responses, further damaging the liver. The virus then eventually infects the microvascular system, which leads to the terminal stages of the virus, which accounts for the hemorrhagic bleeding and shock. (Sullivan et al., 2003)
The Ebola virus genome has been studied and found to be 19 kb in length and containing a virion envelope glycoprotein, as well as nucleoprotein (NP), matrix proteins, nonstructural proteins and viral polymerase. (Sanchez et al., 2001) The glycoproteins in the Ebola virus are synthesized as a secreted form and binds to endothelial cells. (Sanchez et al., 1998) The binding of the Ebola virus glycoprotein was further analyzed and it was indicated that the binding occurred via cell surface receptors. (Chan et al., 2001) These particular viruses are composed of a single glycoprotein that forms what are called peplomers around the surface of the virion. The glycoproteins in the virus are expressed from the glycoprotein gene. (Jeffers et al., 2002)
Previous investigations into the role of glycoprotein have found that the glycoprotein plays a key role in the infection of the Ebola virus. The glycoprotein allows the virus to enter its contents into other cells, which causes cell damage and the release of cytokines, which is further associated with the fever. Therefore, the glycoprotein expression in the cells causes rounding and detachment and is the one of the seven gene products of the Ebola virus that results in this type of effect. (Simmons et al., 2002; Yang et al., 2000) The peplomers on the surface of the virion are made of glycoproteins, which are attached to the membrane in a lipid bilayer. The peplomers are what facilitate the entrance of the virus into the host cells.
The process involves binding to the receptors, entry of the virus, acidification of the cell, and conformational change in the glycoprotein. Recombinant DNA methods have been used in the study of Ebola virus glycoprotein changes. Through these studies, it has been illustrated that mutations to the peptide sequence in the virus, have prevented virus entry and the termination of the cleavage during Ebola virus glycoprotein production, also prevents virus entry. This type of research is important in the study of these viruses, especially as these viruses are a risk for biosafety. (Jeffers et al., 2002)
In the paper written by Jeffers (et al., 2002) the effects of covalent modifications in the glycoprotein, GP1 and GP2, were analyzed. The study included analysis of a genetic mutation of the plasmid DNA on the specific change on the encoded proteins. The mutated sequences were then further observed to study the changes in the bonding and virus entry. Human kidney cells, mouse embryo cells, and retroviral packaging cells were used in order to study the role of the modification of the Ebola virus glycoprotein. It was hypothesized that the elimination of one of the cysteines in the cysteine pairs would cause elimination of the other cysteine, resulting in effects of the glycoprotein processing. In addition, it was indicated that elimination of both of the cysteines would have no additional effect on the glycoprotein processing since eliminating one of the cysteine destroys the other. (Jeffers et al., 2002)
In order to examine the processing of the Ebola virus glycoprotein, the researched used radioimmunoprecipitation assay (RIPAs). Results of the study found that the mutation of the first cysteine in the glycoprotein resulted in glycoprotein molecules secreted into the medium at a higher rate compared to the normal activity of glycoprotein. It was also found that the mutation of the other cysteines in the glycoprotein resulted in decreased levels in the expression of the glycoprotein molecules. Specially, the mutation of the second and fourth cysteine resulted in no glycoprotein production; however, mutations in the third and fifth cysteine resulting in little amounts of glycoprotein. In addition, the study found that the elimination of the terminal end of the amino chain resulted in an increased amount of glycoprotein section into the medium. (Jeffers et al., 2002)
To further analyze the results of the glycoprotein mutations, the correlation between mutant glycoproteins and retroviruses were analyzed. The results showed that with the mutation of the cysteine-53, which was found to be involved with the glycoprotein formation terminated the connection with glycoprotein and the retrovirus, and transduction of the virus. Viruses containing other cysteine glycoproteins also showed decreased processing when mutated. In addition, the effect of mutation of the N-linked glycosylation sites on the virus was analyzed as well. It was found that the processing of the mutant glycoproteins resulted in similar outcomes. The researchers found that the glycoprotein molecules that had mutations at Asn protein levels, resulted in lower levels of glycosylation, thereby modifying the cell.
The purpose of the study was to study the covalent modifications of the glycoprotein in the Ebola virus. In the study conducted by Jeffers (et al. 2002), the results supported the hypothesis that properties of the Ebola virus glycoproteins and retrovirus glycoproteins exist. The results of the study suggest a model for the cysteine bridge for the Ebola virus glycoprotein, which helps determine the specific domain for each cysteines. The importance of this model proposed is that the same five cysteines found in the Ebola virus glycoprotein are also found in other retroviruses, such as the Marburg, in which researchers have found the disulfide bonds on their envelope surface of the cysteines.
In the study conducted by Jeffers (et al., 2002), it was confirmed that the first cysteine in the glycoprotein was linked to the disulfide linkage and involved in the release of glycoprotein into the medium. In addition, it was suggested that the changes resulted from the elimination of the glycoprotein cysteine bridge. Overall, the study provided information in the understanding of the structure and function of the glycoprotein in the Ebola virus. In addition, the researchers found that eliminating the O-linked glycosylation region in the Ebola virus glycoprotein increase the processing, which could lead to applications in gene therapy. (Jeffers et al., 2002)
The results of this study have led to some significant data suggestions for the role of glycoproteins in disease transmission of the Ebola virus. This information is significant to the medical field since the Ebola virus is considered a biosafety threat. In addition, the researchers have further suggested the possibility of an evolutionary basis for the Ebola virus. The data collected in Jeffers (et al., 2002) illustrated a similarity between the filovirus glycoprotein and the oncogenic retrovirus seen in avian species. This further suggests the possibility of the virus may have evolved from an avian species. With this suggestion and research findings, researchers are able to continue their study into the viral glycoprotein in order to pinpoint exact viral entry of the Ebola virus, as well as other viruses from this viral Family. Further understanding of the mechanisms can lead to the formulation of antiviral therapies and vaccines and prevention of transmission of this fatal disease.
Chan, S. Y., C. J. Empig, F. J. Welte, R. F. Speck, A. Schmaljohn, J. F. Kreisberg, and M. A. Goldsmith. (2001). Folate receptor-Î± is a cofactor for cellular entry by Marburg and Ebola viruses. Cell 106:117-126.
Hughes, C. and Varmus, H. (1997). . The Place of Retroviruses in Biology. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press. Available from: http://www.ncbi.nlm.nih.gov/books/NBK19382/
Jeffers, S.A., Sanders, D.A., Sanchez, A. (2002). Covalent Modifications of the Ebola Virus Glycoprotein. Journal of Virology. 76(24): p. 1246312472
Sanchez, A., A. S. Khan, S. R. Zaki, G. J. Nabel, T. G. Ksiazek, and C. J. Peters. (2001). Filoviridae: Marburg and Ebola viruses, p. 1279-1304. In D. M. Knipe and P. M. Howley (ed.), Fields virology. Lippincott, Williams & Wilkins, Philadelphia, Pa.
Sanchez, A., Z. Yang, L. Xu, G. J. Nabel, T. Crews, and C. J. Peters. (1998). Biochemical analysis of the secreted and virion glycoproteins of Ebola virus. J. Virol. 72:6442-6447.
Simmons, G., R. J. Wool-Lewis, F. Baribaud, R. C. Netter, and P. Bates. (2002). Ebola virus glycoproteins induce global surface protein down-modulation and loss of cell adherence. J. Virol. 76:2518-2528.
Sullivan, N., Yang, Z., and Nabel, G.J. (2003). Ebola Virus Pathogenesis: Implications for Vaccines and Therapies. J. Virol. 77(18): 9733-9737.
Yang, Z.-Y., H. J. Duckers, N. J. Sullivan, A. Sanchez, E. G. Nabel, and G. J. Nabel. (2000). Identification of the Ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury. Nat. Med. 6:886-889.
David Sanders Lab. (2007). Putting Ebola Virus to Good Use-Gene Therapy. Retrieved from: http://bilbo.bio.purdue.edu/~viruswww/Sanders_home/research.html.