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Mechanisms of Poxvirus Immune Evasion
Summary: Michele Barry is studying the proteins that interfere with cell death, focusing on the mechanisms human cells use to expel viruses and the ways that viruses fight back in the vaccinia virus. Her research could provide clues for the development of new therapeutic approaches for human diseases.
To survive and replicate within the host, viruses use multiple strategies to circumvent the antiviral immune response. One strategy takes advantage of the cell suicide response, known as apoptosis, which is an essential part of the immune system's method for detecting and destroying virus-infected cells. To ensure their survival, many viruses have developed strategies to avoid cell death. Poxviruses are a family of viruses that infect a wide range of species and encode a remarkable array of proteins that function to evade the host immune response. The most notorious member of the family is variola virus, the causative agent of smallpox. The World Health Organization initiated an aggressive vaccination program that eradicated smallpox from the human population in 1978. This program used vaccinia virus as the vaccinating agent.
Vaccinia virus, the prototypical poxvirus, is the best-characterized member of the Poxviridae family. The ease with which recombinant poxviruses are generated has made them attractive for medical research. As such, poxviruses are used as gene-therapy vectors and vaccines and currently are being developed as oncolytic viruses in order to destroy cancerous cells. In addition, given their extensive array of immune-evasion strategies, poxviruses serve as model organisms for understanding virus-host interactions and elucidating the mechanisms viruses use to evade the immune response.
Our work aims to understand how members of the poxvirus family inhibit the antiviral immune response and specifically how this family of viruses inhibits apoptosis. Apoptosis is a tightly controlled cellular suicide process that was first fully described in the early 1970s. The best-characterized poxvirus anti-apoptotic protein is the caspase inhibitor crmA/SPI-2. We have recently shown, however, that in addition to crmA/SPI-2, vaccinia virus encodes a second novel inhibitor of apoptosis that functions at the mitochondrion to hinder apoptotic death.
The importance of the mitochondrial checkpoint in apoptotic cells led us to speculate that vaccinia virus might use a mechanism to modulate the mitochondrial arm of the apoptotic pathway. We demonstrated that cells infected with the vaccinia virus strain Copenhagen, which is naturally devoid of crmA/SPI-2, a functional caspase 8 inhibitor, remained resistant to apoptosis induced by the addition of anti-Fas or staurosporine. Additionally, vaccinia virus infection interfered with loss of the mitochondrial membrane potential and cytochrome c release, two hallmarks of mitochondrion-associated apoptosis. Given that no open reading frame with homology to anti-apoptotic proteins existed in vaccinia virus, our observations clearly indicated that vaccinia virus employed a novel strategy to regulate the retention of cytochrome c within the mitochondria and inhibit apoptosis.
To identify the viral gene responsible, we used several vaccinia deletion viruses. With this approach, we determined that cells infected with a deletion virus lacking 55 open reading frames were unable to inhibit cytochrome c release and loss of the mitochondrial membrane potential. Using an infection/transfection strategy, we found that expression of one open reading frame, F1L, restored the ability to inhibit apoptosis in infected cells. Furthermore, an N-terminal green fluorescent protein–tagged version of F1L, localized predominantly to mitochondria and the last 26 amino acids of F1L, was necessary and sufficient for mitochondrial localization.
The F1L open reading frame encodes a protein consisting of 226 amino acids. Extensive database searches failed to reveal any cellular proteins with obvious homology to F1L, providing no clue to the mechanism of action of F1L. F1L orthologues are present only within members of the Orthopoxvirus genus, which includes variola virus, ectromelia virus, and monkeypox virus. Members of other poxvirus genera do not encode F1L but instead encode either an M11L protein, which localizes to the mitochondria and inhibits apoptosis, or, in the case of fowlpox virus, an obvious Bcl-2 homologue. These observations lead to questions about the evolution of three distinct anti-apoptotic proteins within the poxvirus family and their respective mechanisms of action.
The Bcl-2 family of proteins tightly coordinate the mitochondrial events that occur during apoptosis. The Bcl-2 family contains anti-apoptotic members, such as Bcl-2 and Bcl-xl, and a large group of pro-apoptotic members. The pro-apoptotic members are subdivided into multidomain pro-apoptotic proteins, such as Bak and Bax, and the BH3-only proteins, which trigger the activation of Bax and Bak. The activation of Bax and Bak must be tightly regulated to suppress death. Using an immunoaffinity column, we recently found that F1L interacts with the pro-apoptotic protein Bak, a pro-apoptotic member of the Bcl-2 family that is located on the outer mitochondrial membrane. Upon receipt of an an apoptotic trigger, Bak undergoes a series of activation events resulting in the release of cytochrome c and the activation of a family of proteases known as caspases. The exact mechanism of cytochrome c release is unknown and an area of active investigation. Bak activation plays a pivotal role in the release of cytochrome c, and F1L interaction with Bak inhibits the pro-apoptotic function of Bak. Given that both Bak and Bax are activated during apoptosis, we hypothesized that, in addition to inhibiting Bak, F1L may also inhibit Bax activity. Expression of F1L inhibited the activity of Bax, but we failed to detect an obvious interaction with Bax, suggesting that, in addition to inhibiting Bak directly, F1L inhibits Bax indirectly. In support of this idea, we have shown that F1L interacts with the BH3-only protein Bim. Whether F1L, or the other poxvirus-encoded anti-apoptotic proteins, interacts with BH3-only proteins other than Bim is unknown. Despite displaying a lack of sequence homology to anti-apoptotic Bcl-2 family members, F1L appears to be a functional homologue. Future structural data will aid in the further characterization of F1L.
Deletion of F1L from vaccinia virus results in a virus that initiates an apoptotic response in cells, suggesting that in the absence of F1L vaccina virus triggers a cellular death switch. Apoptosis induced by the F1L-deleted virus induces caspase activation and cytochrome c release. This observation has led to an interest in defining the cellular apoptotic response to virus infection. Our future work will include elucidating how specific vaccinia virus mutants trigger apoptosis in cells.
Through the study of viral proteins, we hope to learn more about the regulation of apoptosis and how viruses interfere with the process. We anticipate that knowledge gained from our studies will provide insights into cellular antiviral immune responses and clues for the development of anti-viral therapeutic approaches.
Last updated September 2008
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