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Richard W.
Moyer
Ph.D. |
RESEARCH INTERESTS:
Dr. Moyer's long-term interests center about the identification and characterization of genes which contribute to viral (poxvirus) pathogenesis and disease. Towards that end,
he is currently investigating two distinct poxvirus systems, a vertebrate poxvirus (mainly rabbitpox virus) and an insect poxvirus.
Vertebrate poxviruses: In the rabbitpox virus (RPV) system, Dr.
Moyer's laboratory is currently studying specific genes required not for viral growth per se, but rather those designed specifically to control aspects of the host response to infection. Such genes affect cellular processes which include inflammation, chemotaxis of immune cells and apoptosis. Mutations in most such viral genes, while not required for growth of the virus in cell culture, attenuate the virus infection in animals, profoundly influence the ultimate outcome of disease, affect the host range of the virus and also affect the plaque phenotype of the virus in cell culture and the Apock@ color produced upon infection of embryonated eggs.
The laboratory currently focuses on two specific classes of poxvirus encoded genes: (1) three serpin genes encoded by the orthopoxviruses (vaccinia, cowpox virus and and rabbitpox virus and (2) a gene which encodes a chemokine binding protein.
Serpins (serine proteinase inhibitors) are used throughout nature in a multitude of biological processes in which they serve as regulatory proteins. Yet, poxviruses are the only viruses known to encode active serpins. Orthopoxviruses encode three of them. Two (SPI-1 and SPI-2/crmA) while ~50% identical, are completely dissimilar in the critical reactive site loop (RSL) which determines proteinase specificity. SPI-2 (crmA) prevents influx of inflammatory cells into developing viral lesions and is known to inhibit the host interleukin-1β converting enzyme (ICE). ICE, a member of the newly discovered caspase (cysteine proteinases which cleaves after aspartic acid) family. ICE cleaves proIL-1β to form active IL-1β. ICE has also been implicated in the regulation of apoptosis. Cloned SPI-2, when transfected into cells blocks apoptosis in cells otherwise programmed to undergo apoptosis. However, inactivation of the SPI-2 gene within the virus itself does not generally lead to apoptosis of infected cells. Recently,
Dr. Moyer and his fellow researchers have shown that the SPI-2 gene strongly inhibits the action of cytotoxic T-lymphocytes, consistent with the more recently discovered inhibition of granzyme B by SPI-2.
They are now beginning to study two additional poxvirus serpins SERP-2 and SPI-7 from myxoma and swinepox viruses respectively. These serpins, like SPI-2 have aspartic acid as the major determinant in the RSL, yet, both have quite different proteinase inhibitory spectra suggesting novel, yet undiscovered proteinases may be the natural targets of these serpins.
The second serpin, SPI-1 is completely dissimilar in the RSL and has no inhibitory effects on ICE. However, cells infected with viral mutants lacking a functional SPI-1 gene exhibit a reduced host range and surprisingly, infected non-permissive cells exhibit characteristics which are similar to apoptosis.
Recently, Dr. Moyer's laboratory has identified a member of the chymotrypsin family of proteinases which can be inhibited SPI-1.
They have also shown in assays designed to measure killing of cytotoxic T-lymphocytes (CTLs), that both SPI-1 an SPI-2 genes act in concert to inhibit the killing of target cells. How this Anon-caspase@ inhibitor (SPI-1) influences the CTL mediated apoptotic killing of target cells is of keen interest. Recent development of an in vitro system which allows induction of apoptosis in purified nuclei, has made biochemical dissection of the apoptotic pathway and elucidation of the role of each of the SPI genes in regulating apoptosis accessible.
The third serpin, SPI-3, is completely unrelated to either SPI-1 or SPI-2. Originally this gene was shown by us to function to prevent fusion (syncytia) formation in infected cells. We have also shown that this serpin inhibits tPA (tissue plasminogen activator), urokinase, and plasmin. Surprisingly, however, the ability of SPI-3 to inhibit fusion was found to be completely unrelated to proteinase inhibition (serpin) function. This represented the first demonstration of a bifunctional serpin. The relationship between these two activities is currently under investigation. Studies to explore the ability of this protein to control inflammation within a clinical setting are also under investigation. A distantly related homologue of this protein, encoded by myxoma virus (SERP-1) is also under study.
Dr. Moyer and his researchers are also studying the role of a 35 kDa secreted rabbitpox virus encoded protein which binds all C-C chemokines with avidity equal to or exceeding that of typical cytokine receptors. This protein, however, has no recognizable motifs in common with any known cytokine receptor. A homologue of this protein exists in myxoma virus but this protein is only 45% homologous to the corresponding orthopoxvirus protein.
They plan to compare the properties of the two proteins. We have shown that the rabbitpox virus protein binds to C-C chemokines, binding to cellular receptors and subsequent signaling is prevented.
They are now investigating whether this protein can prevent C-C chemokine mediated cellular chemotaxis. We are also studying the specific role of this gene in the virus infection of animals.
Insect poxviruses: Vertebrate poxviruses have been shown to encode many genes which neutralize many aspects of the host immune network. It is this general property of vertebrate poxviruses which served as an initial impetus to begin an examination of the insect poxviruses (entomopoxviruses, EPVs), which are lethal pathogens of their natural insect hosts. Insects, however, lack a typical vertebrate immune system.
Studies have shown that EPVs and in particular the EPV from Amsacta moorei (AmEPV) have enormous potential as biopesticides, expression vectors for foreign proteins, vectors for transient human gene therapies including deployment as recombinant viral vaccine vectors. AmEPV properties consistent with these predictions and applications include: (1) a fatal infection of host insects susceptible to the virus; (2) levels of expression in insect cells which are equal to or exceed that of recombinant baculoviruses and (3) the ability to abortively infect vertebrate cells. Surprisingly, the AmEPV infected vertebrate cells survive the infection and continue to grow, despite entry of the virus into the cell and expression of only early and not late AmEPV genes. It is this last property of the virus which suggests its use as a delivery system for transient gene therapies in humans.
The purpose of Dr. Moyer's current work on AmEPV is to expand molecular knowledge of the virus including further studies of the behavior of the virus in vertebrate cells. These studies are necessary to verify the safety of AmEPV for use as a vector for gene delivery to vertebrates while fully exploiting the biological potential of the virus. Specifically,
Dr. Moyer is interested in why EPVs do not grow in vertebrate hosts as the nature of this block is key to the species barrier of insect viruses. Indeed, the mechanism by which vertebrate cells are susceptible to AmEPV infection, yet continue to grow and divide is also a key to any environmental concerns relating to containment, suitability for gene therapy and the exploitation of the virus as both a
biopesticide.
CURRENT GRANT SUPPORT:
National Institutes of Health
Basic microbiology and infectious diseases (Training Grant)
9/30/96 - 8/31/06
Current Year: $187,328(NCE) Total Award: $767,057
National Institutes of Health/NIAID
Gene Regulation of Mammalian DNA Viruses
2/1/01 - 1/31/06
Current Year: $240,400 Total Award: $1,877,758
National Institutes of Health/NIAID
Pathogenesis and Gene Regulation of Entomopoxvirus
9/27/01 - 7/31/06
Current Year: $274,150 Total Award: $1,450,650
PUBLICATIONS:
1. Barry, M., Heibein, J.A., Lee, Siow-Fong, Moyer, R.W., Green, D.R. and Bleackley, R.C. (2000) Granzyme B short circuits the need for caspase 8 activity during granule-mediated CTL killing by directly cleaving Bid. Mol. And Cell Biol. 20 (11): 3781-94.
2. Turner, P.C., Baquero, M.T., Yuan, S., Thoennes, S.R., Moyer, R.W. (2000). The cowpox virus serpin SPI-3 complexes with and inhibits urokinase-type and tissue-type plasminogen activators and plasmin. Virology 272, 267-280.
3. Wang, Yun Xiang, Turner, P.C., Ness, Traci L., Moon, Kristen B., Schoeb, Trenton R., and
Moyer, R.W. (2000). The cowpox virus SPI-3 and myxoma virus SERP1 seroins are not functionally interchangable despite their similar proteinase inhibition profiles in vitro. *Virology, 272, 281-292.
*Received cover photo for this issue.
4. Bawden A.L., Glassberg, K., Farmerie, W., Diggans, J., Shaw, R., and
Moyer, R.W. (2000). The complete genomic sequence of the Amsacta moorei entomopoxvirus: analysis and comparison with other poxviruses. Virology 274, 120-139.
5. Fang, J. W., and Moyer, R.W. (2000) The effects of the conserved extreme 3' end sequence of hepatitis C virus (HCV) RNA on the in vitro stabilization and translation of the HCV RNA genome. J. Hepatol 33, 632-9.
6. Turner, P.C., and Moyer, R.W. (2001) Serpins enable poxviruses to evade immune defenses. ASM News 67, 201-210.
7. Silverman, G.A., Bird, P.I., Carrell, R.W., Church, F.C., Coughlin, P.B., Gettins, P.G., Irving, J.A., Lomas, D.A., Luke, C.J.,
Moyer, R.W., Pemberton, P.A., Remold-O'Donnell, E., Salvesen, G.S., Travis, J., and Whisstock, J.C. (2001) The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhbition, novel functions, and a revised nomenclature. J Biol Chem 6, 33293-6.
8. Sriskanda, V., Moyer, R.W., and Shuman, S. (2001) NAD+- dependent DNA ligase encolded by a eukaryotic virus. J Biol Chem 276, 36100-9.
9. Turner, P.C. and Moyer, R.W. (2002) Poxvirus Immune Modulators: Functional Insights From Animal Models. Virus Res. Sep; 88 (1-2):35-53.
10. Brum, L.M., Turner, P.C., Devick, H.,m Baquero, T. and Moyer, R.W. (2002) Plasma Membrane Localization and Fusion Inhibitory Activity of the Cowpox Virus Serpin SPI-3 Require a Functional Signal Sequence and the Virus Encoded Hemagglutinin. Virology (In Press)
BOOKS:
Books, Sole Author:
1. Moyer, R.W. (1998) AThe control of apoptosis by poxviruses - an environmental issue@ in Seminars in Virology, (L.K. Miller and E.G. White, eds), Academic Press.
2. Moyer, R.W. (1999) AThe Entomopoxviruses@ in Encyclopedia of Virology, Third Edition (R. Webster and A. Granoff, eds.), Academic Press, pp.474-481.
Books, Co-authored:
3. Turner, P.C. and R.W. Moyer, eds. (1990). "The Molecular Pathogensis of Poxviruses" in The Poxviruses, Springer Verlag, New York, pp. 125-153, 1990.
Books, Contributor of Chapter(s):
4. Moyer ,R. W., and McFadden, G. (2000) APoxvirus Virokines@, (volume 1), in Cytokine Reference, (S. Durum, T. Hirano, J. Vilcek & N. Nicola, eds.), Academic Press, pgs. 285-289, 805-809, 841-847, 1285-1289, 1303-1307, 1429-1436.
5. Bawden, A.L., Y. Li, Glassberg, K., and Moyer, R.W. (2000) AEntomopoxviruses@ in Viral Vectors: Basic Science and Gene Therapy, (A. Cid-Arregui, ed) Eaton Publishing, Natick, MA., p. 341-359.
6. Ness, T. L., and Moyer, R.W. (2000) ASuipoxvirus@ in The Springer Index of Viruses, (C. A. Tidona and G. Darai, eds), Springer Verlag, New York. http://oesys.springer.de/virses/index.HTML.
7. Moyer, R.W. (senior editor), Arif, B.M., Black, D.N., Boyle, D.B., Buller, R.M., D.B., Dumbell, K.R., Esposito, J.J., McFadden, G., Moss, B., Mercer, A.A., Ropp, S., Tripathy, D.N., Upton, C. (1999) APoxviridae@ in Virus Taxonomy, Seventh Report (F.A. Murphy, C.M. Fauquet, D.H.L. Bishop, S.A. Ghabrial, A.W. Jarvis, G.P. Martelli, M.A. Mayo, M.D. Summers, eds.) Academic Press, New York, pp. 137-157.
8. Moyer, R.W. and McFadden, G. (2003) Firus-Encode Caspase Inhibitors. In "Caspases: (Eds. M.Los and H. Walczak) RG Landes Bio Science.
9. McFadden, G. and Moyer, R.W. (2000) APoxvirus Viroceptors@,(volume 2), in Cytokine Reference , (S. Durum, T. Hirano, J. Vilcek & N. Nicola, eds.), Academic Press, pgs. 1601-1605, 1633-1639, 1839-1845, 1855-1859, 2109-2115, 2143-2147.
10. Ness, T.L. and Moyer, R.W. (2002) ASuipoxvirus@ in The Springer Index of Viruses, (C.A. Tidona and G. Darai, eds.), Springer-Verlag, Berlin Heidelberg New York, pp. 902-906.
11. Moyer, R.W. and McFadden, G. (2002) "Poxvirus Virokines", (chapter 35), in Cytokines, (S. Durum, T., Hirano, J. Vilcek & Nicola, ed), Academic Press, in press.
12. McFadden, G. and Moyer, R.W. (2002) "Poxvirus Viroceptors", (chapter 128), in Cytokines, (S. Durum, T. Hirano, J. Vilcek & Nicola, ed), Academic Press, in press.
13. McFadden, G. and Moyer, R.W., (2002) Chapter 18: "Virus-encoded caspase inhibitors" in "Caspases: Their Role in Cell Death and Cell Survival:, edited by Marek Los and Henning Walczak, published by Kluwe, Academic Press.
14. Condit, R.W., Chapter "Poxviruses" for the Textbook: Topley and Wilson' Microbiology and Microbial Infections, 10th Edition published by Edward Arnold Ltd. This is the leading medical student text book in Europe.
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