Vaccinia Virus Semaphorin

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Vaccinia Virus Semaphorin Grant McFadden1,* and Richard Moyer2 1

The John P. Robarts Research Institute and Department of Microbiology and Immunology, The University of Western Ontario, 1400 Western Road, London, Ontario, N6G 2V4, Canada 2 Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, PO Box 100266, Gainesville, FL 32610-0266, USA * corresponding author tel: (519)663-3184, fax: (519)663-3847, e-mail: [email protected] DOI: 10.1006/rwcy.2000.03019.

SUMMARY

Structure

Certain poxviruses express a member of the semaphorin superfamily, which are a large group of cellular proteins that were first characterized in nervous tissue as regulators of guidance of sensory neurons, but members probably have immunomodulatory roles as well. The vaccinia virus semaphorin is encoded by the A39R gene (in strain Copenhagen) and is expressed as a secreted protein that binds to a cellular receptor called VESPR (virus-encoded semaphorin protein receptor), but the biological role of this protein has yet to be defined.

A39R is a contiguous gene lacking introns.

BACKGROUND

Discovery Supernates from cells infected with vaccinia virus (Lister strain) in serum-free media were analyzed by two-dimensional gel electrophoresis. A novel protein of 55 kDa (pI of 10) was noted. N-terminal sequencing showed identity with amino acids 15±24 of the A39R ORF of the sequence vaccinia Copenhagen strain. Subsequent analysis revealed the similarity to semaphorins (Comeau et al., 1998).

Alternative names A39R ORF (vaccinia Copenhagen)

Main activities and pathophysiological roles The protein binds to human monocytes and causes large cellular aggregates, suggesting induction of cell surface activation antigens. Treatment of monocytes with A39R leads to the induction of CD54 (ICAM-1). Human monocyte cultures incubated with A39R induce production of the proinflammatory cytokines IL-8 and perhaps IL-6.

GENE AND GENE REGULATION

Accession numbers M35027

Chromosome location The gene is located in the HindIII A (largest fragment resulting from HindIII digestion of viral DNA) fragment of the vaccinia Copenhagen genome. The designation `R' refers to the direction of transcription, which is from left to right.

1434 Grant McFadden and Richard Moyer

Regulatory sites and corresponding transcription factors

GenPept: 335517

Expression of the A39R gene is proposed to be late and dependent on viral DNA replication, because of the presence of the late poxvirus promoter TAAATG motif and the presence of a single early transcription termination signal (TTTTTNT) within the gene.

Sequence See Figure 1.

Description of protein Cells and tissues that express the gene Vaccinia virus (Copenhagen) expresses a 403 amino acid protein, whereas the corresponding ORF in vaccinia WR is 441 amino acids due to an N-terminal extension of 38 amino acids. A related sequence is found in variola (India) virus but is interrupted to comprise three open reading frames: variola ORFs A42 (amino acids 1±71), A43 (amino acids 146±237), and A44 (amino acids 262±398). A segment of the vaccinia virus Copenhagen A39R gene (comprising amino acids 76±110) is found within a molluscum contagiosum ORF (MC163R) of 620 amino acids.

PROTEIN

Accession numbers GenBank: M35027 Figure 1

The protein contains a cleaved 14 amino acid Nterminal signal sequence. Hence the first 14 amino acids are absent in the secreted protein. There are several potential N-linked glycosylation sites. The protein is a member of the semaphorin family. Semaphorins typically contain an N-terminal signal sequence followed by a large (500 amino acid) region of loosely conserved amino acids called a `Sema' domain (Kolodkin et al., 1993).

Important homologies Interestingly, the prototypic members of the Sema family are cellular proteins originally described as being involved in neuronal development in grasshoppers (Kolodkin et al., 1992) and in avian growth cone guidance development (Luo et al., 1993). However, one of the semaphorins (CD100) and the mouse homolog Sema G serve to regulate immune function (Hall et al., 1996; Elhabazi et al., 1997). CD100, an 862 amino acid protein, is expressed on

Amino acid sequence of vaccinia semaphorin.

Vaccinia Virus Semaphorin 1435 Figure 2 A `best fit' alignment of the vaccinia (VAC) (403 amino acid) and alcelaphine herpesvirus type 1 (AHV) (643 amino acid) semaphorins. The two proteins share only 29% amino acid identity.

the surface of hematopoietic cells and antibodies against CD100 affect T cell activity. The alcelaphine herpesvirus type 1 (AHV) (Ensser and Fleckenstein, 1995) also encodes a semaphorin (Lange et al., 1998). AHV is the causative agent of malignant catarrhal fever in ruminants. The AHV protein is considerably longer (643 amino acids) than the vaccinia protein (403 amino acids). The relationship between the AHV and vaccinia proteins is shown in Figure 2. Both the vaccinia and AHV proteins bind to the virus-encoded semaphorin protein receptor (VESPR) isolated (Comeau et al., 1998) as the receptor for the vaccinia protein. While both proteins bind to the VESPR receptor with high avidity, the vaccinia protein is the preferred ligand.

Posttranslational modifications The A39 ORF contains an N-terminal signal sequence and cleavage site between amino acids 14 and 15. There are also several potential N-glycosylation sites at amino acids 53±56, 126±129, and 240±243, as well as several potential myristolylation sites.

RECEPTOR UTILIZATION One receptor for the vaccinia A39R protein has been isolated from cells of lymphoid origin (Comeau et al., 1998). This virus-encoded semaphorin protein receptor (VESPR) is a 1568 amino acid (200 kDa) type I transmembrane glycoprotein, which is a member of the plexin family. The plexin family members are typically type I membrane proteins and were originally identified in the Xenopus nervous system. The extracellular segments of plexins possess three internal repeats of cysteine clusters that are homologous to the cysteine-rich domain of the c-met proto-oncogene protein product. Plexins are involved in cell adhesion. Homologs of this plexin family have been isolated from mouse, human, and Xenopus tissues, although as yet no ligands have been identified. VESPR contains an N-terminal signal sequence, with a potential cleavage site between amino acids 34 and 35. There are 24 putative N-linked glycosylation sites. The protein contains a transmembrane sequence at amino acids 945±965. The extracellular domain of VESPR contains a region of amino acid homology that is also found within the semaphorin proteins themselves (the

1436 Grant McFadden and Richard Moyer Sema domain). This homology is located at amino acids 380±482 of the VESPR sequence and corresponds to a C-terminal 100 amino acid portion of the semaphorin domain. VESPR is found in a variety of tissues and cells including B and T cells from peripheral blood, monocytes and dendritic cells.

IN VITRO ACTIVITIES

In vitro findings The vaccinia semaphorin binds to monocytes through interaction with VESPR and causes them to aggregate. CD54 expression is upregulated and synthesis of IL-8 and IL-6 is induced. Both the AHV semaphorin and cellular CD100 also cause monocyte aggregation.

References Comeau, M. R., Johnson, R., DuBose, R. F., Petersen, M., Gearing, P., VandenBos, T., Park, L., Farrah, T., Buller, R. M., Cohen, J. I., Strockbine, L. D., Rauch, C., and Spriggs, M. K.

(1998). A poxvirus-encoded semaphorin induces cytokine production from monocytes and binds to a novel cellular semaphorin receptor. VESPR Immunity 8, 473±482. Elhabazi, A., Lang, V., Herold, C., Freeman, F. J., Bensussan, A., Boumsell, L., and Bismuth, G. (1997). The human semaphorinlike leukocyte cell surface molecule CD 100 associates with a serine kinase activity. J. Biol. Chem. 272, 23515±23520. Ensser, A., and Fleckenstein, B. (1995). Alcelaphine herpesvirus type 1 has a semaphorin-like gene. J. Gen. Virol. 76, 1063± 1067. Hall, K. T., Boumsell, L., Schultze, J. L., Boussiotis, V. A., Dorfman, D. M., Cardoso, A. A., Bensussan, A., Nadler, L. M., and Freeman, G. J. (1996). Human CD100, a novel leukocyte semaphorin that promotes B-cell aggregation and differentiation. Proc. Natl Acad. Sci. USA 93, 11780±11785. Kolodkin, A. L., Matthes, D. J., O'Connor, T. P., Patel, N. H., Admon, A., Bentley, D., and Goodman, C. S. (1992). Fasciclin IV: Sequence, expression and function during growth cone guidance in the grasshopper embryo. Neuron 9, 831±845. Kolodkin, A. L., Matthes, D. J., and Goodman, C. S. (1993). The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules. Cell 75, 1389±1399. Lange, C., Liehr, T., Goen, M., Gebhart, E., Fleckenstein, B., and Ensser, A. (1998). New eukaryotic semaphorins with close homology to semaphorins of DNA viruses. Genomics 51, 340± 350. Luo, Y., Raible, D., and Raper, J. A. (1993). Collapsin: a protein in brain that induces the collapse and paralysis of neuronal growth cones. Cell 75, 217±227.