1 Laboratory of Animal Experiment for Disease Model, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan
2 Division of Molecular Immunology, Research Section of Molecular Pathogenesis, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan
3 Gene Techno Science, Sapporo 062-8517, Japan
4 Sankyo Labo Service Corporation, Tokyo 132-0023, Japan
Correspondence
Etsuro Ono
etsuro{at}imm.hokudai.ac.jp
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ABSTRACT |
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MAIN TEXT |
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Interaction between alphaherpesvirus envelope glycoprotein gD and a specific cellular receptor is required for virus entry into mammalian cells (Campadelli-Fiume et al., 1988; Spear et al., 2000
). Binding of alphaherpesviruses to cells occurs primarily through an interaction of virion glycoprotein gC with cell-surface heparan sulfate, whereas fusion between the virion envelope and cell membrane requires glycoproteins gB, gD, gH and gL. To date, five human alphaherpesvirus receptors have been identified: herpesvirus entry mediator (HVEM, also known as HveA and TNFRSF14), a TNF receptor-related protein (Montgomery et al., 1996
); three immunoglobulin superfamily members, HveB (PRR2, nectin-2), HveC (PRR1, nectin-1), and HveD (PVR, CD155); and 3-O-sulfated heparan sulfate (Cocchi et al., 1998
; Geraghty et al., 1998
; Warner et al., 1998
; Shukla et al., 1999
). Several lines of evidence suggest that alphaherpesvirus gD interacts with a cell-surface receptor in addition to heparan sulfate to mediate virus entry and that, in certain cell types, PRV, HSV-1 and BHV-1 can use a common gD receptor for entry (Campadelli-Fiume et al., 1988
; Spear, 1993
).
The nectin family consists of proteins related in sequence and structure to poliovirus receptor (nectin-2/HveB, nectin-1/HveC and nectin-3). They have the same immunoglobulin-like domain structures (one V domain followed by two C2 domains) (Mendelsohn et al., 1989; Eberle et al., 1995
; Lopez et al., 1995
; Reymond et al., 2000
; Satoh-Horikawa et al., 2000
). Human, mouse and porcine nectin-1 have the broadest specificity for mediating alphaherpesvirus entry and are highly conserved in primary sequence. It seems likely that various mammalian forms of nectin-1 will prove to be pan-alphaherpesvirus entry receptors. Nectin-1 and nectin-2 appear to be expressed by a broad range of tissues and cells (Cocchi et al., 1998
; Geraghty et al., 1998
; Warner et al., 1998
). Some isotypes of nectin-1, nectin-2 and nectin-3 can localize to cadherin-based adherens junctions in epithelial cells through binding of their cytoplasmic tails to PDZ domains in afadin, a cytoplasmic protein that binds to actin filaments and components of cadherin-based cell junctions (Mandai et al., 1997
; Asakura et al., 1999
; Takahashi et al., 1999
; Miyahara et al., 2000
; Satoh-Horikawa et al., 2000
).
To construct plasmids expressing soluble forms of porcine HveC (PHveCIg) or murine HveA (MHveAIg), pCXN2 expression vectors containing chimeric genes that encode fusion proteins consisting of an extracellular domain of porcine HveC or murine HveA and the Fc portion of human IgG1 were constructed. The cDNA of the extracellular domain of porcine HveC was amplified as described by Milne et al. (2001). The extracellular domain of murine HveA cDNA was amplified by using RT-PCR, in which an mRNA isolated from concanavalin A (Con A)-stimulated splenocytes obtained from BALB/c mice was used as a template. The PCR primers used to amplify the cDNA were 5'-TAACTCGAGCTCTTGGCCTGAAGTTTC-3' and 5'-TTAAGGATCCGAGGAGCAGGTGGTGTCT-3'. Each cDNA was inserted into the XhoI and BamHI sites of a plasmid carrying IgG1-Fc DNA (Nakagawa et al., 1998
), and the sequence verified. Each plasmid was digested with XbaI and end-filled with Klenow fragments, and then a SalI linker was inserted into the blunt-ended site. Then, the plasmids were digested with XhoI and SalI to obtain the chimeric gene fragments. Each fragment was inserted into an XhoI site of pCXN2 vector (Niwa et al., 1991
). To detect MHveAIg, polyclonal antibodies against MHveAIg were raised in a New Zealand White rabbit immunized with the purified MHveAIg. Vero cells were transfected with each plasmid and stable transformants were selected as described previously (Ono et al., 1995
).
PHveCIg and MHveAIg were expressed under the control of the CAG promoter (cytomegalovirus IE enhancer and chicken -actin promoter; Fig. 1
A) in the transformed cell lines. A total of 35 G418-resistant cells were cloned from different transfection experiments and subsequently grown. A rabbit anti-human HveC antibody-specific band (Krummenacher et al., 1998
) was detected by Western blot analysis in 15 of the 35 cell lines. All of the PHveCIg-expressing cell lines exhibited resistance to PRV infection, indicating an obvious correlation between the resistance to PRV infection and expression of PHveCIg. To examine the resistance to PRV infection in the transformed cell lines, three PHveCIg-expressing cell lines and one non-expressing cell line were chosen (Fig. 1B
). To confirm the PHveCIgPRV gD interaction, immunoprecipitation was performed using the supernatants of Vero cells infected with PRV and the representative cell line expressing PHveCIg (C-A6). Both supernatants were incubated for 1 h, and then protein GSepharose 4B, known to bind to the Fc portion of human IgG1, was added to the reaction mixture to capture the PHveCIggD complex. The results in Fig. 1(C)
show a prominent band reacted with monoclonal antibody against PRV gD (Matsuda et al., 1991
) and a rabbit anti-human HveC antibody-specific band detected by Western blot analysis. In view of the possibility of Fc receptor-mediated binding of the protein to virions having an effect on entry, cell lines expressing MHveAIg were established, and two MHveAIg-expressing cell lines were chosen as control cell lines (Fig. 1B
).
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Consistent with the earlier studies (Geraghty et al., 1998; Krummenacher et al., 1998
), a secreted form of HveC, PHveCIg, blocked infection of HSV-1 in addition to PRV and BHV-1. It is likely that the resistance to alphaherpesvirus infection is mediated at least in part by inhibition of the interaction between gD and simian HveC by the secreted PHveCIg. In the present study, cell-bound PHveCIg is also concerned with the inhibition of PRV infection, in addition to the secreted PHveCIg. Although the mechanism by which cell-bound PHveCIg inhibits alphaherpesvirus infection is unclear, it is likely that PHveCIg sterically interferes with the early assembly of a fusion complex involving multiple components from the alphaherpesvirus envelope and cell plasma membrane. Consequently, the transformed cell lines exhibit the resistance to alphaherpesvirus infection.
In the present study, transformed cell lines expressing PHveCIg showed significant resistance to PRV infection. This resistance was much more striking than that observed in PRV-resistant transformed cell lines expressing transgenes repressing PRV immediate-early gene transcription (Ono et al., 1995, 1998
; Tasaki et al., 2001
). It is noteworthy that PRV entry into the C-A6 cells was almost completely inhibited (Fig. 2B
). The demonstration that PHveCIg expressed in the transformed cell lines is sufficient to compete with PRV infectivity may provide a basis for the future development of agricultural livestock for enhanced disease resistance to pseudorabies.
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ACKNOWLEDGEMENTS |
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Received 2 July 2003;
accepted 1 October 2003.