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Institute for Experimental Pathology, University of Iceland, Keldur, Reykjavík, Iceland
Correspondence
Valgerdur Andrésdóttir
valand{at}hi.is
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Sigurdsson, 1954). VMV establishes a lifelong infection and persists in the host despite a strong immune response. In an VMV infection, a type-specific neutralizing antibody response appears 1–6 months after infection, whilst more broadly reacting antibodies appear up to 4 years later (Andresdottir et al., 2002). A similar antibody response has been reported for human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) (Moog et al., 1997; Nara et al., 1990; Rudensey et al., 1998).
The envelope glycoproteins of the lentiviruses are highly variable proteins with a conserved conformation. The protein structure is maintained, with conserved cysteine residues and glycosylation (Li et al., 1993; Tschachler et al., 1990), and there is some structural similarity between the envelope glycoproteins of all lentiviruses (Gallaher et al., 1995; Hotzel & Cheevers, 2001). There are more than 20 potential N-linked glycosylation sites in the outer glycoprotein of VMV, as well as in those of other lentiviruses. It has been suggested that the role of this extensive glycosylation is to shield the virus from neutralizing antibodies (Reitter et al., 1998; Wei et al., 2003). This was first suggested by Huso et al. (1988), who showed that whilst treatment of caprine arthritis encephalitis virus (CAEV) with neuraminidase did not reduce infectivity, it enhanced the kinetics of neutralization of the virus by goat antibodies. Indeed, the lentiviruses seem to have evolved many mechanisms of immune evasion (Frost et al., 2005; Kwong et al., 2002; Wei et al., 2003; Wyatt & Sodroski, 1998). One way for the lentiviruses to escape the immune response may be by continuous change of epitopes through mutation, as first proposed for VMV by Gudnadottir (1974) and further confirmed and extended by Narayan et al. (1977, 1978, 1981). A wealth of genetic, immunological and structural studies of HIV-1 envelope glycoproteins have revealed remarkable diversity and conformational flexibility of these molecules that may result in neutralization escape, either by mutation of the neutralization epitopes or indirectly by conformational masking of epitopes or shielding by glycosylation (Huang et al., 2005; Kwong et al., 2002; Wei et al., 2003; Wyatt & Sodroski, 1998; Wyatt et al., 1998).
The initial type-specific neutralizing antibodies detected in the sera of HIV-1-infected humans are mostly directed to the V3 regions on gp120, hence the term ‘principal neutralization domain’ (PND) (Javaherian et al., 1989). The V3 region plays a central role in determining coreceptor usage and viral tropism (reviewed by Hartley et al., 2005). Early antibodies are also directed to the V1/V2 variable loops, whereas later, more broadly reacting antibodies are probably directed mostly to receptor-binding site surfaces (Wyatt & Sodroski, 1998). In SIV, the V1 and V4 variable regions seem to contain the principal neutralizing determinants (Kinsey et al., 1996; Rudensey et al., 1998). We have mapped mutations altering an early type-specific neutralization response of the VMV strain KV1772 to the fourth variable region of VMV (Skraban et al., 1999).
The Icelandic VMV strains K1514 and K1772 induce a much stronger neutralizing antibody response than other strains of VMV or CAEV (Cheevers et al., 1991, 1993; Narayan et al., 1984). These virus strains are therefore well suited for determining the effect of neutralizing antibodies in a VMV infection. In a previous study from this laboratory, 20 sheep were inoculated with VMV strain K1514, and virus was isolated from blood and cerebrospinal fluid over a period of 7.5 years. All of the sheep mounted a strong, strain-specific neutralizing antibody response 2–5 months after infection, but more broadly reacting neutralizing antibodies appeared up to 4 years later. Most virus strains that were isolated from the sheep up to 7 years after infection were neutralized by the early, type-specific antiserum. However, 10 of 61 virus isolates from blood were antigenic variants (Lutley et al., 1983). The neutralization domain was found to be mutated in nine of the ten antigenic variants (Andresdottir et al., 2002). In this study, we investigated the importance of a conserved cysteine and potential glycosylation changes occurring in the antigenic variants in replication kinetics and neutralization phenotype.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Skraban et al., 1999). Sheep choroid plexus (SCP) cells were grown at 37 °C in a humidified atmosphere of 5 % CO2 in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 2 mM glutamine, 100 IU penicillin ml–1, 100 IU streptomycin ml–1 and either 10 % (growth medium) or 1 % (maintenance medium) lamb serum. Macrophage cultures were established from heparinized blood by sedimentation on Histopaque-1077 (Sigma) as described previously (Skraban et al., 1999).
Reverse transcriptase (RT) assay.
Virus particles from 0.5 ml cell-free supernatant from infected cells were pelleted at 14 000 r.p.m. at 4 °C for 1 h in a microcentrifuge. RT activity was determined by measuring the incorporation of [3H]TTP in the extension of oligo(dT), using poly(rA) as a template as described previously (Andresdottir et al., 1998).
Neutralization assay.
Neutralization assays were carried out with monolayers of SCP cells in 96-well tissue-culture plates as described previously (Skraban et al., 1999). Briefly, 100 TCID50 virus in 0.1 ml DMEM with 2 % normal lamb serum was mixed with 0.1 ml of a twofold dilution of serum sample from infected sheep. The mixtures were incubated at room temperature for 20 h and each dilution was inoculated in quadruplicate onto monolayers of SCP cells. Control cultures were inoculated with virus in the absence of antiserum. Cytopathic effect was monitored after 7, 14, 21 and 28 days. Neutralization titre was calculated as the reciprocal of the serum dilution that caused complete neutralization in 50 % of inoculated cultures.
Construction of mutant viruses.
The molecular clone KV1772 is contained in two plasmids, as has been described previously (Skraban et al., 1999). Mutations altering the potential glycosylation sites and introducing the C–Y change in the neutralization domain were generated by PCR-mediated site-directed mutagenesis. The presence of the mutations was confirmed by DNA sequencing.
DNA transfections were performed with monolayers of ovine fetal synovial (FOS) cells. Lipofectamine 2000 was used as specified by the manufacturer (Invitrogen). Supernatants from transfected cells were tested periodically for RT activity.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, as identified in CAEV by Valas et al. (2000). The positions of V4 and V5 are in good agreement with results that we and others have obtained from studies of variation in VMV, whereas the positions of V1–V3 differ somewhat between studies (Andresdottir et al., 1998; Gjerset et al., 2007; Mwaengo et al., 1997; Sargan et al., 1991). We previously mapped a type-specific neutralization epitope to a region of 39 aa in the fourth variable domain of gp135 (Skraban et al., 1999). To determine whether this region defined an immunodominant epitope, a number of early sera from sheep that had been inoculated with either the virus strain K1514 or the homologous, molecularly cloned virus KV1772 were tested for neutralization of the ‘wild-type’ KV1772 or strain VR1, which has multiple mutations within the 39 aa epitope region in a KV1772 background. We found that whilst KV1772 was neutralized by all sera at a high titre, VR1 escaped all early sera and was only partly neutralized by a serum taken 30 months after inoculation. However, VR1 was neutralized readily by autologous antiserum (Table 1). It thus appears that early sera are directed against this epitope.
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) (Andresdottir et al., 2002). The region contains two conserved cysteines previously suggested to form a cysteine loop (Fig. 1
) (Skraban et al., 1999). Two of the antigenic variants had alterations of one of the conserved cysteines. In one strain, 1518-108, there was a deletion of the cysteine, and in strain 1521-165, a C–Y change had occurred. In both strains, the cysteine change was accompanied by other mutations. To assess the importance of the cysteine in replication proficiency and in the ability of the virus to escape neutralization, a mutation where the cysteine was changed to tyrosine was generated in the molecular clone KV1772. This mutant was called BS4 (Fig. 2). Replication of the mutant virus was examined in blood-derived macrophages and compared with that of parental KV1772. The mutation did not have any detectable effect on the replication proficiency of the virus (Fig. 3). Next, the neutralization sensitivity of the virus was tested. We use SCP cells routinely for testing neutralization and we have shown that the neutralization specificity is unchanged whether we use SCP cells or macrophages as host cells (Skraban et al., 1999). As shown in Table 2, the C–Y mutation rendered the virus completely resistant to neutralization by the type-specific antiserum. We have previously shown that the neutralization epitope is conformational and it is likely that, by changing the cysteine, a disulfide bridge is disrupted (Skraban et al., 1999).
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). The mutation from isolate 1548-123, S–R, in the potential glycosylation site NES was generated in the backbone of KV1772. This mutant was called BS5. As shown in Table 2, there was no change in neutralization phenotype. It appears likely that the mutation acts in concert with other mutation(s) outside the region that was sequenced in the antigenic variants.
Another frequently occurring mutation in the antigenic variants created an additional potential glycosylation site, sometimes accompanying a mutation abolishing the conserved glycosylation site. This resulted in either two glycosylation sites 7 aa apart or a shift of the glycosylation site by 7 aa (Fig. 2). The additional potential glycosylation site was generated by introducing a K–N substitution in the sequence KCS, generating an NCS glycosylation site. This was done on the backbone of both KV1772 (resulting in two glycosylation sites) and the mutant BS5 (resulting in a shift of glycosylation site). These strains were called BS16 and BS17, respectively (Fig. 2). Table 2 shows the neutralization phenotypes of the three glycosylation mutants BS5, BS16 and BS17. The strain with the conserved glycosylation site mutated was neutralized fully by type-specific serum, and so was BS17, with the glycosylation site shifted. However, the mutant virus with two glycosylation sites (BS16) escaped neutralization. The replication proficiency of BS16 was tested and compared with that of the parental strain KV1772 and BS17. Sheep blood-derived macrophages were inoculated with the virus strains and replication kinetics were examined. As shown in Fig. 4, the mutant viruses replicated with similar kinetics to the parental KV1772 strain.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The epitope region contains two conserved cysteines and a conserved potential glycosylation site. We previously reported frequent mutations in one of the conserved cysteines and in the glycosylation site in antigenic variants (Andresdottir et al., 2002). By introducing these mutations into the backbone of the molecular clone KV1772, we have shown that the cysteine is important for the structure of the epitope. We chose to change the cysteine to tyrosine, as this was a mutation found in one of the antigenic variants. We have previously suggested that this cysteine participates in a disulfide bridge, forming a loop that may have a function analogous to that of V3 in HIV-1. We cannot discern whether the neutralization escape was caused by a disrupted disulfide bridge or by changing the amino acid per se. However, two independent escape mutations of this cysteine may indicate that it is important in the conformation of the epitope.
It has been shown by a number of studies with both HIV and SIV that neutralization resistance can be conferred by glycosylation masking the epitopes (Back et al., 1994; Cheng-Mayer et al., 1999; Reitter et al., 1998; Wei et al., 2003). In seven of the ten antigenic variants, we found mutations modulating glycosylation sites of the neutralization domain. The region contains one potential glycosylation site that is well conserved in all sequenced strains of VMV and CAEV (Valas et al., 1997). Yet, this site was mutated in five of the ten antigenic variants. However, disruption of this glycosylation site in a KV1772 background had no effect on neutralization phenotype. This mutation must therefore act in concert with other mutation(s), either within the region or at a distant site. Shifting the glycosylation site by 7 aa, a mutation that occurred in three of the antigenic variants, also had no effect by itself. Two antigenic variants had the two potential glycosylation sites 7 aa apart. This mutant virus was fully replication-proficient in macrophages, but escaped neutralization by the KV1772 strain-specific antiserum. Although it has not been shown that both glycosylation sites are used, by analogy with HIV-1, it seems likely that added glycosylation masks the epitope. The surface glycoprotein of KV1772 has 24 potential glycosylation sites distributed non-randomly, similar to HIV (Fig. 1). The gp120 core of HIV is divided into an inner domain and an outer domain with a short bridging sheet. The inner domain binds antibodies that are non-neutralizing and is referred to as the non-neutralizing face; part of the outer domain is heavily glycosylated and is probably recognized by the host immune system as ‘self’ and is not immunogenic, whilst the surface that interacts with neutralizing antibodies involves parts of both domains and the bridging sheet and includes the V1/V2 and V3 variable loops (Wyatt & Sodroski, 1998). As the secondary/tertiary structure or the receptor-binding site(s) of VMV gp135 are not known, we can only speculate about the relationship of the neutralization domain to the receptor-binding site. Hotzel & Cheevers (2003, 2005) have provided evidence for similarities in the structures of VMV gp135 and HIV-1 gp120. The heavily glycosylated region on gp135 probably forms the outer region, as suggested by Hotzel & Cheevers (2005), and we suggest that the neutralization region constitutes a loop that shields a receptor-binding site analogous to V3 in HIV.
In summary, our studies provide evidence for the importance of the conformational structure of the PND as antigen for neutralizing antibodies. Furthermore, our studies support a role of glycosylation and a changing ‘glycan shield’ in VMV antibody escape. It thus appears that all lentiviruses may use similar strategies for immune evasion and that the Icelandic VMV strains that persist in the host despite high titres of neutralizing antibodies may serve as a useful model for the study of lentiviral persistence in the face of strong immune responses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Andresdottir, V., Skraban, R., Matthiasdottir, S., Lutley, R., Agnarsdottir, G. & Thorsteinsdottir, H. (2002). Selection of antigenic variants in maedi-visna virus infection. J Gen Virol 83, 2543–2551.
Andresson, O. S., Elser, J. E., Tobin, G. J., Greenwood, J. D., Gonda, M. A., Georgsson, G., Andresdottir, V., Benediktsdottir, E., Carlsdottir, H. M. & other authors (1993). Nucleotide sequence and biological properties of a pathogenic proviral molecular clone of neurovirulent visna virus. Virology 193, 89–105.[CrossRef][Medline]
Back, N. K., Smit, L., De Jong, J. J., Keulen, W., Schutten, M., Goudsmit, J. & Tersmette, M. (1994). An N-glycan within the human immunodeficiency virus type 1 gp120 V3 loop affects virus neutralization. Virology 199, 431–438.[CrossRef][Medline]
Cheevers, W. P., Knowles, D. P., Jr & Norton, L. K. (1991). Neutralization-resistant antigenic variants of caprine arthritis-encephalitis lentivirus associated with progressive arthritis. J Infect Dis 164, 679–685.[Medline]
Cheevers, W. P., McGuire, T. C., Norton, L. K., Cordery-Cotter, R. & Knowles, D. P. (1993). Failure of neutralizing antibody to regulate CAE lentivirus expression in vivo. Virology 196, 835–839.[CrossRef][Medline]
Cheng-Mayer, C., Brown, A., Harouse, J., Luciw, P. A. & Mayer, A. J. (1999). Selection for neutralization resistance of the simian/human immunodeficiency virus SHIVSF33A variant in vivo by virtue of sequence changes in the extracellular envelope glycoprotein that modify N-linked glycosylation. J Virol 73, 5294–5300.
Frost, S. D., Wrin, T., Smith, D. M., Kosakovsky Pond, S. L., Liu, Y., Paxinos, E., Chappey, C., Galovich, J., Beauchaine, J. & other authors (2005). Neutralizing antibody responses drive the evolution of human immunodeficiency virus type 1 envelope during recent HIV infection. Proc Natl Acad Sci U S A 102, 18514–18519.
Gallaher, W. R., Ball, J. M., Garry, R. F., Martin-Amedee, A. M. & Montelaro, R. C. (1995). A general model for the surface glycoproteins of HIV and other retroviruses. AIDS Res Hum Retroviruses 11, 191–202.[Medline]
Gjerset, B., Jonassen, C. M. & Rimstad, E. (2007). Natural transmission and comparative analysis of small ruminant lentiviruses in the Norwegian sheep and goat populations. Virus Res 125, 153–161.[CrossRef][Medline]
Gorrell, M. D., Brandon, M. R., Sheffer, D., Adams, R. J. & Narayan, O. (1992). Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes. J Virol 66, 2679–2688.
Gudnadottir, M. (1974). Visna-maedi in sheep. Prog Med Virol 18, 336–349.[Medline]
Hartley, O., Klasse, P. J., Sattentau, Q. J. & Moore, J. P. (2005). V3: HIV's switch-hitter. AIDS Res Hum Retroviruses 21, 171–189.[CrossRef][Medline]
Hotzel, I. & Cheevers, W. P. (2001). Conservation of human immunodeficiency virus type 1 gp120 inner-domain sequences in lentivirus and type A and B retrovirus envelope surface glycoproteins. J Virol 75, 2014–2018.
Hotzel, I. & Cheevers, W. P. (2003). Caprine arthritis-encephalitis virus envelope surface glycoprotein regions interacting with the transmembrane glycoprotein: structural and functional parallels with human immunodeficiency virus type 1 gp120. J Virol 77, 11578–11587.
Hotzel, I. & Cheevers, W. P. (2005). Mutations increasing exposure of a receptor binding site epitope in the soluble and oligomeric forms of the caprine arthritis-encephalitis lentivirus envelope glycoprotein. Virology 339, 261–272.[CrossRef][Medline]
Huang, C. C., Tang, M., Zhang, M. Y., Majeed, S., Montabana, E., Stanfield, R. L., Dimitrov, D. S., Korber, B., Sodroski, J. & other authors (2005). Structure of a V3-containing HIV-1 gp120 core. Science 310, 1025–1028.
Huso, D. L., Narayan, O. & Hart, G. W. (1988). Sialic acids on the surface of caprine arthritis-encephalitis virus define the biological properties of the virus. J Virol 62, 1974–1980.
Javaherian, K., Langlois, A. J., McDanal, C., Ross, K. L., Eckler, L. I., Jellis, C. L., Profy, A. T., Rusche, J. R., Bolognesi, D. P. & other authors (1989). Principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein. Proc Natl Acad Sci U S A 86, 6768–6772.
Kinsey, N. E., Anderson, M. G., Unangst, T. J., Joag, S. V., Narayan, O., Zink, M. C. & Clements, J. E. (1996). Antigenic variation of SIV: mutations in V4 alter the neutralization profile. Virology 221, 14–21.[CrossRef][Medline]
Kwong, P. D., Doyle, M. L., Casper, D. J., Cicala, C., Leavitt, S. A., Majeed, S., Steenbeke, T. D., Venturi, M., Chaiken, I. & other authors (2002). HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites. Nature 420, 678–682.[CrossRef][Medline]
Li, Y., Luo, L., Rasool, N. & Kang, C. Y. (1993). Glycosylation is necessary for the correct folding of human immunodeficiency virus gp120 in CD4 binding. J Virol 67, 584–588.
Lutley, R., Petursson, G., Palsson, P. A., Georgsson, G., Klein, J. & Nathanson, N. (1983). Antigenic drift in visna: virus variation during long-term infection of Icelandic sheep. J Gen Virol 64, 1433–1440.
Moog, C., Fleury, H. J., Pellegrin, I., Kirn, A. & Aubertin, A. M. (1997). Autologous and heterologous neutralizing antibody responses following initial seroconversion in human immunodeficiency virus type 1-infected individuals. J Virol 71, 3734–3741.[Abstract]
Mwaengo, D. M., Grant, R. F., DeMartini, J. C. & Carlson, J. O. (1997). Envelope glycoprotein nucleotide sequence and genetic characterization of North American ovine lentiviruses. Virology 238, 135–144.[CrossRef][Medline]
Nara, P. L., Smit, L., Dunlop, N., Hatch, W., Merges, M., Waters, D., Kelliher, J., Gallo, R. C., Fischinger, P. J. & Goudsmit, J. (1990). Emergence of viruses resistant to neutralization by V3-specific antibodies in experimental human immunodeficiency virus type 1 IIIB infection of chimpanzees. J Virol 64, 3779–3791.
Narayan, O., Griffin, D. E. & Chase, J. (1977). Antigenic shift of visna virus in persistently infected sheep. Science 197, 376–378.
Narayan, O., Griffin, D. E. & Clements, J. E. (1978). Virus mutation during ‘slow infection’: temporal development and characterization of mutants of visna virus recovered from sheep. J Gen Virol 41, 343–352.
Narayan, O., Clements, J. E., Griffin, D. E. & Wolinsky, J. S. (1981). Neutralizing antibody spectrum determines the antigenic profiles of emerging mutants of visna virus. Infect Immun 32, 1045–1050.
Narayan, O., Sheffer, D., Griffin, D. E., Clements, J. & Hess, J. (1984). Lack of neutralizing antibodies to caprine arthritis-encephalitis lentivirus in persistently infected goats can be overcome by immunization with inactivated Mycobacterium tuberculosis. J Virol 49, 349–355.
Reitter, J. N., Means, R. E. & Desrosiers, R. C. (1998). A role for carbohydrates in immune evasion in AIDS. Nat Med 4, 679–684.[CrossRef][Medline]
Rudensey, L. M., Kimata, J. T., Long, E. M., Chackerian, B. & Overbaugh, J. (1998). Changes in the extracellular envelope glycoprotein of variants that evolve during the course of simian immunodeficiency virus SIVMne infection affect neutralizing antibody recognition, syncytium formation, and macrophage tropism but not replication, cytopathicity, or CCR-5 coreceptor recognition. J Virol 72, 209–217.
Sargan, D. R., Bennet, I. D., Cousens, C., Roy, D. J., Blacklaws, B. A., Dalziel, R. G., Watt, N. J. & McConnell, I. (1991). Nucleotide sequence of EV1, a British isolate of maedi-visna virus. J Gen Virol 72, 1893–1903.
Sigurdsson, B. (1954). Observations on three slow infections of sheep (I). Br Vet J 110, 255–270.
Skraban, R., Matthiasdottir, S., Torsteinsdottir, S., Agnarsdottir, G., Gudmundsson, B., Georgsson, G., Meloen, R. H., Andresson, O. S., Staskus, K. A. & other authors (1999). Naturally occurring mutations within 39 amino acids in the envelope glycoprotein of maedi-visna virus alter the neutralization phenotype. J Virol 73, 8064–8072.
Tschachler, E., Buchow, H., Gallo, R. C. & Reitz, M. S., Jr (1990). Functional contribution of cysteine residues to the human immunodeficiency virus type 1 envelope. J Virol 64, 2250–2259.
Valas, S., Benoit, C., Guionaud, C., Perrin, G. & Mamoun, R. Z. (1997). North American and French caprine arthritis-encephalitis viruses emerge from ovine maedi-visna viruses. Virology 237, 307–318.[CrossRef][Medline]
Valas, S., Benoit, C., Baudry, C., Perrin, G. & Mamoun, R. Z. (2000). Variability and immunogenicity of caprine arthritis-encephalitis virus surface glycoprotein. J Virol 74, 6178–6185.
Wei, X., Decker, J. M., Wang, S., Hui, H., Kappes, J. C., Wu, X., Salazar-Gonzalez, J. F., Salazar, M. G., Kilby, J. M. & other authors (2003). Antibody neutralization and escape by HIV-1. Nature 422, 307–312.[CrossRef][Medline]
Wyatt, R. & Sodroski, J. (1998). The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science 280, 1884–1888.
Wyatt, R., Kwong, P. D., Desjardins, E., Sweet, R. W., Robinson, J., Hendrickson, W. A. & Sodroski, J. G. (1998). The antigenic structure of the HIV gp120 envelope glycoprotein. Nature 393, 705–711.[CrossRef][Medline]
Received 29 August 2007;
accepted 17 November 2007.
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) compared with those of the parental strain KV1772 (
) in sheep blood-derived macrophages.
, BS17) compared with those of the parental strain KV1772 (