Mutational analysis of a principal neutralization domain of visna/maedi virus envelope glycoprotein Benedikta S. Haflidado ´ ttir, Sigrı ´dur Matthı ´asdo ´ ttir, Gudru ´ n Agnarsdo ´ ttir, Sigurbjorg Torsteinsdo ´ ttir, Gudmundur Pe ´ tursson, O ´ lafur S. Andre ´ sson and Valgerdur Andre ´ sdo ´ ttir Correspondence Valgerdur Andre ´ sdo ´ ttir valand@hi.is Institute for Experimental Pathology, University of Iceland, Keldur, Reykjavı ´k, Iceland Received 29 August 2007 Accepted 17 November 2007 We have shown previously that a type-specific neutralization domain is located within a 39 aa sequence in the fourth variable domain of gp135 in visna/maedi virus. We now show that neutralizing antibodies detected early in infection are directed to this epitope, suggesting an immunodominant nature of this domain. Ten antigenic variants were previously analysed for mutations in this region, and all but one were found to be mutated. To assess the importance of these mutations in replication and neutralization, we reconstructed several of the mutations in an infectious molecular clone and tested the resulting viruses for neutralization phenotype and replication. Mutation of a conserved cysteine was shown to alter the neutralization epitope, whilst the replication kinetics in macrophages were unchanged. Mutations modulating potential glycosylation sites were found in seven of the ten antigenic variants. A frequently occurring mutation, removing a potential glycosylation site, had no effect on its own on the neutralization phenotype of the virus. However, adding an extra potential glycosylation site in the region resulted in antigenic escape. The results indicate that the conserved cysteine plays a role in the structure of the epitope and that glycosylation may shield the principal neutralization site. INTRODUCTION Visna/maedi virus (VMV) is a macrophage-tropic lenti- virus causing slowly progressive diseases that mainly affect the lungs and the central nervous system of sheep (Gorrell et al., 1992; 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 glyco- protein 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, immuno- logical and structural studies of HIV-1 envelope glycopro- teins have revealed remarkable diversity and con- formational 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 Journal of General Virology (2008), 89, 716–721 DOI 10.1099/vir.0.83410-0 716 0008-3410 G 2008 SGM Printed in Great Britain