The signature of purifying selection suggests that mutations in p

The signature of purifying selection suggests that mutations in p15E had been deleterious for KoRV, and would explain why p15E remains conserved among different proviruses. The sequences on which these tests of selection were based may be derived from either exogenous or endogenous KoRVs. However, in a recent study of LTRs in endogenous KoRVs, it was determined that all of the detectable mutations present across endogenous KoRVs had occurred while the virus was still exogenous, and that not enough time had elapsed since endogenization for any mutations to have occurred across the ca. 10,000bp of enKoRV sequences examined (Ishida et al., 2014). Thus the variation detected among KoRVs in our dataset is likely to reflect mutations and selection pressure that occurred only among exogenous KoRVs, even if these were detected among proviruses that had subsequently endogenized.
Despite a high degree of conservation and purifying selection, we found some mutations that may alter the structure and function of specific p15E domains, which are also likely to represent changes present before KoRV endogenized (Ishida et al., 2014). Protein modeling suggested that two radical substitutions in the immunosuppressive domain may affect the local protein conformation and alter the surface charge. These drastic changes could potentially alter the function of the highly conserved immunosuppressive domain. Because these variants were also detected in museum specimens, they are unlikely to represent cloning errors although each of the variants was detected by our study in only a single clonal sequence. There were four other variants in the trimer interface and the two viral ectodomains that affected amino acids of known function. These variants were predicted to alter the protein structure locally and may affect the trimerization, fusion, and infection of the viruses. Additionally, in some other cases the location of the R-group was altered and surface amino acids were exchanged for buried residues and vice versa. These changes could have important functional implications, because they may generate new or alleviate pre-existing antigenic sites, which may result in differences in immune response. Moreover, we found that certain amino Ketorolac tromethamine salt variations (Fig. S2) were predicted to drastically change the local surface charge. Such changes in the KoRV p15E domain could have important biological implications, as has been shown for other viruses, where changes in charge surrounded pockets and electrostatic interactions with small molecules were found to affect trimer assembly, membrane fusion, and viral entry (Ramsdale et al., 1996; Zhu et al., 1998). Some of the variants were also identified in museum specimens (Fig. 2) suggesting that the variants persisted over time. Some variants, especially the ones that were not in frame, may represent proviruses that are not replication competent. Additionally some of the non-synonymous mutations may have occurred in endogenous KoRVs, after the proviruses integrated into the host germ line, in which case sequences with unusual mutations may not be expressed, and thus would have little or no relevance to the utility of a vaccine geared towards koalas unaffected by KoRV.
The transmembrane envelope protein p15E has been used in immunizing animals against gammaretroviruses (Denner et al., 2012; Kaulitz et al., 2011; Langhammer et al., 2011; Waechter et al., 2013). With the exception of FeLV in cats, studies of p15E as a vaccine candidate have not been conducted using the viral host species. Vaccines for FeLV currently rely on inactivated whole virus or on the non-glycosylated surface envelope protein, although neither type provides full protection, and p15E has been proposed as being of potential benefit for vaccination (Fiebig et al., 2003; Langhammer et al., 2006).
The current study found that p15E was highly conserved among KoRV proviruses in koalas from northern and southern Australia and in KoRV variants reported to be exogenous; that functional domains including the immunosuppressive domain and several epitopes were highly conserved between KoRV and similar retroviruses; and that none of the non-synonymous variation detected within KoRV in the two epitopes would greatly affect protein structure. KoRV p15E has been shown to induce neutralizing antibodies (Fiebig et al., 2006), suggesting that KoRV p15E may be a potential candidate for vaccine development. Of course, before widespread use, vaccine trials would be necessary to demonstrate their efficacy in providing protective immunity against KoRV in koalas. Vaccines would also have to be tested for safety, to ensure that they do not trigger unexpected side effects. These may be of particular concern in the case of KoRV since many koalas carry endogenous copies of KoRV that are present in every nucleated cell. In any future studies, it would be important to consider that the effects of any potential vaccine may vary depending on whether or not an individual koala carries endogenous copies of KoRV.