Please use this identifier to cite or link to this item: http://hdl.handle.net/1946/4024
The Antiretroviral APOBEC3 Proteins of Artiodactyls
Retróveiruhindrinn APOBEC3 í klaufdýrum
The APOBEC3 proteins are a mammalian specific family of nucleic acid cytosine deaminases. A number of family members, including the most studied family member human APOBEC3G, have been shown to be able to inhibit retroviral infection by causing C to U deamination of retroviral DNA during reverse transcription. Some lentiviruses, including HIV-1, can counteract this defense through the viral protein Vif (viron infectivity factor) that triggers APOBEC3 protein ubiquitination and degradation. Because of our prior work with maedi-visna virus our goal was to identify the APOBEC3 genes and proteins of sheep (Ovis aries). This was done by searching for sheep material in databases using conserved motifs from known APOBEC3 proteins. This was challenging since there was very little sheep sequence available and APOBEC3 proteins are under strong positive selection which makes for less conservation of sequence. By utilizing available sequences from related artiodactyl species, we could identify and characterize double domain APOBEC3 proteins from three artiodactyls (cloven hoof ungulates), cattle (Bos taurus), pig (Sus scrofa) as well as sheep. Expression of all of these proteins in E. coli triggered a mutator phenotype. They were also capable of blocking the replication of HIV-1 and to a lesser extent, MLV a distantly related gammaretrovirus of mice, indicating that both the deaminase and the anti-retroviral activites were conserved. The dinucleotide preferences of these proteins were broader than the highly specific preferences of the well studied human APOBEC3F and APOBEC3G. The artiodactyl double-domain APOBEC3 proteins were resistant to inhibition by HIV-1 Vif. Suprisingly, unlike the well characterized human proteins, the deamination activity of these 3 artiodactyl double domain proteins resides in the N-terminal deamination domain.
The genomic sequences of mice and humans revealed a dramatic expansion in the number of APOBEC3 genes – from one in mice to seven in humans. Other primates also appear to have seven APOBEC3 genes. An obvious question was therefore whether this expansion occurred gradually during the evolution of mammals or whether it occurred recently. To begin to address this question, we used degenerate PCR, RACE and database searching to assess the number of APOBEC3 genes in the artiodactyl species we had worked with cattle, sheep and pigs. Several APOBEC3 sequences were recovered but these approaches proved far from definitive. We therefore used APOBEC3-specific hybridization to identify pig and sheep BACs harboring this locus. The full DNA sequence spanning this genomic region was determined by sequencing BAC fragments. Interestingly, sheep proved to have three APOBEC3 genes, whereas pigs had only two. During the course of this work, the cattle genome project released a draft sequence, which also indicated the presence of three APOBEC3 genes. In all three species, read-through transcription and alternative splicing also produced a catalytically active double domain protein, bolstering the innate antiretroviral defenses. These data therefore suggest that the common ancestor of primates and artiodactyls had three APOBEC3 genes and that during mammalian evolution the suidae lineage lost one. These data further suggest the presence of an ongoing selective pressure that has facilitated the gradual expansion of APOBEC3 gene number during the evolution of mammals.
An emerging theme in APOBEC3 research is species specificity. APOBEC3 proteins seem to have a broad inhibitory effect of retroviruses, but each retrovirus seems to have evolved counter-measures that are specific to the APOBEC3 proteins of their host. The usage of cross-species expression might therefore be beneficial in raising barriers to impede cross species viral transmissions or zoonosis. The shortage of allogenic material for transplantation has led to the possible use of materials of non-human origin for transplantation purposes (Xenotransplantation). An unprecedented opportunity for zoonosis occurs when live cells, tissues or organs are transplanted from one species to another. Several xenotransplantation procedures have shown preclinical promise for treating a number of human conditions. Pigs are favorable xenotransplantation sources because of their human-like physiology, large litters, short gestation period and genetic malleability. However, pig to human virus transmission has been a concern since it was shown that porcine endogenous retroviruses (PERVs) could infect human cells in culture. Methods that reduce PERV transmission and possible xenozoonotic infections are therefore desirable. We conceived that introducing a foreign APOBEC3 protein might impede the transmission of viruses from pigs to humans. Long-term co-culture experiments were used to show that PERV transmission form pig to human cells is reduced to nearly undetectable levels by expressing human APOBEC3G in virus-producing pig kidney cells. APOBEC3G interferes with PERV replicaion before the virus immortalizes by integrating into human genomic DNA. In contrast the endogenous APOBEC3 proteins of pigs appeared insufficient to inhibit PERV. These studies indicate that cross-species restriction strategies may help provide safer xenotransplantation resources.