Isidro Hötzel

The Artiodactyl APOBEC3 Innate Immune Repertoire Shows Evidence for a Multi-Functional Domain Organization that Existed in the Ancestor of Placental Mammals

BackgroundAPOBEC3 (A3) proteins deaminate DNA cytosines and block the replication of retroviruses and retrotransposons. Each A3 gene encodes a protein with one or two conserved zinc-coordinating motifs (Z1, Z2 or Z3). The presence of one A3 gene in mice (Z2–Z3) and seven in humans, A3A-H (Z1a, Z2a-Z1b, Z2b, Z2c-Z2d, Z2e-Z2f, Z2g-Z1c, Z3), suggests extraordinary evolutionary flexibility. To gain insights into the mechanism and timing of A3 gene expansion and into the functional modularity of these genes, we analyzed the genomic sequences, expressed cDNAs and activities of the full A3 repertoire of three artiodactyl lineages: sheep, cattle and pigs.ResultsSheep and cattle have three A3 genes, A3Z1, A3Z2 and A3Z3, whereas pigs only have two, A3Z2 and A3Z3. A comparison between domestic and wild pigs indicated that A3Z1 was deleted in the pig lineage. In all three species, read-through transcription and alternative splicing also produced a catalytically active double domain A3Z2-Z3 protein that had a distinct cytoplasmic localization. Thus, the three A3 genes of sheep and cattle encode four conserved and active proteins. These data, together with phylogenetic analyses, indicated that a similar, functionally modular A3 repertoire existed in the common ancestor of artiodactyls and primates (i.e., the ancestor of placental mammals). This mammalian ancestor therefore possessed the minimal A3 gene set, Z1-Z2-Z3, required to evolve through a remarkable series of eight recombination events into the present day eleven Z domain human repertoire.ConclusionThe dynamic recombination-filled history of the mammalian A3 genes is consistent with the modular nature of the locus and a model in which most of these events (especially the expansions) were selected by ancient pathogenic retrovirus infections.

Host Range of Small-Ruminant Lentivirus Cytopathic Variants Determined with a Selectable Caprine Arthritis- Encephalitis Virus Pseudotype System

ABSTRACT The small-ruminant lentiviruses ovine maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) cause encephalitis, progressive pneumonia, arthritis, and mastitis in sheep and goats. Icelandic MVV strains, which are lytic in tissue culture, have a wide species distribution of functional receptors, which includes human cells. In contrast, functional receptors for the nonlytic CAEV CO are absent from human cells. To determine if the wide species distribution of functional receptors is a common property of MVV strains or related to cytopathic phenotype, we tested the infectivity of viruses pseudotyped with the envelope glycoproteins of MVV K1514, CAEV CO, and lytic and nonlytic North American MVV strains to cells of different species. Replication-defective CAEV proviral constructs lacking the env, tat, andvif genes and carrying the neomycin phosphotransferase gene in the vif-tat region were developed for the infectivity assays. Cotransfection of human 293T cells with these proviral constructs and plasmids expressing CAEV, MVV, or vesicular stomatitis virus envelope glycoproteins produced infectious pseudotyped virus which induced resistance of infected cells to G418. Using these pseudotypes, we confirmed the wide species distribution of Icelandic MVV receptors and the narrow host range of CAEV. However, functional receptors for the two North American MVV strains tested, unlike the Icelandic MVV and similar to CAEV, were limited to cells of ruminant species, regardless of cytopathic phenotype. The results indicate a differential receptor recognition by MVV strains which is unrelated to cytopathic phenotype.

Immunization with plasmid DNA expressing the caprine arthritis-encephalitis virus envelope gene : quantitative and qualitative aspects of antibody response to viral surface glycoprotein

Saanen goats were vaccinated intradermally with plasmid DNA expressing caprine arthritis-encephalitis virus (CAEV) rev-env (pENV) or tat-rev-env (pTAT-ENV) or vaccinia virus expressing CAEV env (rWR-63). Sera from all vaccinated goats immunoprecipitated CAEV surface (SU) and transmembrane (TM) glycoproteins with a dominant response to SU. Antibody response to CAEV SU induced by plasmid DNA was relatively biased toward IgG2, whereas vaccinia rWR-63 induced predominantly IgG1 antibodies to SU. Differential IgG isotype bias established by immunization with plasmid or vaccinia vectors was maintained following subcutaneous boost with purified CAEV SU in Freunds incomplete adjuvant (FIA). Goats injected with pUC18 control plasmid followed by immunization with SU-FIA also had IgG2 biased responses, whereas SU-FIA immunization of a goat primed with vaccinia rWR-SC11 without the CAEV env gene induced a predominant IgG1 response. We conclude that pUC based plasmids expressing the CAEV env gene promote stable type 1 biased immune responses to plasmid encoded SU. IgG2 biased response may be due to innate type 1 priming capacity of immunostimulatory CpG motifs in the pUC ampicillin resistance gene.

Caprine Arthritis-Encephalitis Virus Envelope Surface Glycoprotein Regions Interacting with the Transmembrane Glycoprotein: Structural and Functional Parallels with Human Immunodeficiency Virus Type 1 gp120

ABSTRACT A sequence similarity between surface envelope glycoprotein (SU) gp135 of the lentiviruses maedi-visna virus and caprine arthritis-encephalitis virus (CAEV) and human immunodeficiency virus type 1 (HIV-1) gp120 has been described. The regions of sequence similarity are in the second and fifth conserved regions of gp120, and the similarity is highest in sequences coinciding with β-strands 4 to 8 and 25, which are located in the most virion-proximal region of the gp120 inner domain. A subset of this structure, formed by gp120 β-strands 4, 5, and 25, is conserved in most or all lentiviruses. Because of the orientation of gp120 on the virion, this highly conserved virion-proximal region of the gp120 core may interact with the transmembrane glycoprotein (TM) together with the amino and carboxy termini of full-length gp120. Therefore, interactions between SU and TM of lentiviruses may be structurally related. Here we tested whether the amino acid residues in the putative virion-proximal region of CAEV gp135 comprising putative β-strands 4, 5, and 25, as well as its amino and carboxy termini, are important for stable interactions with TM. An amino acid change at gp135 position 119 or 521, located in the turn between putative β-strands 4 and 5 and near β-strand 25, respectively, specifically disrupted the epitope recognized by monoclonal antibody 29A. Thus, similar to the corresponding gp120 regions, these gp135 residues are located in close proximity to each other in the folded protein, supporting the hypothesis of a structural similarity between the gp120 virion-proximal inner domain and gp135. Amino acid changes in the amino- and carboxy-terminal and putative virion-proximal regions of gp135 increased gp135 shedding from the cell surface, indicating that these gp135 regions are involved in interactions with TM. Our results indicate structural and functional parallels between CAEV gp135 and HIV-1 gp120 that may be more broadly applicable to the SU of other lentiviruses.

Plasmid DNA encoding caprine interferon gamma inhibits antibody response to caprine arthritis-encephalitis virus (CAEV) surface protein encoded by a co-administered plasmid expressing CAEV env and tat genes

This study characterized immune responses of Saanen goats co-immunized with pUC18 based plasmids expressing caprine arthritis-encephalitis virus (CAEV) rev-env (pENV) or tat-rev-env (pTAT-ENV) and a second plasmid encoding caprine interferon gamma (IFNgamma). A previous study reported that immunization with pENV or pTAT-ENV induces IgG2 biased antibody responses to plasmid encoded CAEV surface envelope protein (SU). We show here that regional lymph nodes (LN) of pENV and pTAT-ENV immunized goats contain a dominant subset of SU activated IFNgamma+ Th1 lymphocytes. Co-immunization with pENV or pTAT-ENV and a caprine IFNgamma cDNA expression plasmid (pcIFNgamma) did not potentiate activation of SU responsive Th1 lymphocytes. However, the antibody response to SU encoded by pTAT-ENV was inhibited by co-immunization with pcIFNgamma. Results indicate that synergistic effects of CAEV Tat and IFNgamma suppress the primary adaptive B cell response to plasmid encoded SU.

Sequence similarity between the envelope surface unit (SU) glycoproteins of primate and small ruminant lentiviruses.

Sequence similarity has been previously described in the transmembrane domain unit of envelope glycoproteins of primate and non-primate lentiviruses but similarity between the surface unit (SU) glycoprotein of these viruses is less clear or absent. Here we describe a consistent and significant sequence-similarity between the ovine/caprine lentivirus surface glycoprotein gp135 and the primate lentivirus gp120 in the region between variable loops V2 and V3. The biological relevance of this sequence similarity was indicated by clustering of conserved motifs in regions of structural importance in the human immunodeficiency virus type 1 gp120, conservation of cysteine residue pairs forming disulfide bonds and similar patterns of sequence variation in gp135 and gp120 between strains. The results indicate that SU glycoproteins from primate and small ruminant lentiviruses have structurally related domains.

Dimorphic sequences of rap-1 genes encode B and CD4+ T helper lymphocyte epitopes in the Babesia bigemina rhoptry associated protein-1.

The rhoptry-associated protein-1 (RAP-1) of Babesia bigemina induces protective immune responses in cattle and contains neutralization-sensitive B cell epitopes. RAP-1 variants containing blocks of sequence dimorphism in the amino and carboxy terminal ends are encoded by four nonallelic genes in B. bigemina. Epitopes recognized by RAP-1 specific monoclonal antibodies (MAbs) and bovine CD4+ T cell clones were mapped to determine whether these epitopes are localized in the amino and carboxy terminal dimorphic regions. Four B cell epitopes, including a neutralization-sensitive epitope, required both the amino terminal variant type 1 (NT-1) and non-dimorphic sequences for conformation. Intrachain disulfide bonds were required for at least one of these epitopes, since reduction and alkylation of cysteine residues abolished MAb binding. A fifth B cell epitope was mapped to the carboxy terminal variant type 1 (CT-1). As expected, the neutralizing MAb and two other MAbs requiring NT-1 for epitope binding recognized only the two RAP-1 variants with the NT-1 sequence, while the MAb binding an epitope in CT-1 did not bind RAP-1 variants with CT-2. In contrast, the fourth MAb requiring NT-1 for binding recognized all rap-1 gene products, indicating that dimorphic residues are not part of the epitope recognized by this MAb. Bovine CD4+ T cell clones characterized previously as responding in a strain dependent fashion recognized at least one epitope in CT-1, and did not cross-react with CT-2. A second group of bovine CD4+ T cell clones that responded to multiple parasite strains recognized an epitope in a non-dimorphic region of RAP-1. These data indicate that dimorphic regions of RAP-1 encode unique B and T helper lymphocyte epitopes and may be required for enhanced protective immune responses in cattle.

Immune response to caprine arthritis-encephalitis virus surface protein induced by coimmunization with recombinant vaccinia viruses expressing the caprine arthritis-encephalitis virus envelope gene and caprine interleukin-12.

The objective of this study was to determine if interleukin (IL)-12 can focus an antigen specific type 1 immune response characterized by activation of Th1 lymphocytes and production of IgG2 antibodies in vivo. Saanen goats co-immunized with recombinant vaccinia viruses expressing caprine IL-12 (rRB-IL12) and the caprine arthritis-encephalitis virus (CAEV) envelope (env) gene (rWR-63) were evaluated for development of immune responses to the CAEV env encoded surface glycoprotein (SU). Immune responses were defined by: (i) SU antibody titers; (ii) the ratio of SU IgG1 and IgG2 antibodies; (iii) interferon gamma (IFNgamma) and IL-4 gene expression and proliferative response of SU stimulated lymph node mononuclear cells (LNMC). Apart from enhancement of IFNgamma and IL-4 gene expression in SU stimulated LNMC, rRB-IL12 did not affect the immune response to rWR-63 encoded SU. Thus, localized production of exogenous species specific IL-12 at the site of immunization did not focus initial priming of antigen reactive Th lymphocytes. These results are in contrast to previous studies using inbred mice and raise questions regarding the use of cytokine adjuvants to focus immune responses in outbred animals.

Rapid evolution of two discrete regions of the caprine arthritis-encephalitis virus envelope surface glycoprotein during persistent infection.

Five major regions of sequence diversity between strains (V1-V5) have been described in the caprine arthritis-encephalitis lentivirus (CAEV) envelope surface unit glycoprotein (SU). To determine which of these variable regions is important in persistent infection in vivo, we evaluated SU sequence diversity in five neutralization variants from two goats and proviral DNA from five additional goats infected with CAEV-63 for up to 7 years. Overall amino acid sequence divergence in the SU encoded by provirus and neutralization variants compared to parental CAEV-63 ranged from 1.1 to 4%. However, most of the amino acid substitutions and all of the deletions and insertions were present in two discrete regions designated HV1 and HV2. The HV2 region was variable in all neutralization variants and provirus sequences from most animals. This region overlapped the V4 domain of CAEV SU and the neutralization domain of the closely related ovine maedi-visna lentivirus. HV1 was located in a region of SU strictly conserved in all small ruminant lentivirus strains except CAEV-63. This region only varied in a subset of neutralization variants and proviruses, all derived from goats with arthritis. In contrast, sequences in the V1,V2,V3, and V5 regions were stable in neutralization variants and proviruses from infected goats, indicating that sequence diversity between strains in these regions is not due to selection of variants in persistently infected animals. Our results define two discrete regions of CAEV SU that undergo rapid sequence variation in persistently infected goats which may have important roles in virus-host interactions.

Conservation of Human Immunodeficiency Virus Type 1 gp120 Inner-Domain Sequences in Lentivirus and Type A and B Retrovirus Envelope Surface Glycoproteins

ABSTRACT We recently described a sequence similarity between the small ruminant lentivirus surface unit glycoprotein (SU) gp135 and the second conserved region (C2) of the primate lentivirus gp120 which indicates a structural similarity between gp135 and the inner proximal domain of the human immunodeficiency virus type 1 gp120 (I. Hötzel and W. P. Cheevers, Virus Res. 69:47–54, 2000). Here we found that the seven-amino-acid sequence of the gp120 strand β25 in the C5 region, which is also part of the inner proximal domain, was conserved in the SU of all lentiviruses in similar or identical positions relative to the carboxy terminus of SU. Sequences conforming to the gp135-gp120 consensus for β-strand 5 in the C2 region, which is antiparallel to β25, were then sought in the SU of other lentiviruses and retroviruses. Except for the feline immunodeficiency virus, sequences similar to the gp120-gp135 consensus for β5 and part of the preceding strand β4 were present in the SU of all lentiviruses. This motif was highly conserved among strains of each lentivirus and included a strictly conserved cysteine residue in β4. In addition, the β4/β5 consensus motif was also present in the conserved carboxy-terminal region of all type A and B retroviral envelope surface glycoproteins analyzed. Thus, the antiparallel β-strands 5 and 25 of gp120 form an SU surface highly conserved among the lentiviruses and at least partially conserved in the type A and B retroviral envelope glycoproteins.