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1.
Virology ; 399(1): 87-97, 2010 Mar 30.
Article in English | MEDLINE | ID: mdl-20097401

ABSTRACT

SIVagm does not induce disease in its African green monkey (AGM) host. In comparison, the hybrid simian-human immunodeficiency virus SHIV89.6P that carries the HIV env gene induces disease in rhesus macaques more rapidly than the SIVmac parent virus. To address the possibility that this enhancement of disease by HIV env would also occur when present in SIVagm, a full-length SIVagm/89.6Penv chimeric lentivirus genome (termed SHIV-MP) was constructed. SHIV-MP replicated similarly to SIVagm in simian peripheral blood mononuclear cells (PBMCs). In inoculated AGMs, rhesus macaques and pig-tailed (PT) macaques the absolute number of CD4(+) T lymphocytes remained at normal levels. The peak levels of productively infected cells in SHIV-MP-infected monkeys ranged from 10(1) to 10(2) per 10(6) PBMCs, while in SIVagm infected macaques the levels were 10-100-fold higher. The env gene of SHIV89.6P therefore appears insufficient to confer acute pathogenicity to a non-pathogenic primate lentivirus due to poor in vivo replication.


Subject(s)
Chlorocebus aethiops/virology , Genes, env/physiology , Simian Immunodeficiency Virus/pathogenicity , Animals , Cell Line , Chlorocebus aethiops/immunology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Genes, env/genetics , HIV-1/physiology , Humans , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/physiology , Viral Load , Virus Replication/genetics , Virus Replication/physiology
2.
J Virol ; 83(15): 7547-59, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19458006

ABSTRACT

The mammalian APOBEC3 (A3) proteins comprise a multigene family of cytidine deaminases that act as potent inhibitors of retroviruses and retrotransposons. The A3 locus on the chromosome 28 of the horse genome contains multiple A3 genes: two copies of A3Z1, five copies of A3Z2, and a single copy of A3Z3, indicating a complex evolution of multiple gene duplications. We have cloned and analyzed for expression the different equine A3 genes and examined as well the subcellular distribution of the corresponding proteins. Additionally, we have tested the functional antiretroviral activity of the equine and of several of the human and nonprimate A3 proteins against the Equine infectious anemia virus (EIAV), the Simian immunodeficiency virus (SIV), and the Adeno-associated virus type 2 (AAV-2). Hematopoietic cells of horses express at least five different A3s: A3Z1b, A3Z2a-Z2b, A3Z2c-Z2d, A3Z2e, and A3Z3, whereas circulating macrophages, the natural target of EIAV, express only part of the A3 repertoire. The five A3Z2 tandem copies arose after three consecutive, recent duplication events in the horse lineage, after the split between Equidae and Carnivora. The duplicated genes show different antiviral activities against different viruses: equine A3Z3 and A3Z2c-Z2d are potent inhibitors of EIAV while equine A3Z1b, A3Z2a-Z2b, A3Z2e showed only weak anti-EIAV activity. Equine A3Z1b and A3Z3 restricted AAV and all equine A3s, except A3Z1b, inhibited SIV. We hypothesize that the horse A3 genes are undergoing a process of subfunctionalization in their respective viral specificities, which might provide the evolutionary advantage for keeping five copies of the original gene.


Subject(s)
Cytidine Deaminase/metabolism , Equine Infectious Anemia/enzymology , Infectious Anemia Virus, Equine/physiology , Multigene Family , APOBEC Deaminases , Animals , Cell Line , Cytidine Deaminase/genetics , Cytosine Deaminase/genetics , Cytosine Deaminase/metabolism , Equine Infectious Anemia/virology , Gene Expression , HeLa Cells , Horses , Humans , Infectious Anemia Virus, Equine/genetics , Molecular Sequence Data
3.
Genome Biol ; 9(3): R48, 2008.
Article in English | MEDLINE | ID: mdl-18315870

ABSTRACT

BACKGROUND: Over the past years a variety of host restriction genes have been identified in human and mammals that modulate retrovirus infectivity, replication, assembly, and/or cross-species transmission. Among these host-encoded restriction factors, the APOBEC3 (A3; apolipoprotein B mRNA-editing catalytic polypeptide 3) proteins are potent inhibitors of retroviruses and retrotransposons. While primates encode seven of these genes (A3A to A3H), rodents carry only a single A3 gene. RESULTS: Here we identified and characterized several A3 genes in the genome of domestic cat (Felis catus) by analyzing the genomic A3 locus. The cat genome presents one A3H gene and three very similar A3C genes (a-c), probably generated after two consecutive gene duplications. In addition to these four one-domain A3 proteins, a fifth A3, designated A3CH, is expressed by read-through alternative splicing. Specific feline A3 proteins selectively inactivated only defined genera of feline retroviruses: Bet-deficient feline foamy virus was mainly inactivated by feA3Ca, feA3Cb, and feA3Cc, while feA3H and feA3CH were only weakly active. The infectivity of Vif-deficient feline immunodeficiency virus and feline leukemia virus was reduced only by feA3H and feA3CH, but not by any of the feA3Cs. Within Felidae, A3C sequences show significant adaptive selection, but unexpectedly, the A3H sequences present more sites that are under purifying selection. CONCLUSION: Our data support a complex evolutionary history of expansion, divergence, selection and individual extinction of antiviral A3 genes that parallels the early evolution of Placentalia, becoming more intricate in taxa in which the arms race between host and retroviruses is harsher.


Subject(s)
Alternative Splicing , Cytosine Deaminase/genetics , Cytosine Deaminase/metabolism , Evolution, Molecular , Genetic Variation , Amino Acid Sequence , Animals , Base Sequence , Cats/metabolism , Cats/virology , Cytosine Deaminase/chemistry , Gene Amplification , Gene Expression , Gene Order , Genome , Molecular Sequence Data , Phylogeny , Retroviridae/physiology , Virus Replication
4.
Nucleic Acids Res ; 35(11): 3784-96, 2007.
Article in English | MEDLINE | ID: mdl-17517765

ABSTRACT

APOBEC3G (A3G), a member of the recently discovered family of human cytidine deaminases, is expressed in peripheral blood lymphocytes and has been shown to be active against HIV-1 and other retroviruses. To gain new insights into the transcriptional regulation of this restriction factor, we cloned and characterized the promoter region of A3G. Transcriptional start sites were identified by 5'-rapid amplification of cDNA ends analysis. Luciferase reporter assays demonstrated that a 1025 bp A3G promoter sequence (from -959 to +66 relative to the major transcriptional start site) displayed constitutive promoter activity. In T cells, the A3G promoter was not inducible by mitogenic stimulation, interferon treatment or expression of HIV-1 proteins. Using a series of 5' deletion promoter constructs in luciferase reporter assays, we identified a 180 bp region that was sufficient for full promoter activity. Transcriptional activity of this A3G core promoter was dependent on a GC-box (located at position -87/-78 relative to the major transcriptional start site) and was abolished after mutation of this DNA element. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that the identified GC-box represented a binding site for the ubiquitous transcription factors specificity protein (Sp) 1 and Sp3.


Subject(s)
Gene Expression Regulation , Nucleoside Deaminases/genetics , Promoter Regions, Genetic , Repressor Proteins/genetics , Sp1 Transcription Factor/metabolism , Sp3 Transcription Factor/metabolism , APOBEC-3G Deaminase , Base Sequence , Binding Sites , Cell Line , Cytidine Deaminase , Humans , Molecular Sequence Data , Polymerase Chain Reaction/methods , RNA Interference , Sp1 Transcription Factor/antagonists & inhibitors , Sp1 Transcription Factor/genetics , Sp3 Transcription Factor/antagonists & inhibitors , Sp3 Transcription Factor/genetics , T-Lymphocytes/metabolism , Transcription Initiation Site , Transcription, Genetic
5.
J Virol ; 81(13): 7048-60, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17459941

ABSTRACT

The productive replication of human immunodeficiency virus type 1 (HIV-1) occurs exclusively in defined cells of human or chimpanzee origin, explaining why heterologous animal models for HIV replication, pathogenesis, vaccination, and therapy are not available. This lack of an animal model for HIV-1 studies prompted us to examine the susceptibility of feline cells in order to evaluate the cat (Felis catus) as an animal model for studying HIV-1. Here, we report that feline cell lines harbor multiple restrictions with respect to HIV-1 replication. The feline CD4 receptor does not permit virus infection. Feline T-cell lines MYA-1 and FeT-1C showed postentry restrictions resulting in low HIV-1 luciferase reporter activity and low expression of viral Gag-Pol proteins when pseudotyped vectors were used. Feline fibroblastic CrFK and KE-R cells, expressing human CD4 and CCR5, were very permissive for viral entry and HIV-long terminal repeat-driven expression but failed to support spreading infection. KE-R cells displayed a profound block with respect to release of HIV-1 particles. In contrast, CrFK cells allowed very efficient particle production; however, the CrFK cell-derived HIV-1 particles had low specific infectivity. We subsequently identified feline apolipoprotein B-editing catalytic polypeptide 3 (feAPOBEC3) proteins as active inhibitors of HIV-1 particle infectivity. CrFK cells express at least three different APOBEC3s: APOBEC3C, APOBEC3H, and APOBEC3CH. While the feAPOBEC3C did not significantly inhibit HIV-1, the feAPOBEC3H and feAPOBEC3CH induced G to A hypermutations of the viral cDNA and reduced the infectivity approximately 10- to approximately 40-fold.


Subject(s)
Disease Models, Animal , HIV Infections/metabolism , HIV-1/metabolism , Virus Replication , Animals , Base Sequence , CD4 Antigens/metabolism , Cats , Cell Line , Cytosine Deaminase/metabolism , Evaluation Studies as Topic , Fusion Proteins, gag-pol/biosynthesis , Humans , Molecular Sequence Data , Pan troglodytes , Receptors, CCR5/metabolism , Species Specificity , Terminal Repeat Sequences
6.
Proc Natl Acad Sci U S A ; 102(22): 7982-7, 2005 May 31.
Article in English | MEDLINE | ID: mdl-15911774

ABSTRACT

Genome hypermutation of different orthoretroviruses by cellular cytidine deaminases of the APOBEC3 family during reverse transcription has recently been observed. Lentiviruses like HIV-1 have acquired proteins preventing genome editing in the newly infected cell. Here we show that feline foamy virus (FFV), a typical member of the foamy retrovirus subfamily Spumaretrovirinae, is also refractory to genome deamination. APOBEC3-like FFV genome editing in APOBEC3-positive feline CRFK cells only occurs when the accessory FFV Bet protein is functionally inactivated. Editing of bet-deficient FFV genomes is paralleled by a strong decrease in FFV titer. In contrast to lentiviruses, cytidine deamination already takes place in APOBEC3-positive FFV-producing cells, because edited proviral DNA genomes are consistently present in released particles. By cloning the feline APOBEC3 orthologue, we found that its homology to the second domain of human APOBEC3F is 48%. Expression of feline APOBEC3 in APOBEC3-negative human 293T cells reproduced the effects seen in homologous CRFK cells: Bet-deficient FFV displayed severely reduced titers, high-level genome editing, reduced particle release, and suppressed Gag processing. Although WT Bet efficiently preserved FFV infectivity and genome integrity, it sustained particle release and Gag processing only when fe3 was moderately expressed. Similar to lentiviral Vif proteins, FFV Bet specifically bound feline APOBEC3. In particles from Bet-deficient FFV, feline APOBEC3 was clearly present, whereas its foamy viral antagonist Bet was undetectable in purified WT particles. This is the first report that, in addition to lentiviruses, the foamy viruses also developed APOBEC3-counter-acting proteins.


Subject(s)
Cytidine Deaminase/metabolism , Genome , Proviruses/genetics , Retroviridae Proteins/metabolism , Spumavirus/genetics , Amino Acid Sequence , Animals , Blotting, Western , Cats , Cell Line , Cloning, Molecular , Codon/genetics , Cytidine Deaminase/genetics , DNA Primers , DNA, Complementary/genetics , Humans , Immunoprecipitation , Molecular Sequence Data , Mutation/genetics , Plasmids/genetics , Retroviridae Proteins/genetics , Sequence Alignment , Spumavirus/metabolism , Virion/metabolism
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