Host Restriction Factors APOBEC3G/3F and Other Interferon-Related Gene Expressions Affect Early HIV-1 Infection in Northern Pig-Tailed Macaque (Macaca leonina).

The northern pig-tailed macaques (NPMs) lack TRIM5α, an antiviral restriction factor, and instead have TRIM5-CypA. In our previous study, we demonstrated that HIV-1NL4-3 successfully infected NPMs and formed a long-term viral reservoir in vivo. However, the HIV-1-infected NPMs showed relatively high viremia in the first 6 weeks of infection, which declined thereafter suggesting that HIV-1 NL4-3 infection in these animals was only partly permissive. To optimize HIV-1 infection in NPMs therefore, we generated HIV-1NL4-R3A and stHIV-1sv, and infected NPMs with these viruses. HIV-1NL4-R3A and stHIV-1sv can replicate persistently in NPMs during 41 weeks of acute infection stage. Compared to the HIV-1NL4-R3A, stHIV-1sv showed a notably higher level of replication, and the NPMs infected with the latter induced a more robust neutralizing antibody but a weaker cellular immune response. In addition, IFN-I signaling was significantly up-regulated with the viral replication, and was higher in the stHIV-1sv infected macaques. Consequently, the sequences of pro-viral env showed fewer G-A hyper-mutations in stHIV-1sv, suggesting that vif gene of SIV could antagonize the antiviral effects of APOBEC3 proteins in NPMs. Taken together, NPMs infected with HIV-1NL4-R3A and stHIV-1sv show distinct virological and immunological features. Furthermore, interferon-related gene expression might play a role in controlling primary HIV-1NL4-R3A and stHIV-1sv replication in NPMs. This result suggests NPM is a potential HIV/AIDS animal model.

Regulatory elements of stx2 gene and the expression level of Shiga-like toxin 2 in Escherichia coli O157:H7.

BACKGROUND/PURPOSE: Shiga-like toxin (Stx) is an important factor in the pathogenesis of Escherichia coli O157:H7 infection and is responsible for some severe complications. Stx2 is usually associated with hemolytic uremic syndrome in humans. Its expression is regulated by elements located upstream of the stx2 gene, including stx2-promoter sequence, ribosome binding site, and the antiterminator q gene. The present study aimed to find the correlation between regulatory elements and the expression level of Stx2 in two local isolates of E. coli O157:H7. METHODS: Two local E. coli O157:H7 strains SM-25(1) and KL-48(2), originating from human and cattle feces, respectively, and an E. coli reference strain, ATCC 43894, were investigated. The complete stx2 gene covering the sequences of promoter, ribosome binding site, and open reading frame and q gene of each strain was analyzed. The magnitude of Stx2 production was detected with a reverse passive latex agglutination method and Stx mediated cellular damage was determined with the Vero cell assay. RESULTS: A comparison of the complete stx2 gene contained stx2-promoter, ribosome binding site, and q genes of two local strains KL-48(2) and SM25(1), and the E. coli ATCC 43894 showed that the amino acid sequences were identical. Both local isolates were Stx negative in the reverse passive latex agglutination test and nontoxic in the Vero cell assay. CONCLUSION: The expression level of Shiga-like toxin of the two local isolates of E. coli O157:H7 did not only depend on the regulatory elements of the stx2 gene.

The Role of the Antiviral APOBEC3 Gene Family in Protecting Chimpanzees against Lentiviruses from Monkeys.

Cross-species transmissions of viruses from animals to humans are at the origin of major human pathogenic viruses. While the role of ecological and epidemiological factors in the emergence of new pathogens is well documented, the importance of host factors is often unknown. Chimpanzees are the closest relatives of humans and the animal reservoir at the origin of the human AIDS pandemic. However, despite being regularly exposed to monkey lentiviruses through hunting, chimpanzees are naturally infected by only a single simian immunodeficiency virus, SIVcpz. Here, we asked why chimpanzees appear to be protected against the successful emergence of other SIVs. In particular, we investigated the role of the chimpanzee APOBEC3 genes in providing a barrier to infection by most monkey lentiviruses. We found that most SIV Vifs, including Vif from SIVwrc infecting western-red colobus, the chimpanzee's main monkey prey in West Africa, could not antagonize chimpanzee APOBEC3G. Moreover, chimpanzee APOBEC3D, as well as APOBEC3F and APOBEC3H, provided additional protection against SIV Vif antagonism. Consequently, lentiviral replication in primary chimpanzee CD4(+) T cells was dependent on the presence of a lentiviral vif gene that could antagonize chimpanzee APOBEC3s. Finally, by identifying and functionally characterizing several APOBEC3 gene polymorphisms in both common chimpanzees and bonobos, we found that these ape populations encode APOBEC3 proteins that are uniformly resistant to antagonism by monkey lentiviruses.

Multilocus genotypic characterization of Escherichia coli O157:H7 recovered from food sources.

Escherichia coli O157:H7 strains (n = 33) recovered from different food sources in Egypt were characterized using molecular assays to identify strain genotypes associated with various levels of pathogenic potential. Genotypic characterization included: lineage-specific polymorphism assay (LSPA-6), Shiga-toxin-encoding bacteriophage insertion site assay (SBI), clade 8 typing, Tir (A255 T) polymorphism, and variant analysis of Shiga toxin 2 gene (Stx 2a and Stx 2c), and anti-terminator Q genes (Q 933 and Q 21). Genotypes LI/II (76%), SBI 1 (60·6%), clade 8 (69·7%), Tir (255 T) (72·7%) and Stx 2c (45·5%) were found to be significantly more frequent compared to other genetic markers in the strains analysed. Multivariable analysis revealed a significant association between LPSA-6 and clade types as well as Tir (A255 T). To the best of our knowledge, this is the first study to report the characterization of these genetic markers in E. coli O157:H7 strains in the Middle East and Africa.

HIV Restriction Factors and Mechanisms of Evasion.

Retroviruses have long been a fertile model for discovering host-pathogen interactions and their associated biological principles and processes. These advances have not only informed fundamental concepts of viral replication and pathogenesis but have also provided novel insights into host cell biology. This is illustrated by the recent descriptions of host-encoded restriction factors that can serve as effective inhibitors of retroviral replication. Here, we review our understanding of the three restriction factors that have been widely shown to be potent inhibitors of HIV-1: namely, APOBEC3G, TRIM5α, and tetherin. In each case, we discuss how these unrelated proteins were identified, the mechanisms by which they inhibit replication, the means used by HIV-1 to evade their action, and their potential contributions to viral pathogenesis as well as inter- and intraspecies transmission.

The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells.

The cytidine deaminase APOBEC3G (A3G) enzyme exerts an intrinsic anti-human immunodeficiency virus (HIV) defense by introducing lethal G-to-A hypermutations in the viral genome. The HIV-1 viral infectivity factor (Vif) protein triggers degradation of A3G and counteracts this antiviral effect. The impact of A3G on the adaptive cellular immune response has not been characterized. We examined whether A3G-edited defective viruses, which are known to express truncated or misfolded viral proteins, activate HIV-1-specific (HS) CD8+ cytotoxic T lymphocytes (CTLs). To this end, we compared the immunogenicity of cells infected with wild-type or Vif-deleted viruses in the presence or absence of the cytidine deaminase. The inhibitory effect of A3G on HIV replication was associated with a strong activation of cocultivated HS-CTLs. CTL activation was particularly marked with Vif-deleted HIV and with viruses harboring A3G. Enzymatically inactive A3G mutants failed to enhance CTL activation. We also engineered proviruses bearing premature stop codons in their genome as scars of A3G editing. These viruses were not infectious but potently activated HS-CTLs. Therefore, the pool of defective viruses generated by A3G represents an underestimated source of viral antigens. Our results reveal a novel function for A3G, acting not only as an intrinsic antiviral factor but also as an inducer of the adaptive immune system.

APOBEC3G induces a hypermutation gradient: purifying selection at multiple steps during HIV-1 replication results in levels of G-to-A mutations that are high in DNA, intermediate in cellular viral RNA, and low in virion RNA.

BACKGROUND: Naturally occurring Vif variants that are unable to inhibit the host restriction factor APOBEC3G (A3G) have been isolated from infected individuals. A3G can potentially induce G-to-A hypermutation in these viruses, and hypermutation could contribute to genetic variation in HIV-1 populations through recombination between hypermutant and wild-type genomes. Thus, hypermutation could contribute to the generation of immune escape and drug resistant variants, but the genetic contribution of hypermutation to the viral evolutionary potential is poorly understood. In addition, the mechanisms by which these viruses persist in the host despite the presence of A3G remain unknown. RESULTS: To address these questions, we generated a replication-competent HIV-1 Vif mutant in which the A3G-binding residues of Vif, Y(40)RHHY(44), were substituted with five alanines. As expected, the mutant was severely defective in an A3G-expressing T cell line and exhibited a significant delay in replication kinetics. Analysis of viral DNA showed the expected high level of G-to-A hypermutation; however, we found substantially reduced levels of G-to-A hypermutation in intracellular viral RNA (cRNA), and the levels of G-to-A mutations in virion RNA (vRNA) were even further reduced. The frequencies of hypermutation in DNA, cRNA, and vRNA were 0.73%, 0.12%, and 0.05% of the nucleotides sequenced, indicating a gradient of hypermutation. Additionally, genomes containing start codon mutations and early termination codons within gag were isolated from the vRNA. CONCLUSION: These results suggest that sublethal levels of hypermutation coupled with purifying selection at multiple steps during the early phase of viral replication lead to the packaging of largely unmutated genomes, providing a mechanism by which mutant Vif variants can persist in infected individuals. The persistence of genomes containing mutated gag genes despite this selection pressure indicates that dual infection and complementation can result in the packaging of hypermutated genomes which, through recombination with wild-type genomes, could increase viral genetic variation and contribute to evolution.

Construction of a minimal HIV-1 variant that selectively replicates in leukemic derived T-cell lines: towards a new virotherapy approach.

T-cell acute lymphoblastic leukemia is a high-risk type of blood-cell cancer. We analyzed the possibility of developing virotherapy for T-cell acute lymphoblastic leukemia. Virotherapy is based on the exclusive replication of a virus in leukemic cells, leading to the selective removal of these malignant cells. We constructed a minimized derivative of HIV-1, a complex lentivirus encoding multiple accessory functions that are essential for virus replication in untransformed cells, but dispensable in leukemic T cells. This mini-HIV virus has five deletions (vif, vpR, vpU, nef, and U3) and replicated in the SupT1 cell line, but did not replicate in normal peripheral blood mononuclear cells. The stripped down mini-HIV variant was also able to efficiently remove leukemic cells from a mixed culture with untransformed control cells. In contrast to wild-type HIV-1, we did not observe bystander killing in mixed culture experiments with the mini-HIV variant. Furthermore, viral escape was not detected in long-term cultures. The mini-HIV variant that uses CD4 and CXCR4 for cell entry could potentially be used against CXCR4-expressing malignancies such as T-lymphoblastic leukemia/lymphoma, natural killer leukemia, and some myeloid leukemias.

Guanine-adenine bias: a general property of retroid viruses that is unrelated to host-induced hypermutation.

The recently discovered mammalian enzymes, APOBEC3G and 3F, induce guanine-to-adenine hypermutation in retroviruses. However, the preference of adenine over guanine in retroviral codon usage is not correlated with the presence or absence of APOBEC3G or its viral inhibitor (Vif), and its pattern does not reflect the biochemical properties of APOBEC3G action. The guanine-adenine bias of retroviruses is thus probably not a result of host-induced mutational pressure, but rather reflects a general predisposition associated with reverse transcription.

Cytidine deamination of retroviral DNA by diverse APOBEC proteins.

The human cytidine deaminase APOBEC3G edits both nascent human immunodeficiency virus (HIV) and murine leukemia virus (MLV) reverse transcripts, resulting in loss of infectivity. The HIV Vif protein is able to protect both viruses from this innate restriction to infection. Here, we demonstrate that a number of other APOBEC family members from both humans and rodents can mediate anti-HIV effects, through cytidine deamination. Three of these, rat APOBEC1, mouse APOBEC3, and human APOBEC3B, are able to inhibit HIV infectivity even in the presence of Vif. Like APOBEC3G, human APOBEC3F preferentially restricts vif-deficient virus. Indeed, the mutation spectra and expression profile found for APOBEC3F indicate that this enzyme, together with APOBEC3G, accounts for the G to A hypermutation of proviruses described in HIV-infected individuals. Surprisingly, although MLV infectivity is acutely reduced by APOBEC3G, no other family member tested here had this effect. It is especially interesting that although both rodent APOBECs markedly diminish wild-type HIV infectivity, MLV is resistant to these proteins. This implies that MLV may have evolved to avoid deamination by mouse APOBEC3. Overall, our findings show that although APOBEC family members are highly related, they exhibit significantly distinct antiviral characteristics that may provide new insights into host-pathogen interactions.

APOBEC3F properties and hypermutation preferences indicate activity against HIV-1 in vivo.

APOBEC3G (CEM15 ) deaminates cytosine to uracil in nascent retroviral cDNA. The potency of this cellular defense is evidenced by a dramatic reduction in viral infectivity and the occurrence of high frequencies of retroviral genomic-strand G --> A transition mutations. The overwhelming dinucleotide hypermutation preference of APOBEC3G acting upon a variety of model retroviral substrates is 5'-GG --> -AG. However, a distinct 5'-GA --> -AA bias, which is difficult to attribute to APOBEC3G alone, prevails in HIV-1 sequences derived from infected individuals (e.g., ). Here, we show that APOBEC3F is also a potent retroviral restrictor but that its activity, unlike that of APOBEC3G, is partially resistant to HIV-1 Vif and results in a clear 5'-GA --> -AA retroviral hypermutation preference. This bias is also apparent in a bacterial mutation assay, suggesting that it is an intrinsic APOBEC3F property. Moreover, APOBEC3F and APOBEC3G appear to be coordinately expressed in a wide range of human tissues and are independently able to inhibit retroviral infection. Thus, APOBEC3F and APOBEC3G are likely to function alongside one another in the provision of an innate immune defense, with APOBEC3F functioning as the major contributor to HIV-1 hypermutation in vivo.

Characterization of pol, vif, vpr, and vpu genes of HIV type 1 in AIDS patients with high viral load and stable CD4+ T cell counts on combination therapy.

The success of combination therapy has also led to AIDS patients who exhibit elevated viral load without a corresponding decline in CD4+ T cells. In this study, we characterized changes in the pol gene and accessory genes vif, vpr, and vpu of HIV-1 isolated from the plasma of patients receiving highly active antiretroviral therapy. From each patient three sequences were obtained and compared with the sequence of HIV-1 from nontreated patients, revealing many substitutions that were similar in most cases. Protease and reverse transcriptase genes showed many mutations that were due to antiviral drugs. Premature termination was observed in the vif gene of one patient, leading to a protein truncated after 187 amino acids. In another patient the entire vpr open reading frame was missing, with no synthesis of Vpu protein because the 5' end of the gene was missing, including the start codon. In the same patient, the Vif protein was also truncated because of the deletion of 100 nucleotides at the 3' end of the vif gene.

Cloning, characterization, and tissue expression pattern of mouse Nma/BAMBI during odontogenesis.

Degenerate oligonucleotides to consensus serine kinase functional domains previously identified a novel, partial rabbit tooth cDNA (Zeichner-David et al., 1992) that was used in this study to identify a full-length mouse clone. A 1390-base-pair cDNA clone was isolated encoding a putative 260-amino-acid open reading frame containing a hydrophobic 25-amino-acid potential transmembrane domain. This clone shares some homology with the TGF-beta type I receptor family, but lacks the intracellular kinase domain. DNA database analysis revealed that this clone has 86% identity to a newly isolated human gene termed non-metastatic gene A and 80% identity to a Xenopus cDNA clone termed BMP and activin membrane bound inhibitor. Here we report the mouse Nma/BAMBI cDNA sequence, the tissue expression pattern, and confirmed expression in dental cell lines. This study demonstrates that Nma/BAMBI is a highly conserved protein across species and is expressed at high levels during odontogenesis.

Characterization and phylogenetic analysis of South African HIV-1 subtype C accessory genes.

To acquire new knowledge about the genetic diversity and potential impact on vaccine strategies of HIV-1 subtype C in South Africa, we have characterized the vif, vpr, and vpu genes of 15 isolates. Phylogenetic analysis of the genomic fragment encompassing these genes revealed subtype C subclusters, suggesting close relatedness with subtype C strains from other geographic locations and excluded isolation of South African strains. The putative T155 phosphorylation site in the C terminal of Vif was absent in all subtype C sequences. Variation in the predicted amino acid sequences of the three genes further showed strong correlation with other subtype C sequences.

Roles of the auxiliary genes and AP-1 binding site in the long terminal repeat of feline immunodeficiency virus in the early stage of infection in cats.

To examine the roles of auxiliary genes and the AP-1 binding site in the long terminal repeat of feline immunodeficiency virus (FIV) in vivo, three mutant viruses, which are defective in the vif gene ([delta]vif), ORF-A gene (deltaORF-A), and AP-1 binding site (deltaAP-1), and wild-type virus as a positive control were separately inoculated into three specific-pathogen-free cats. These cats were assessed by measuring the number of proviral DNA copies in peripheral blood mononuclear cells (PBMCs), the CD4/CD8 ratio and antibody responses to FIV for 16 weeks and then examining histological changes at necropsy. Although viral DNAs were detected in PBMCs from all 12 cats to various degrees until 16 weeks postinoculation, no virus was recovered from PBMCs of cats infected with (delta)vif virus during the observation period. However, a very weak antibody response was induced in one cat infected with the (delta)vif virus. In contrast, despite the successful recovery of virus from both groups of cats infected with deltaORF-A and deltaAP-1 virus, antibody responses and decrease in the CD4/CD8 ratio in the groups were milder than those in cats infected with wild-type virus. Furthermore, the numbers of proviral DNA copies in PBMCs from the two groups were not able to reach the level in cats infected with wild-type virus during the observation period. From these results, we conclude that these mutant viruses are still infectious for cats but failed in efficient viral replication and suggest that these auxiliary genes and enhancer element are important or essential to full viral replication kinetics and presumably to full pathogenicity during the early stage of infection in vivo.

A high frequency of defective vif genes in peripheral blood mononuclear cells from HIV type 1-infected individuals.

We studied the heterogeneity and intactness of the vif gene in six HIV-1-infected individuals at various clinical stages. The proviral vif sequences in peripheral blood mononuclear cells were amplified by PCR, followed by cloning and sequencing of 45 vif clones. The intraindividual diversity of the vif genes ranged from 0.45 to 3.3% and was not correlated with disease stage. Although the vif gene has been shown to be essential for infection of HIV-1 in vitro, a high frequency (31%) of defective vif genes was observed. In one patient, six vif clones carried double nonsense mutations at the same positions, five of which were clustered in the phylogenetic tree, suggesting that these vif-defective viruses may have replicated in vivo. Phylogenetic analysis revealed that the vif sequences from each individual were clustered into a separate group and that all of them belong to subtype B.

Complementation of human immunodeficiency virus type 1 vif mutants in some CD4+ T-cell lines.

The viral infectivity factor gene, vif of human immunodeficiency virus type 1 (HIV-1), is required for full infectivity in most T-cell lines. The replication kinetics exhibited by these mutants has been shown to be cell type-dependent. In H9 cells as well as primary lymphocytes, vif mutants are incapable of establishing infection. This has led to classification of these cell types as non-permissive for vif mutant replication. The T-cell lines Sup T1 and C8166 are able to replicate the vif mutant virus, leading to their classification as permissive for vif mutant replication. In this study, four cell lines (Sup T1, C8166, Molt 4 Clone 8, and A3.01) were tested for their ability to replicate vif mutant virus derived from two different strains of HIV-1 (HXB2 and NL4-3) that had been passaged on various cell lines. Although the kinetics of initial infection was delayed in all cells, by the second passage of vif mutant virus on Sup T1 or Molt 4 cells the kinetics of replication were identical to wild type virus. In contrast, mutant virus displayed delayed replication kinetics in C8166 and A3.01 cells in both initial and subsequent passages. In addition, the levels of viral DNA in infected Sup T1 cells were similar for delta vif and wild type virus, but in C8166 cells delta vif virus DNA levels were reduced compared to wild type virus. These results argue that in Sup T1 and Molt 4 cells there is a factor present that is able to complement the defect in vif mutant viruses which is absent or inefficient in its activity in C8166 and A3.01 cells.

Integration is essential for efficient gene expression of human immunodeficiency virus type 1.

A mutant of human immunodeficiency virus type 1 which carries a frameshift insertion in the integrase/endonuclease region of pol gene was constructed in vitro. Upon transfection into cells, although this mutant exhibited a normal phenotype with respect to expression of gag, pol, and env genes and to generation of progeny virions, no replication-competent virus in CD4-positive cells emerged. An assay for the single-step replication of a defective viral genome dependent on trans complementation by rev protein was established and used to monitor the early phase of viral infection process. Viral clones with a mutation in the vif, vpr, or vpu gene displayed no abnormality in the early phase. In contrast, the integrase mutant did not direct a marker gene expression after infection. Together with an observation that the mutant lacked the ability to integrate, these results indicated that the integration was required for efficient viral gene expression and productive infection of human immunodeficiency virus type 1.

Transcomplementation of VIF- HIV-1 mutants in CEM cells suggests that VIF affects late steps of the viral life cycle.

The vif gene of HIV-1 has previously been claimed to be essential for the ability of cell-free virus preparations to infect cells. Here we report that the CEM T-cell-line, stably transfected with and expressing vif, supports the replication of vif- HIV-1 viruses to the same extent as wild-type HIV1. Cell entry and early replication stages are the same for vif- and vif+ HIV-1 passaged in CEM, as measured both by a PCR-based cell entry assay and by fusogenic potential. These findings indicate that vif does not affect viral infectivity on CEM cells, but seems to act at a later stage of virus replication/maturation. We also show that the VIF proteins of two different HIV-1 strains can transcomplement different vif- HIV-1 mutants.