Biomedical subjects

Simon Wain-Hobson

Publications and source records attributed to Simon Wain-Hobson.

At least 19 recordsLinked to original sources

PubMed2007

In HIV-1 pathogenesis the die is cast during primary infection.

The chronic stage of HIV-1 infection has been extensively described as a slowly evolving phase, in which the virus induces T-cell death slightly faster than the human body is able to recover. In contrast, T-cell and viral replication dynamics during primary infection have been less well studied. Recent studies in the SIV-macaque model and in HIV-positive patients during the acute infection period have highlighted the massive and irreversible depletion of CD4 memory T cells in the mucosa, particularly in the gut. Hence, gut-associated lymphoid tissue (GALT) plays a central role in the early stages of HIV-1 pathogenesis. Due to its particular cytokine expression pattern, GALT may favour the differential replication of certain HIV-1 subtypes during primary infection, particularly of subtype C. This could enhance the chance of a successful transmission. Moreover, these early events taking place in GALT during primary infection have major implications for therapy and vaccine design.

Acute Disease↗

PubMed2006

Twin gradients in APOBEC3 edited HIV-1 DNA reflect the dynamics of lentiviral replication.

The human immunodeficiency virus (HIV) Vif protein blocks incorporation of two host cell cytidine deaminases, APOBEC3F and 3G, into the budding virion. Not surprisingly, on a vif background nascent minus strand DNA can be extensively edited leaving multiple uracil residues. Editing occurs preferentially in the context of TC (GA on the plus strand) and CC (GG) depending on the enzyme. To explore the distribution of APOBEC3F and -3G editing across the genome, a product/substrate ratio (AA + AG)/(GA + GG) was computed for a series of 30 edited genomes present in the data bases. Two highly polarized gradients were noted each with maxima just 5' to the central polypurine tract (cPPT) and LTR proximal polypurine tract (3'PPT). The gradients are in remarkable agreement with the time the minus strand DNA remains single stranded. In vitro analyses of APOBEC3G deamination of nascent cDNA spanning the two PPTs showed no pronounced dependence on the PPT RNA:DNA heteroduplex ruling out the competing hypothesis of a PPT orientation effect. The degree of hypermutation varied smoothly among genomes indicating that the number of APOBEC3 molecules packaged varied considerably.

APOBEC Deaminases↗

PubMed2006

Optimisation of secretion of recombinant HBsAg virus-like particles: Impact on the development of HIV-1/HBV bivalent vaccines.

The hepatitis B surface antigen (HBsAg) assembles into virus-like particles (VLPs) that can be used as carrier of immunogenic peptides for the development of bivalent vaccine candidates. It is shown here that by respecting certain qualitative features of mammalian preS1 and preS2 protein domains upstream of HBsAg, foreign sequences can be inserted in their place while maintaining efficient secretion of VLPs. A polyepitope bearing HIV-1 epitopes restricted to the HLA-A*0201 class I allele was optimised for secretion as an HBsAg fusion protein by counterbalancing the generally hydrophobic class I epitopes with hydrophilic spacers, eliminating epitopes bearing cysteine residues, limiting the number of internal methionine residues to a minimum and adopting Homo sapiens codon usage. The optimised HIV-1 polyepitope-HBsAg recombinant protein with up to 138 residues assembled into efficiently secreted recombinant VLPs. DNA immunisation in HLA-A*0201 and HLA-A*0201/HLA-DR1 transgenic mice resulted in the recovery of humoral response against the carrier and enhanced levels of HIV-1 specific CD8(+) T lymphocyte activation. Efficient self-assembly of recombinant HBsAg VLPs opens up the possibility of making efficient bivalent HBV/HIV vaccine candidates, which is particularly apposite given that the two viruses are frequently associated.

AIDS Vaccines↗

PubMed2006

The HIV-1 clade C promoter is particularly well adapted to replication in the gut in primary infection.

OBJECTIVE: Coinfection of rhesus macaques with human/simian immunodeficiency virus chimeras harbouring the minimal core-promoter/enhancer elements from HIV-1 clade B, C and E viral prototypes (STR-B, STR-C and STR-E) revealed a remarkable dichotomy in terms of spatio-temporal viral replication. The clade C chimera (STR-C) predominated in primary infection. The present study was aimed at identifying the origin of STR-C plasma viraemia at this infection phase. DESIGN: By competing isogenic viruses differing only in their promoters, it was possible to identify subtle phenotypical differences in viral replication kinetics and compartmentalization in vivo. METHODS: Two rhesus macaques were coinfected by the three STR chimeras and the relative colonization of different compartments, particularly blood and stool, was determined for each chimera. Moreover, growth competition experiments in thymic histocultures enriched in interleukin (IL)-7 were performed and relative percentages of chimeras were estimated in supernatants and thymocytes lysates at different time points. RESULTS: It is demonstrated here that at the peak of primary infection, preferential replication of STR-C was supported by the gut-associated lymphoid tissue (GALT), an IL-7 rich microenvironment. This was shown by the correlation of the RNA viral genotype in blood and stools, compartments directly draining virions from the GALT. Thymic histocultures confirmed that replication of STR-C is particularly susceptible to this cytokine, compared to its STR-B and STR-E counterparts. CONCLUSIONS: These data show that the GALT cytokine network may well favour HIV-1 clade C replication during primary infection. This could result in enhanced transmission.

Animals↗

PubMed2006

The third Bond.

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Crystallography↗

PubMed2006

Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication.

Hypermutations in hepatitis B virus (HBV) DNA by APOBEC3 cytidine deaminases have been detected in vitro and in vivo, and APOBEC3G (A3G) and APOBEC3F (A3F) have been shown to inhibit the replication of HBV in vitro, but the presumably low or even absent hepatic expression of these enzymes has raised the question as to their physiological impact on HBV replication. We show that normal human liver expresses the mRNAs of APOBEC3B (A3B), APOBEC3C (A3C), A3F, and A3G. In primary human hepatocytes, interferon alpha (IFN-alpha) stimulated the expression of these cytidine deaminases up to 14-fold, and the mRNAs of A3G, A3F, and A3B reached expression levels of 10%, 3%, and 3%, respectively, relative to GAPDH mRNA abundance. On transfection, the full-length protein A3B(L) inhibited HBV replication in vitro as efficiently as A3G or A3F, whereas the truncated splice variant A3B(S) and A3C had no effect. A3B(L) and A3B(S) were detected predominantly in the nucleus of uninfected cells; however, in HBV-expressing cells both proteins were found also in the cytoplasm and were associated with HBV viral particles, similarly to A3G and A3F. Moreover, A3G, A3F, and A3B(L), but not A3B(S), induced extensive G-to-A hypermutations in a fraction of the replicated HBV genomes. In conclusion, the editing enzymes A3B(L), A3F, and most markedly A3G, which are expressed in liver and up-regulated by IFN-alpha in hepatocytes, are candidates to contribute to the noncytolytic clearance of HBV.

Base Sequence↗

PubMed2006

Restriction of foamy viruses by APOBEC cytidine deaminases.

Foamy viruses (FVs) are nonpathogenic retroviruses infecting many species of mammals, notably primates, cattle, and cats. We have examined whether members of the apolipoprotein B-editing catalytic polypeptide-like subunit (APOBEC) family of antiviral cytidine deaminases restrict replication of simian FV. We show that human APOBEC3G is a potent inhibitor of FV infectivity in cell culture experiments. This antiviral activity is associated with cytidine editing of the viral genome. Both molecular FV clones and primary uncloned viruses were susceptible to APOBEC3G, and viral infectivity was also inhibited by murine and simian APOBEC3G homologues, as well as by human APOBEC3F. Wild-type and bet-deleted viruses were similarly sensitive to this antiviral activity, suggesting that Bet does not significantly counteract APOBEC proteins. Moreover, we did not detect FV sequences that may have been targeted by APOBEC in naturally infected macaques, but we observed a few G-to-A substitutions in humans that have been accidentally contaminated by simian FV. In infected hosts, the persistence strategy employed by FV might be based on low levels of replication, as well as avoidance of cells expressing large amounts of active cytidine deaminases.

APOBEC-3G Deaminase↗

PubMed2005

Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo.

Because the replication of hepatitis B virus (HBV) proceeds via an obligatory reverse transcription step in the viral capsid, cDNA is potentially vulnerable to editing by cytidine deaminases of the APOBEC3 family. To date only two edited HBV genomes, referred to as G --> A hypermutants, have been described in vivo. Recent work suggested that HBV replication was indeed restricted by APOBEC3G but by a mechanism other than editing. The issue of restriction has been explored by using a sensitive PCR method allowing differential amplification of AT-rich DNA. G --> A hypermutated HBV genomes were recovered from transfection experiments involving APOBEC3B, -3C, -3F, and -3G indicating that all four enzymes were able to extensively deaminate cytidine residues in minus-strand DNA. Unexpectedly, three of the four enzymes (APOBEC3B, -3F, and -3G) deaminated HBV plus-strand DNA as well. From the serum of two of four patients with high viremia, G --> A hypermutated genomes were recovered at a frequency of approximately 10(-4), indicating that they are, albeit relatively rare, part of the natural cycle of HBV infection. These findings suggest that human APOBEC3 enzymes can impact HBV replication via cytidine deamination.

APOBEC-3G Deaminase↗

PubMed2005

Recovery of APOBEC3-edited human immunodeficiency virus G->A hypermutants by differential DNA denaturation PCR.

Virus genomes from the same family may exhibit a wide range in their DNA GC content, whereas viral hypermutants differ substantially in GC content from their parental genomes. As AT-rich DNA melts at lower temperatures than GC-rich DNA, use of a lower denaturation temperature during PCR should allow differential amplification of AT-rich genomes or variants within a quasispecies. The latter situation has been explored explicitly in a two-step process by using a series of well-defined viral sequences differing in their AT content. Firstly, the lowest denaturation temperature (T(p)) that allowed amplification of the parental sequence was determined. Secondly, differential amplification of AT-rich viral variants was obtained by using a denaturation temperature 1-3 degrees C lower than T(p). Application of this sensitive method to two different viruses allowed us to identify human immunodeficiency virus type 1 G-->A hypermutants in a situation where none were expected and to amplify AT-rich variants selectively within a spectrum of poliovirus mutants.

Base Sequence↗

PubMed2005

Extensive editing of a small fraction of human T-cell leukemia virus type 1 genomes by four APOBEC3 cytidine deaminases.

In the absence of the human immunodeficiency virus type 1 (HIV-1) Vif protein, the host-cell cytidine deaminases APOBEC3F and -3G are co-packaged along with virion RNA. Upon infection of target cells, nascent single-stranded DNA can be edited extensively, invariably giving rise to defective genomes called G-->A hypermutants. Although human T-cell leukemia virus type 1 (HTLV-1) replicates in the same cell type as HIV-1, it was shown here that HTLV-1 is relatively resistant to the antiviral effects mediated by human APOBEC3B, -3C, -3F and -3G. Nonetheless, a small percentage of genomes (0.1<f<5 %) were edited extensively: up to 97 % of cytidine targets were deaminated. In contrast, hypermutated HTLV-1 genomes were not identified in peripheral blood mononuclear cell DNA from ten patients with non-malignant HTLV-1 infection. Thus, although HTLV-1 DNA can indeed be edited by at least four APOBEC3 cytidine deaminases in vitro, they are conspicuously absent in vivo.

APOBEC-3G Deaminase↗

PubMed2005

The majority of human immunodeficiency virus type 1 particles present within splenic germinal centres are produced locally.

In most stages of human immunodeficiency virus (HIV) infection, cell-free viral particles can be detected in germinal centres (GCs) that are principally retained, in the form of immune complexes, on the surface of follicular dendritic cells (FDCs). The source of this virus remains unknown, although it is agreed that the FDCs themselves are not infected productively. By sequencing HIV viral DNA, genomic RNA and spliced mRNA isolated from individual splenic white pulps, it was shown here that the majority of HIV-1 viral particles are produced locally within the supporting lymphoid structure and do not result from trapping of circulating viruses or immune complexes. These findings underline the exquisite spatial organization of HIV-1 replication in vivo, suggesting a local origin for viruses trapped in splenic GCs.

DNA, Viral↗

PubMed2005

A genetic-algorithm approach to simulating human immunodeficiency virus evolution reveals the strong impact of multiply infected cells and recombination.

It has been previously shown that the majority of human immunodeficiency virus type 1 (HIV-1)-infected splenocytes can harbour multiple, divergent proviruses with a copy number ranging from one to eight. This implies that, besides point mutations, recombination should be considered as an important mechanism in the evolution of HIV within an infected host. To explore in detail the possible contributions of multi-infection and recombination to HIV evolution, the effects of major microscopic parameters of HIV replication (i.e. the point-mutation rate, the crossover number, the recombination rate and the provirus copy number) on macroscopic characteristics (such as the Hamming distance and the abundance of n-point mutants) have been simulated in silico. Simulations predict that multiple provirus copies per infected cell and recombination act in synergy to speed up the development of sequence diversity. Point mutations can be fixed for some time without fitness selection. The time needed for the selection of multiple mutations with increased fitness is highly variable, supporting the view that stochastic processes may contribute substantially to the kinetics of HIV variation in vivo.

Algorithms↗

PubMed2005

Antigenic stimulation specifically reactivates the replication of archived simian immunodeficiency virus genomes in chronically infected macaques.

Human immunodeficiency virus/simian immunodeficiency virus (SIV) diversification is a direct consequence of viral replication and occurs principally in secondary lymphoid organs where CD4(+) T cells are activated and proliferate. However, the evolution of viral quasispecies may also be driven by various nonexclusive mechanisms, including adaptation to specific immune responses and modification of viral fitness. Analysis of viral quasispecies in SIV-infected macaques subjected to repeated antigenic stimulations allowed us to demonstrate transient expansions of SIV populations that were highly dependent upon activation of antigen-specific T cells. T-cell clones expanded in response to a particular antigen were infected by a specific viral population and persisted for prolonged periods. Upon a second stimulation by encounter with the same antigen, these specific genomes were at the origin of a new burst of replication, leading to rapid but transient replacement of the viral quasispecies in blood. Finally, longitudinal analysis of SIV sequence variation during and between antigenic stimulations revealed that viral evolution is mostly constrained to periods of strong immunological activity.

Amino Acid Sequence↗

PubMed2005

HIV-1 clade promoters strongly influence spatial and temporal dynamics of viral replication in vivo.

Although the primary determinant of cell tropism is the interaction of viral envelope or capsid proteins with cellular receptors, other viral elements can strongly modulate viral replication. While the HIV-1 promoter is polymorphic for a variety of transcription factor binding sites, the impact of these polymorphisms on viral replication in vivo is not known. To address this issue, we engineered isogenic SIVmac239 chimeras harboring the core promoter/enhancer from HIV-1 clades B, C, and E. Here it is shown that the clade C and E core promoters/enhancers bear a noncanonical activator protein-1 (AP-1) binding site, absent from the corresponding clade B region. Relative ex vivo replication of chimeras was strongly dependent on the tissue culture system used. Notably, in thymic histocultures, replication of the clade C chimera was favored by IL-7 enrichment, which suggests that the clade C polymorphism in the AP-1 and NF-kappaB binding sites is involved. Simultaneous infection of rhesus macaques with the 3 chimeras revealed a strong predominance of the clade C chimera during primary infection. Thereafter, the B chimera dominated in all tissues. These data show that the clade C promoter is particularly adapted to sustain viral replication in primary viremia and that clade-specific promoter polymorphisms constitute a major determinant for viral replication.

Animals↗

PubMed2004

A universal primer set for PCR amplification of nuclear histone H4 genes from all animal species.

To control the quality of genomic DNA of samples from a wide variety of animals, a heminested PCR assay specifically targeting a nuclear gene has been developed. The histone H4 gene family comprises a small number of genes considered among the most conserved genes in living organisms. Tissue samples from necropsies and from cells belonging to 43 different species were studied, eight samples from invertebrates and 35 samples from vertebrates covering all classes. Ancient DNA samples from three Siberian woolly mammoths (Mammuthus primigenius) dating between 40,000 and 49,000 years before present were also tested for PCR amplification. Performance of HIST2H4 amplification were also compared with those of previously published universal PCRs (28S rRNA, 18S rRNA, and cytochrome b). Overall, 95% of species studied yielded an amplification product, including some old samples from gorilla and chimpanzees. The data indicate that the HIST2H4 amplimers are, thus, suitable for both DNA quality testing as well as species identification in the animal kingdom.

Animals↗

PubMed2004

Anti-termination by SIV Tat requires flexibility of the nascent TAR structure.

Substitution of the SIVmac239 promoter/enhancer by the strong EF1alpha promoter results in a severe replication defect due to a failure to respond to Tat. Revertant viruses with minimal promoter sequences (two Sp1 sites and a TATA box) were obtained that had fully restored their replicative potential. Comparison of the different LTRs indicated that structural alterations in the TAR stem due to a 31bp exon of the EF1alpha promoter rather than the mere presence of transcription factor binding sites within U3 were responsible for the attenuation. Structural models based on genuine RNA sequences combined with a refined algorithm to calculate the probability of the looping-mediated interaction between protein complexes bound to nucleic acid polymers indicated that the local concentration of TAR-bound Tat close to the RNA polymerase II complex was reduced more than 100-fold for the mutant as compared to SIVmac239. These results show that HIV/SIV replication requires only a minimal set of cis-acting elements in the promoter and suggest a hitherto unrecognised requirement of flexibility for the nascent TAR structure to allow anti-termination by Tat.

Amino Acid Sequence↗

PubMed2004

APOBEC3G is a single-stranded DNA cytidine deaminase and functions independently of HIV reverse transcriptase.

In the absence of the viral vif gene, human immunodeficiency virus (HIV) may be restricted by the APOBEC3G gene on chromosome 22. The role of the HIV Vif protein is to exclude host cell APOBEC3G from the budding virion. As APOBEC3G shows sequence homology to cytidine deaminases, it is presumed that in the absence of Vif, cytidine residues in the cDNA are deaminated yielding uracil. It is not known if additional proteins mediate APOBEC3G function or if deamination occurs in concert with reverse transcription. This report describes an in vitro assay showing that Baculovirus derived APOBEC3G alone extensively deaminates cDNA independently of reverse transcriptase. It reproduces the dinucleotide context typical of G --> A hypermutants derived from a Delta(vif) virus. By using an RNaseH- form of reverse transcriptase, it was shown that the cDNA has to be free of its RNA template to allow deamination. APOBEC3G deamination of dC or dCTP was not detected. In short, APOBEC3G is a single-stranded DNA cytidine deaminase capable of restricting retroviral replication.

APOBEC-3G Deaminase↗

PubMed2004

Simian immunodeficiency virus promoter exchange results in a highly attenuated strain that protects against uncloned challenge virus.

Among the many simian immunodeficiency virus (SIV) immunogens, only live attenuated viral vaccines have afforded strong protection to a natural pathogenic isolate. Since the promoter is crucial to the tempo of viral replication in general, it was reasoned that promoter exchange might confer a novel means of attenuating SIV. The core enhancer and promoter sequences of the SIV macaque 239nefstop strain (NF-kappaB/Sp1 region from -114 bp to mRNA start) have been exchanged for those of the human cytomegalovirus immediate-early promoter (CMV-IE; from -525 bp to mRNA start). During culture of the resulting virus, referred to as SIVmegalo, on CEMx174 or rhesus macaque peripheral blood mononuclear cells, deletions arose in distal regions of the CMV-IE sequences that stabilized after 1 or 2 months of culture. However, when the undeleted form of SIVmegalo was inoculated into rhesus macaques, animals showed highly controlled viremia during primary and persistent infection. Compared to parental virus infection in macaques, primary viremia was reduced by >1,000-fold to undetectable levels, with little sign of an increase of cycling cells in lymph nodes, CD4(+) depletion, or altered T-cell activation markers in peripheral blood. Moreover, in contrast to wild-type infection in most infected animals, the nef stop mutation did not revert to the wild-type codon, indicating yet again that replication was dramatically curtailed. Despite such drastic attenuation, antibody titers and enzyme-linked immunospot reactivity to SIV peptides, although slower to appear, were comparable to those seen in a parental virus infection. When animals were challenged intravenously at 4 or 6 months with the uncloned pathogenic SIVmac251 strain, viremia was curtailed by approximately 1,000-fold at peak height without any sign of hyperactivation in CD4(+)- or CD8(+)-T-cell compartment or increase in lymph node cell cycling. To date, there has been a general inverse correlation between attenuation and protection; however, these findings show that promoter exchange constitutes a novel means to highly attenuate SIV while retaining the capacity to protect against challenge virus.

AIDS Vaccines↗