V1 Sequence Research Articles

Evolution of the human immunodeficiency virus type 2 envelope in the first years of infection is associated with the dynamics of the neutralizing antibody response

BackgroundDifferently from HIV-1, HIV-2 disease progression usually takes decades without antiretroviral therapy and the majority of HIV-2 infected individuals survive as elite controllers with normal CD4+ T cell counts and low or undetectable plasma viral load. Neutralizing antibodies (Nabs) are thought to play a central role in HIV-2 evolution and pathogenesis. However, the dynamic of the Nab response and resulting HIV-2 escape during acute infection and their impact in HIV-2 evolution and disease progression remain largely unknown. Our objective was to characterize the Nab response and the molecular and phenotypic evolution of HIV-2 in association with Nab escape in the first years of infection in two children infected at birth.ResultsCD4+ T cells decreased from about 50% to below 30% in both children in the first five years of infection and the infecting R5 viruses were replaced by X4 viruses within the same period. With antiretroviral therapy, viral load in child 1 decreased to undetectable levels and CD4+ T cells recovered to normal levels, which have been sustained at least until the age of 12. In contrast, viral load increased in child 2 and she progressed to AIDS and death at age 9. Beginning in the first year of life, child 1 raised high titers of antibodies that neutralized primary R5 isolates more effectively than X4 isolates, both autologous and heterologous. Child 2 raised a weak X4-specific Nab response that decreased sharply as disease progressed. Rate of evolution, nucleotide and amino acid diversity, and positive selection, were significantly higher in the envelope of child 1 compared to child 2. Rates of R5-to-X4 tropism switch, of V1 and V3 sequence diversification, and of convergence of V3 to a β-hairpin structure were related with rate of escape from the neutralizing antibodies.ConclusionOur data suggests that the molecular and phenotypic evolution of the human immunodeficiency virus type 2 envelope are related with the dynamics of the neutralizing antibody response providing further support for a model in which Nabs play an important role in HIV-2 pathogenesis.

Lava accretion system around mid‐ocean ridges: Volcanic stratigraphy in the Wadi Fizh area, northern Oman ophiolite

Detailed lithological study combined with geochemical variations of lavas reveals the across‐axis accretionary process at Wadi Fizh in the northern Oman ophiolite. The >900 m thick V1 sequence is divided into the lower V1 (LV1), middle V1 (MV1) and upper V1 (UV1) sequence by 0.4 m and 0.8 m thick umbers at 410 mab (meters above the base of the extrusive rocks) and 670 mab, respectively. The lowest part of the LV1 (LV1a) consists of lobate sheet and pillow lava flows extruded on the relatively flat ridge crest. Elongate pillows at 230 mab are flows draping downslope from the ridge crest and characterize the lithofacies on the ridge flank. Just above a jasper layer at 270 mab, 130 m thick evolved lavas were transported from the crest and emplaced on the ridge flank (LV1b). Off‐axial accretionary processes recorded in the MV1 resulted in alternating flows of less evolved, depleted lava and evolved lava, suggesting that the MV1 off‐axial lava sequence comprises flows emanated from both on‐ and off‐axis source vents. The less evolved and depleted UV1 flows suggest independent sources distinct from the axial lavas. The Lasail Unit is regarded as a subunit of the V1 because it is comparable to the UV1 in the geological, petrological, and geochemical characteristics. The broad compositional range of the V1 sequence endorses a view that the Wadi Fizh area corresponds to a segment end of the Oman paleospreading system accompanied by off‐axis volcanism as in segment boundaries of the present East Pacific Rise.

Is the syllabification of Irish a typological exception? An experimental study

We examined whether Irish speakers syllabify intervocalic consonants as codas (e.g., póca ‘pocket’ /po:k.ə/ CVC.V), as claimed by many authors, but contrary to claims in phonological theory of a universal preference for syllables with onsets. We conducted a perception experiment using a part-repetition task and presented auditory stimuli consisting of VCV items with a single medial consonant (Cm), varying in the length of V1 and the manner of articulation of Cm (e.g., póca /po:kə/ ‘pocket’, lofa /lofə/ ‘rotten’), as well as VCCV items varying in the length of V1 and consonant sequence type (e.g., masla /maslə/ ‘insult’, canta /ka:ntə/ ‘hunk’). Response patterns were in line with many, though not all, of the findings in the literature for other languages: Listeners preferred syllables with onsets, often treated Cm as ambisyllabic, syllabified Cm as a coda more often when V1 was short, and dispreferred stops as codas. The results, however, did not completely support the Syllable Onset Segmentation Hypothesis (SOSH), which proposes differing roles for syllable onsets and offsets in word segmentation. For VCCV items, listeners show a great deal of variability in decisions not only about where the first syllable ends, but also about where the second syllable begins, a variability that could not be explained by the number of legal onsets possible for a given consonant sequence. We examined the hypotheses that variability in perception can be accounted for by (1) variability in production (in the signal) and (2) phoneme pattern frequency. We searched pattern frequencies in an electronic dictionary of Irish, but found no support for an account in which language-specific syllabification patterns reflect patterns of word-initial phoneme sequences. Our investigation of potential acoustic cues to syllable boundaries showed a gradient effect of vowel length on syllabification judgments: the longer the V1 duration, the less likely a closed syllable, in line with results for other languages. For Irish, though, this pattern interestingly holds for all consonant manners except stops. The phonetic analyses point to other language-specific differences in phonetic patterns that cue syllable boundaries. For Irish, unlike English, consonant duration was not a more important cue to syllable boundaries than vowel duration, and there was no evidence that relative duration between the two consonants of a medial sequence signals syllable boundaries. The findings have implications not only for the syllable structure of Irish and theories of syllabification more generally. They are relevant to all theoretical and applied work on Irish that makes reference to the syllable.

Intragenomic diversity of the V1 regions of 16S rRNA genes in high-alkaline protease-producing Bacillus clausii spp

Alkaliphilic Bacillus sp. strain KSM-K16, which produces high-alkaline M-protease, was characterized phenotypically, biochemically and genetically. This strain was identified as Bacillus clausii based on the results of taxonomic studies, including sequencing of the 16S rRNA gene and DNA-DNA hybridization. Seven rRNA operons in the genome were identified by pulsed-field gel electrophoresis. Sequencing of cloned 16S rRNA genes revealed two distinct types of variable region V1. Moreover, some cloned 16S rRNA genes in some of the reference strains of B. clausii had a V1 region of yet another type. The B. clausii strains could clearly be divided into at least two subgroups based on the frequencies of the types of cloned V1 sequence. Bacillus sp. strain KSM-K16 was found to be in a different phylogenetic position from other high-alkaline protease-producing strains of B. clausii.

A recombinant bacterial cell surface (S-layer)-major birch pollen allergen-fusion protein (rSbsC/Bet v1) maintains the ability to self-assemble into regularly structured monomolecular lattices and the functionality of the allergen.

The mature crystalline bacterial cell surface (S-layer) protein SbsC of Bacillus stearothermophilus ATCC 12980 comprises amino acids 31-1099 and assembles into an oblique lattice type. As the deletion of up to 179 C-terminal amino acids did not interfere with the self-assembly properties of SbsC, the sequence encoding the major birch pollen allergen (Bet v1) was fused to the sequence encoding the truncated form rSbsC(31-920). The S-layer fusion protein, termed rSbsC/Bet v1, maintained the ability to self-assemble into flat sheets and open-ended cylinders. The presence and the functionality of the fused Bet v1 sequence was proved by blot experiments using BIP1, a monoclonal antibody against Bet v1 and Bet v1-specific IgE-containing serum samples from birch pollen allergic patients. The location and accessibility of the allergen moiety on the outer surface of the S-layer lattice were demonstrated by immunogold labeling of the rSbsC/Bet v1 monolayer, which was obtained by oriented recrystallization of the S-layer fusion protein on native cell wall sacculi. Thereby, the specific interactions between the N-terminal part of SbsC and a distinct type of secondary cell wall polymer were exploited. This is the first S-layer fusion protein described that had retained the specific properties of the S-layer protein moiety in addition to those of the fused functional peptide sequence.

Characterization of human polymorphic DNA repair methyltransferase.

The O6-methylguanine-DNA methyltransferase (MGMT) is a critical defence against alkylation-induced mutagenesis and carcinogenesis. More than a 20-fold interindividual difference in the MGMT activity is known to exist among human cultured fibroblasts. We previously reported three allelic variants of the human MGMT gene, namely V1, V2, and V3. Both V1 and V2 carry amino acid substitutions, Leu84Phe and Trp65Cys, respectively, while V3 has a silent mutation. In order to reveal the pharmacogenetic and ecogenetic significance of polymorphism in the human MGMT gene, we investigated the in-vivo characteristics of V1 and V2 methyltransferase enzyme. Escherichia coli strain KT233 (ogt-, ada-) and mer- HeLa MR cells carrying a V1 sequence exhibited almost the same level of sensitivity against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as did those with a wild-type sequence. The level of methyltransferase protein in those cells was essentially the same as for the wild-type and V1 samples. On the other hand, E. coli and human cells expressing V2 cDNA showed a significantly reduced level of survival. In these cells, V2 protein was hardly detected, even though mRNA was produced normally. An in-vitro translation experiment revealed that the V2 sequence had the potential to produce methyltransferase protein, as did the wild-type and V1 sequences. There was also evidence for a small amount of V2 protein being produced but rapidly degraded, thus implying that the V2 molecule is unstable in vivo. Using purified recombinant proteins, we estimated the kinetic values of wild-type and variant form of enzymes, which would support these views. From these results, we concluded that the wild-type and V1 protein have similar enzymatic and physicochemical properties, while V2 protein is considered to be unstable and rare.

Effect of natural HIV-1 envelope V1-V2 sequence diversity on the binding of V3-specific and non-V3-specific antibodies.

In past years, much attention has been paid to the HIV-1 envelope (env) protein variable region 3 (V3), termed the principal neutralizing determinant. HIV-1 vaccines were often designed to target V3, and vaccine efficacy was often measured with V3-based assays. Thus, some disappointment resulted when volunteers in first clinical vaccine trials generated V3-specific antibodies that could not protect against V3-similar viruses. We describe an analysis of V1 and V2 sequence effects on antibody binding to V3 and non-V3 determinants. This study involved the preparation of seven full-length (gp160), chimeric env proteins in a vaccinia virus (VV) expression system. Chimeric proteins displayed different V1-V2 sequences but were otherwise identical. A panel of 12 monoclonal antibodies was then tested for binding activities toward the seven chimeras. Results showed that V1-V2 sequences affected antibody binding to env, both in V3 and non-V3 positions. These data demonstrate the enormous complexity of HIV-1 env protein conformation and antigenic determinants. Respect for the complexity of antibody-antigen interactions encourages the design of sophisticated immunoglobulin and protein cocktails for use in HIV-1 therapies and vaccines, respectively.

Regulation of the neural-specific gene VGF in PC12 cells. Identification of transcription factors interacting with NGF-responsive elements.

Nerve growth factor (NGF) is an important regulator of differentiation and survival in both the peripheral and central nervous systems. We have begun to analyze the mechanism by which NGF induces the expression of a neural specific gene, VGF, in PC12 cells. Using DNase I footprinting and transient transfection analysis, we identified two VGF promoter regions, V1 and V2, that are required for basal promoter expression as well as gene induction by NGF, epidermal growth factor (EGF), and cAMP. The V1 element is essential for VGF promoter function, but it is not sufficient to confer NGF responsiveness to a heterologous promoter. In contrast, the V2 element can independently stimulate the expression of a linked herpes simplex virus (HSV) thymidine kinase promoter in response to NGF. We showed that the V2 region also contains a sequence that acts as a promoter-specific negative regulator of basal VGF gene expression. As determined by gel mobility shift and Southwestern analysis, the V1 sequence is recognized by a novel PC12 nuclear protein of about 110-kDa molecular mass. Using oligonucleotide competition and antibody supershift assays, we demonstrated that the cAMP-response element (CRE) motif within the V2 element interacted specifically with proteins related to cAMP-response element binding (CREB), JunB, and JunD transcription factors. The JunB-related binding activities were transiently induced by NGF, suggesting that part of the mechanism utilized by NGF to activate VGF transcription includes increased synthesis of a V2 binding protein. Taken together, our analysis suggests that the VGF promoter is regulated by a complex mechanism, and its activation requires combinatorial action of several transcription factors interacting with multiple promoter elements.

Distinct but related human immunodeficiency virus type 1 variant populations in genital secretions and blood.

For a HIV vaccine to be effective, it will be essential that it protect against the virus variants to which individuals are most frequently exposed. HIV-1 is predominantly a sexually acquired virus, thus, variants in genital secretions are a potentially important reservoir of viruses that are transmitted. Because there are no data available on variants in the genital mucosa, we analyzed this provirus population and compared it to the proviruses in the blood of individuals chronically infected with HIV-1. A major genetic difference between variants within a patient were insertions, which were apparently created by duplication of adjacent sequences, that resulted in acquisition of new potential glycosylation sites in V1 and V2. Comparisons of mucosal and PBMC variants suggest that these tissues harbor distinct, but related populations of HIV-1 variants. In two of three patients, the mucosal variants were most closely related to a minor variant genotype in blood. In a third individual, viruses in both tissues were surprisingly homogeneous, but the majority of variants in the cervix encoded a V1 sequence with a predicted glycosylation pattern similar to a minor variant in blood. The V3 sequence patterns of the mucosal isolates indicate they may be predominantly macrophage-tropic viruses.

Identification of a brain-specific protein kinase Cζ pseudogene (ΨPKCζ) transcript

Protein kinase C (PKC), a widely-distributed enzyme implicated in the regulation of many physiological processes, consists of a family of at least twelve isoenzymes which differ in tissue distribution, subcellular localization, regulatory properties, etc. In addition to this heterogeneity at the protein level, we identify here for the first time a PKC zeta pseudogene (psi PKC zeta) transcript, specifically expressed in the brain, which is identical with PKC zeta except for sequence divergence within the first variable domain (V1). The authenticity of this unique V1 sequence (V1') in mRNA was confirmed by RNase protection and reverse transcriptase PCR (RT-PCR) analysis. When translated in-frame with PKC zeta, a stop codon is located 28 amino acids towards the N-terminus of the divergence point and the intervening sequence lacks an expected initiating methionine. psi PKC zeta is non-functional in terms of protein synthesis since Western blotting with an antibody directed against the C-terminus of PKC zeta failed to reveal a protein smaller than PKC zeta, and synthetic psi PKC zeta RNA failed to support protein synthesis in a translation system in vitro. PCR amplification of rat genomic DNA demonstrated lack of an intron at the junction between V1' and the first constant domain (the V1'-C1 border), and genomic DNA Southern blot analysis using PKC zeta and psi PKC zeta-specific probes indicated that they have different loci. psi PKC zeta, therefore, is not derived from the PKC zeta gene by alternative splicing, but rather is the product of a distinct gene. In Northern blot analysis, brain PKC zeta mRNA was identified as a low-abundance 3.1 kb transcript, while the abundant 2.5 and 4.7 kb mRNAs previously reported to encode PKC zeta are, in fact, psi PKC zeta transcripts. Analysis of rat brain, heart, lung, liver, kidney and skeletal muscle revealed psi PKC zeta mRNA only in brain. PKC zeta transcripts were most abundant in lung and kidney (2.7 and 4.7 kb mRNAs), correlating with the tissue profile of PKC zeta immunoreactivity in Western blots. Probes complementary to the common V5 and C1 domains detected both PKC zeta and psi PKC zeta transcripts. Interestingly, the C1 probe also detected an abundant novel 1.75 kb mRNA in brain and heart, suggesting the existence of an additional PKC zeta-related species. This work, therefore, also emphasizes the importance of careful choice of oligonucleotide and cDNA probes to study PKC zeta mRNA.

Humoral response of cynomolgus macaques to human soluble CD4: Antibody reactivity restricted to xeno-human determinants

The CD4 cell surface glycoprotein which is expressed primarily by a subset of T lymphocytes plays a key role in normal immune responses. In the immunopathogenesis of AIDS, it serves as the high-affinity receptor for HIV, facilitating viral attachment and entry into CD4 + cells. As such, the truncated soluble form of this molecule (sT4) has been proposed as a therapeutic drug for the treatment of AIDS whereby it would act as decoy for viral entry into cells or facilitate elimination of soluble viral envelope glycoprotein. In a study designed to look at the effect of sT4 on immune function, sT4 was administrated to experimentally naive primates. In this report, we show that administration of sT4 to cynomolgus macaque monkeys over a period of up to 3 weeks results in antibody responses with specificities for human CD4 molecules. Antisera thus generated bound sT4 and cell surface CD4 expressed on human T lymphocytes but failed to bind to cynomolgus lymphocytes. These antibodies caused no apparent adverse effects on normal immune functions of the cynomolgus macaques. We conclude from these data that the antibody response to soluble CD4 in cynomolgus monkeys is directed at determinants present on human CD4 but absent on monkey CD4. The restricted xenogeneic specificity of the antibody response indicates that soluble CD4 may not be highly immunogenic in syngeneic hosts. The present study also shows that these antibodies can block HIV-induced syncytium formation indicating that the antibodies bind to regions on the CD4 molecule close to the HIV-env gp120 binding site. The gp120 binding site, which resides within the N-terminal V1 domain of CD4, encompasses a region which corresponds to the complementarity determining regions (CDRs) of immunoglobulins. The CDR-like regions of CD4-V1 manifest the greatest species divergence, are tolerant to experimental in vitro mutagenesis, and generate the predominant antibody response in mice immunized with human CD4 indicating that differences in the V1 sequence between human and other nonhuman primates may localize to this regions.