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PubMed2026

Pervasive HIV recombination limits the utility of circulating recombinant form nomenclature.

The naming of HIV-1 circulating recombinant forms (CRFs)-descendent viruses from the same intersubtype recombination events, is along with the designation of 'subtypes' and 'groups', routinely used to track HIV-1 diversity. However, we argue that continuing to designate all detected CRFs as distinct entities is biologically unjustified, as many represent recombinants of limited epidemiological significance. Indeed, the mechanistic underpinning of HIV-1 recombination highlights the arbitrary nature of naming these incidental recombinants, the majority of which are rarely detected again. This underlines the need to prioritise taxonomically meaningful clades, with a focus on biological significance such as emergence events associated with significant epidemiological spread, phenotypic properties or transmission advantage.

HIV-1↗

PubMed2005

Expression of recombinant forms of human 21.5 kDa myelin basic protein and proteolipid protein in CHO cells.

MBP and PLP are major structural protein components of myelin. Both proteins play a functional role in formation of myelin sheath and in maintenance of its compaction. Immune responses to MBP and PLP have been implicated in the pathogenesis of multiple sclerosis (MS), an auto-immune disease of the central nervous system. Recombinant forms of both proteins isolated and purified from bacterial or insect cell systems are commonly used to study the specificity of auto-response in MS. We have prepared recombinant forms of MBP and PLP stably expressed in CHO cells. Several clones with proper cytoplasmic MBP or surface PLP localization were obtained and characterized by flow cytometry and indirect immunostaining. CHO cells expressing the recombinant forms of MBP and PLP can be very useful in studies on the autoimmune mechanism of MS.

Animals↗

PubMed2004

HIV type 1 circulating recombinant form CRF09_cpx from west Africa combines subtypes A, F, G, and may share ancestors with CRF02_AG and Z321.

Two HIV-1 intersubtype recombinant forms are circulating widely in populations and have become important strains in the pandemic: CRF01_AE in Southeast Asia and CRF02_AG in West and West Central Africa, respectively. Several other circulating recombinant forms (CRF) have also been identified, but with fewer numbers of infections and/or more limited geographic spread. Here we expand knowledge of HIV-1 CRF using clinical samples, principally from West Africa, that were difficult to classify by partial genome sequencing. DNA was extracted from primary patient peripheral blood mononuclear cells (PBMC). The virtually complete HIV-1 genome was amplified by polymerase chain reaction (PCR) and directly sequenced. Additional strains were characterized by partial envelope sequencing. Phylogenetic analysis was used to identify and map intersubtype recombination breakpoints. Four virtually complete genome sequences and two partial envelope sequences represent CRF09_cpx, a newly identified complex recombinant HIV-1 whose principal focus seems to be in West Africa. This recombinant includes segments of subtypes A, F, G, and unclassified genetic material. It shares unique unclassified regions with the early Zaire strain Z321. There are similarities in structure, but considerable genetic distances, between CRF09_cpx and CRF02_AG IbNG. In conclusion, it is possible that this CRF shared common ancestors with both Z321 and CRF02_AG in the course of the pandemic, perhaps arising by recombination between earlier forms of these strains. Although newly identified, at least one infection with CRF09_cpx has already occurred outside of Africa.

Adult↗

PubMed2000

Recombinant forms of the neurotrophic factor pigment epithelium-derived factor activate cellular metabolism and inhibit proliferation of the RAW macrophage cell line.

Recombinant forms of the neurotrophic factor pigment epithelium-derived factor (PEDF) activate metabolism of RAW macrophage cells while simultaneously inhibiting their proliferation. The recombinant forms (rPEDF) acted with EC(50)s of 0.1-1 nM while full-length native bovine PEDF was inactive. Urea, which is the buffer used to extract recombinant PEDF, stimulated RAW cell proliferation, the first report of an effect of urea on non-kidney cells. PEDF acted within 12 h and its effects persisted up to 72 h with continuous exposure. Although rPEDF had no direct action on glioma cell lines, it increased the amount of a soluble factor released by RAW cells which was capable of blocking glioma cell division. Thus PEDF may function as a neuroimmune modulator, affecting both neural and immune system cells.

Animals↗

PubMed2006

Genetic analysis of HIV-1 strains in rural eastern Cameroon indicates the evolution of second-generation recombinants to circulating recombinant forms.

The HIV-1 genetic diversity in most parts of Cameroon is well described and shown to be very broad. However, little is known about the composition of the HIV-1 epidemic in the rural parts of eastern Cameroon. Therefore, we investigated 25 specimens from this region for their subtypes in gag, pol, and env gene fragments. Along with genetic material of subtypes A1, C, G, CRF01_AE, CRF02_AG, and CRF11_cpx, we also identified a large number (24%, 6/25) of distinct env sequences within the subtype A radiation. CRF02_AG was the predominant genetic form in all genes studied. Half of the specimens studied were considered "pure" based on concordant subtypes in the genes studied, whereas the other half were unique recombinant forms (URFs). Except for 1 URF, all were second-generation recombinants (SGRs), 90% of which contained genetic material of CRF02_AG in at least 1 gene. Notably, we identified individuals from 3 different villages infected with CRF01_AE(gag)CRF02_AG(pol)A(env) strains, which is indicative of the evolution of this URF to a circulating recombinant form (CRF). In addition, we identified a CRF02_AG(pol)C(env) recombinant infecting a man and a woman living in the same village, suggesting horizontal transmission of this recombinant. The current study emphasizes the power of HIV-1 recombination through the generation of SGRs and the evolution of URFs into CRFs. These findings suggest that, in a region where a predominant HIV-1 strain cocirculates among several subtypes, recombination could eventually decrease the proportion of this strain over time, such as CRF02_AG in Cameroon.

Cameroon↗

PubMedDate not supplied

[Structural-functional study of recombinant forms of onconase].

A method for expression of an onconase gene leading to a soluble form of the protein was developed. The enzymatic and cytotoxic properties of the protein's recombinant forms were studied. Recombinant onconase with an additional N-terminal Met residue isolated in nondenaturing conditions did not substantially differ from the native enzyme in ribonucleolytic activity. The addition of a 33-mer peptide containing auxiliary elements for the simplification of isolation and detection of the recombinant protein did not affect the enzyme properties of onconase. The method proposed is useful for the onconase structure-function relation studies and enables construction of onconase-based fusion proteins for anticancer therapy.

Animals↗

PubMed2005

HIV-1 subtypes and circulating recombinant forms (CRFs) from HIV-infected patients residing in two regions of central and southern Italy.

A total of 347 pol gene sequences from 88 Tuscan and 259 Apulian subjects (including 52 non-Italians and 9 children) were analyzed phylogenetically. Forty-four (12.6%) non-B subtypes were found, including 3.4% C, 1.4% F1, 0.8% G, and 0.3% each for J and A pure subtypes, and 3.7% CRF02_AG, 1.4% CRF01_AE, 0.6% BF, and 0.3% CRF06-cpx recombinant forms. An additional sample close-matched the pol gene of an unique recombinant form (URF AGK 99GR303). The non-B subtypes were from 40 adults and 4 children; 12 of these 44 patients were epidemiologically linked. Thirty-three of the 44 non-B viruses pertained to non-Italian immigrants and 11 to Italians, signifying that 63.4% immigrants and 3.7% Italians harbored non-B subtypes. The overall frequency of non-B subtypes was higher in Tuscany than in Apulia (18.1% vs. 10.8%). Moreover, 6.1% and 3.0% non-B subtypes were found among Italians from Florence and Apulia, respectively, while 52.1% and 72.4% of immigrants living in Tuscany and Apulia harbored non-B subtypes. Women infected by means of sexual contact prevailed among non-Italian adults; the majority of Italians were males and admitted high-risk sexual behavior. Four Italians had a history of extensive travel in countries of high endemicity. Social and epidemiological changes are responsible for an increasing circulation of non-B subtypes in Italy. Although non-B subtypes principally infect non-Italian patients, in Italy they can no longer be considered exclusively restricted to subjects from endemic areas.

Adolescent↗

PubMed2006

Identification of a novel circulating recombinant form (CRF33_01B) disseminating widely among various risk populations in Kuala Lumpur, Malaysia.

A molecular epidemiological investigation was conducted among various risk populations (n = 184) in Kuala Lumpur, Malaysia, in 2003 to 2005, on the basis of nucleotide sequences of protease and reverse transcriptase regions. In addition to circulating HIV-1 strains, including CRF01_AE (57.1%), subtype B (20.1%), and subtype C (0.5%), we detected a candidate with a new circulating recombinant form (CRF). We determined four near-full-length nucleotide sequences with identical subtype structure from epidemiologically unlinked individuals of different risk and ethnic groups. In this chimera, two short subtype B segments were inserted into the gag-RT region in a backbone of CRF01_AE. The recombinant structure was distinct from previously identified CRF15_01B in Thailand. In agreement with the current HIV nomenclature system, this constitutes a novel CRF (CRF33_01B). The overall prevalence of CRF33_01B is 19.0% (35/184). Although the prevalence of CRF33_01B is particularly high among injecting drug users (42.0%, 21/50), it is also detected in a substantial proportion of homo-/bisexual males (18.8%, 3/16) and heterosexuals (9.8%, 9/92). Moreover, unique recombinant forms composed of CRF01_AE and subtype B that have a significant structural relationship with CRF33_01B were detected in 1.6% (3/184) of study subjects, suggesting an ongoing recombination process in Malaysia. This new CRF seems to be bridging viral transmission between different risk populations in this country.

Adult↗

PubMed2002

In vitro intersubtype recombinants of human immunodeficiency virus type 1: comparison to recent and circulating in vivo recombinant forms.

The increased prevalence of human immunodeficiency virus type 1 (HIV-1) intersubtype recombinants (ISRs) is shaping HIV-1 evolution throughout the world and will have an impact on both therapeutic and vaccine strategies. This study was designed to generate and compare in vitro ISRs to those isolated from HIV-infected individuals throughout the world. Human peripheral blood mononuclear cells were dually infected with seven pairs of HIV-1 isolates from different subtypes (i.e., A to F). Recombinant crossover sites were mapped to specific regions in the envelope (env) gene by using a cloning-hybridization technique and subtype-specific probes. In vitro intersubtype recombination was at least twofold more frequent in the V1-to-V3 region than in any other env fragment, i.e., C1 to V1, V3 to V5, or V5 to gp41. Sequence and recombination site analyses suggested the C2 env domain as a "hot region" for recombination and selection of replication-competent ISRs during the 15-day incubation. In addition to these regional preferences for env recombination, homopolymeric nucleotide tracts, i.e., sequences known to pause reverse transcriptase and promote template switching, were found in most in vitro crossover sites. ISRs, originating from recent dual infections and limited transmission events, partly retained this in vitro regional or sequence preference for recombination sites. However, a shift to crossover sites flanking the gp120-coding sequence was evident in the stable circulating recombinant forms of HIV-1. Based on these findings, HIV-1 recombinants generated from these dual infections may be used as a model for in vivo intersubtype recombination and for the design of various diagnostic assays and vaccine constructs.

Base Sequence↗

PubMed1995

Analysis of the proteolytic activity of a recombinant form of apolipoprotein(a).

We have analyzed the proteolytic activity of a recombinant form of apolipoprotein(a) [r-apo-(a)]. A mutant 17-kringle from of r-apo(a) was engineered that contained a serine to arginine substitution which reinstates the tissue-type plasminogen activator (tPA) as determined by SDS-PAGE and fluorography and by Western blot analysis. However, tPA cleavage did not result in an active protease as both wildtype r-apo(a) and the mutant, either free or incorporated into r-Lp(a) particles, were uniformly inactive against a variety of chromogenic serine protease tripeptide substrates. To assess whether the large number of kringle IV repeats present in apo(a) inhibits proteolytic activity, we generated truncated forms of the Ser-->Arg proteolytic activity, we generated truncated forms of the Ser-->Arg mutant containing one or 10 kringle IV repeats. These truncated versions of r-apo(a) were susceptible to cleavage by tPA but were inactive against the plasmin substrate S-2251. Treatment of the Ser-->Arg mutant of the 17-kringle r-apo(a) with tPA and diisopropylflurophosphate (DFP) did not result in modification of the mutant protease domain by DFP. Finally, we incubated r-apo(a) or r-Lp(a) particles formed in vitro with purified human LDL; no degradation of LDL was observed after 16 h at 37 degrees C. The results of this study suggest that one or more of the substitutions present in the protease domain of apo(a), in addition to the Arg-->Ser substitution, render apo(a) proteolytically inactive.

Amino Acid Sequence↗

PubMed1997

The major dog allergens, Can f 1 and Can f 2, are salivary lipocalin proteins: cloning and immunological characterization of the recombinant forms.

Canis familiaris allergen 1 (Can f 1) and Canis familiaris allergen 2 (Can f 2) are the two major allergens present in dog dander extracts. We now report the isolation of cDNAs encoding both proteins and present their nucleotide and deduced amino acid sequences. Can f 1, produced by tongue epithelial tissue, has homology with the von Ebner's gland (VEG) protein, a salivary protein not previously thought to have allergenic properties. Can f 2, produced by tongue and parotid gland, has homology with mouse urinary protein (MUP), a known allergen. Both VEG protein and MUP are members of the lipocalin family of small ligand-binding proteins. Recombinant forms of Can f 1 and Can f 2 were produced and tested for immunoglobulin E (IgE) reactivity. Among dog-allergic subjects, 45% had IgE directed exclusively to rCan f 1, and 25% had IgE to both rCan f 1 and rCan f 2. In addition, both recombinant proteins were able to cross-link IgE and elicit histamine release from peripheral blood leucocytes in vitro. These findings confirm that Can f 1 and Can f 2 are major and minor dog allergens, respectively, and demonstrate that recombinant forms of dog allergens retain at least some IgE-binding epitopes.

Allergens↗

PubMed1991

Apolipoprotein(a): expression and characterization of a recombinant form of the protein in mammalian cells.

We have stably expressed a recombinant form of apo(a) in a human embryonic kidney cell line. The engineered protein (predicted mass of 250 kDa) contains 17 copies of the apo(a) domain, which resembles kringle 4 of plasminogen, followed by the plasminogen-like kringle 5 and protease-like domain of apo(a). The recombinant protein [r-apo(a)] was isolated from cell culture media by immunoaffinity chromatography, and its physical properties were studied. As is the case for apo(a) isolated from plasma-derived Lp(a), r-apo(a) is highly glycosylated (23% by weight), containing both N- and O-linked glycans, which results in an observed molecular mass of 500 kDa by SDS-PAGE. The high sialic acid content was reflected in a pI of 4.3 for the r-apo(a). Two subpopulations of r-apo(a) secreted by the permanent cell line were identified with respect to lysine-Sepharose binding; the majority of the r-apo(a) bound specifically to this matrix and was eluted with epsilon-aminocaproic acid (epsilon-ACA). When the r-apo(a) plasmid was used to transfect a human hepatoma cell line, lipoprotein particles were secreted containing the disulfide-linked complex of apoB-100 and the r-apo(a). The density of these particles was shown to be heterogeneous, with the majority of the r-Lp(a) floating in the density range of plasma-derived Lp(a).

Apolipoproteins↗

PubMed1997

Glycosylation of bile-salt-stimulated lipase from human milk: comparison of native and recombinant forms.

Bile-salt-stimulated lipase (BSSL) is an enzyme present in human milk. BSSL is important for fat digestion in infants. It contains one site for N-glycosylation and a serine/threonine-rich domain which is highly O-glycosylated. Both N- and O-linked sugar chains were studied on native BSSL from three donors and compared to the glycosylation of recombinant BSSL produced in Chinese hamster ovary or mouse fibroblast (C-127) cell lines. The carbohydrate composition of oligosaccharides was mapped using sugar and methylation analyses, enzyme-linked immunosorbant assay, and different separation techniques. Native BSSL was found to be highly glycosylated (19-26%). It contained a high amount of fucosylated oligosaccharides and expressed both Lewis a and Lewis b blood group antigens. None of the recombinant BSSL forms contained fucose. N-linked structures on native BSSL were identified as mainly mono- and disialylated biantennary complex type structures with or without fucose substitution. High-pH anion-exchange chromatography analysis indicated that the recombinant forms of BSSL contained similar types of N-glycan structures differing mainly in their content of sialic acid and by the absence of fucose residues. Native BSSL contained predominantly large O-linked oligosaccharides. This was in contrast to the recombinant forms of BSSL which contained mainly short type O-glycans with a high content of sialic acid. Interestingly, the estimated number of O-glycans attached to native BSSL was lower than that for the recombinant forms.

Antigens↗

PubMed2001

Mediatorless biosensor for H(2)O(2) based on recombinant forms of horseradish peroxidase directly adsorbed on polycrystalline gold.

Four forms of horseradish peroxidase (HRP) have been used to prepare peroxidase-modified gold electrodes for mediatorless detection of peroxide: native HRP, wild type recombinant HRP, and two recombinant forms containing six-His tag at the C-terminus and at the N-terminus, respectively. The adsorption of the enzyme molecules on gold was studied by direct mass measurements with electrochemical quartz crystal microbalance. All the forms of HRP formed a monolayer coverage of the enzyme on the gold surface. However, only gold electrodes with adsorbed recombinant HRP forms exhibited high and stable current response to H(2)O(2) due to its bioelectrocatalytic reduction based on direct electron transfer between gold and HRP. The sensitivity of the gold electrodes modified with recombinant HRPs was in the range of 1.4-1.5 A M(-1) cm(-2) at -50 mV versus Agmid R:AgCl. The response to H(2)O(2) in the concentration range 0.1-40 microM was not dependent on the presence of a mediator (i.e. catechol) giving strong evidence that the electrode currents are diffusion limited. Lower detection limit for H(2)O(2) detection was 10 nM at the electrodes modified with recombinant HRPs.

Adsorption↗

PubMed1998

p66/p51 and p51/p51 recombinant forms of reverse transcriptase from human immunodeficiency virus type 1--interactions with primer tRNA(Lys3), initiation of cDNA synthesis, and effect of inhibitors.

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) initiates reverse transcription from tRNA(Lys3). HIV-1 RT is a heterodimer consisting of two polypeptides, p66 and p51. In this work, the possible role of each subunit of RT in the interaction with its natural primer tRNA(Lys3) was studied. Two recombinant forms of HIV-1 RT, heterodimer p66/p51 and homodimer p51/p51, were used. Previously we have expressed and purified recombinant RT p51/p51 which possesses DNA polymerase activity [El Dirani-Diab, R., Andreola, M. L., Nevinsky, G., Tharaud, D., Barr, P. J., Litvak, S. & Tarrago-Litvak, L. (1992) FEBS Lett. 301, 23-28]. Here we show that HIV-1 RT p51/p51 displays certain properties very similar to the p66/p51 recombinant enzyme. The homodimer was able to anneal tRNA(Lys3) to the primer-binding site of the HIV-1 RNA template leading to a functional complex capable of synthesizing cDNA. Further, the p51/p51 enzyme behaved like RT p66/p51 concerning the strong inhibition produced by a non-nucleoside RT inhibitor. These data show that for RT p51/p51, one of the subunits of the homodimer adopts a conformation similar to the catalytic subunit (p66) present in the heterodimeric form. Part of this work was devoted to the study of the complex between the recombinant forms of HIV-1 RT and its primer tRNA. Each enzymatic form was cross-linked to tRNA(Lys3) in the presence of a platinum derivative, giving different ribonucleoprotein complexes of molecular masses higher than 100 kDa, suggesting that primer tRNA may interact with both subunits in the heterodimeric enzyme. After RNase A treatment of the complex RT p66/p51 x tRNA, the label was mainly found to migrate with the p66 subunit, although some cross-linking was also found associated to the p51 subunit. These results show that the p66 and p51 subunits of RT interact with tRNA(Lys3). Moreover, cross-linking of tRNA(Lys3) with HIV-1 RT p66/p51 in the presence of a DNA template containing the primer-binding-site sequence yielded an enzymatically active complex.

Base Sequence↗

PubMed1998

Characterization of native and recombinant forms of an unusual cobalt-dependent proline dipeptidase (prolidase) from the hyperthermophilic archaeon Pyrococcus furiosus.

Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co2+ ions (Kd, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co2+ could be replaced by Mn2+ (resulting in a 25% decrease in activity) but not by Mg2+, Ca2+, Fe2+, Zn2+, Cu2+, or Ni2+. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100 degrees C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, the Km values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater Vmax value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.

Amino Acid Sequence↗

PubMed2000

Production and functional characterization of a soluble recombinant form of mouse CD59.

This report describes the engineering, expression, purification and functional characterization of a soluble recombinant form of murine CD59 (srMoCD59). We report the expression in Chinese hamster ovary (CHO) cells of a modified mouse CD59 cDNA that had been truncated at D-74, resulting in the loss of the glycosylphosphatidyl inositol (GPI) anchor, and containing six additional C-terminal histidines. The expressed srMoCD59 was purified from tissue culture supernatant by means of its poly-histidine tag using immobilized metal affinity chromatography. In comparison with CD59 on mouse erythrocytes, the srMoCD59 had a reduced molecular weight (18-20 000 as compared with 20-28 000 for GPI-anchored srMoCD59). The terminal complement inhibitory capacity of this soluble recombinant protein was assessed using two methods: a cobra venom factor (CVF)-triggered 'reactive-lysis' system and a C5b-7 site assay. In both assays, srMoCD59 inhibited lysis by the sera from all three species tested in the rank order mouse > rat >> human. The amount of srMoCD59 required to produce 50% inhibition of lysis in the C5b-7 site assay, using purified terminal components to develop lysis, was 10-fold less than that required in the same assay when EDTA serum was used as a source of C8 and C9, or in the CVF reactive lysis system. These data indicate that the presence of serum markedly interfered with the activity of srMoCD59 and have important implications for the use of recombinant soluble CD59 analogues as therapeutic agents in complement-mediated diseases.

Animals↗

PubMed2003

Circulating recombinant form CRF02_AG in South America.

With the objective of monitoring the distribution of HIV-1 subtypes and circulating recombinant forms (CRFs)in South America, population-based surveillance studies were performed in seven countries. Peripheral blood mononuclear cell, filter paper, fresh blood, and cocultivation samples were collected from HIV-positive patients from Colombia, Ecuador, Peru, Bolivia, Chile, Argentina, and Uruguay, during a 7-year period(1995-2001). DNA was prepared and HIV envelope subtypes were determined by heteroduplex mobility as-say and DNA sequencing from 1289 HIV-positive samples. While subtypes B and F were the most commonly observed subtypes, two CRF02_AG strains were detected, in Ecuador. This is the first report of the existence of this CRF in South America.

Adult↗