Reactions of Ugandan antisera with peptides encoded by V3 loop epitopes of human immunodeficiency virus type 1.

The specificities of antibodies reacting with peptides encoded by V3 loop apical epitopes were determined for sera from 230 seropositive Ugandans, including asymptomatic persons and AIDS patients, sampled between 1986 and 1992. Most (71%) of the sera reacted with the peptide encoded by HIV-MN, 59% reacted with a peptide containing a consensus sequence for Ugandan variants of the HIV-1 global subtype A (referred to as the Uganda A consensus), 59% reacted with a peptide containing a consensus sequence for Ugandan variants of the global subtype D (the Uganda D consensus); 19% of the sera also reacted with peptides encoded by the divergent Ugandan variant U31. There was no obvious correlation between the specificities of antibody binding and the V3 loop sequence of the corresponding virus isolate or provirus. Competitive inhibition and antibody adsorption experiments indicated that the MN peptide, the Uganda A consensus peptide, the Uganda D consensus peptide, and the U31 peptide were recognized by different sets of antibodies. Eighteen percent of the sera from AIDS patients and 26% of the sera from asymptomatic persons were monospecific for one of the MN, Uganda A, or Uganda D peptides. Whereas all except one of the singly reactive AIDS sera were specific for MN, 39% of the singly reactive asymptomatic sera were specific for MN, 39% for the Uganda A peptide, and 21% for the Uganda D peptides. We conclude that analysis of the specificities of antibodies against the V3 loop epitopes in sera from asymptomatic persons could provide useful epidemiological data about the prevalence of viral subtypes within a population.

PIP: The specificities of antibodies reacting with peptides encoded by V3 loop apical epitopes were determined for sera from 230 HIV seropositive Ugandans, including 123 asymptomatic persons and 107 AIDS patients, mostly mothers attending prenatal clinics, sampled between 1986 and 1992. 71% of the sera reacted with the peptide encoded by HIV-MN, 59% reacted with a peptide containing a consensus sequence for Ugandan variants of the HIV-1 global subtype A (referred to as the Uganda A consensus); 59% reacted with a peptide containing a consensus sequence for Ugandan variants of the global subtype D (the Uganda D consensus); and 19% of the sera also reacted with peptides encoded by the divergent Ugandan variant U31. Although 70% of the 1986 sera reacted with the Uganda A consensus peptide, only 49% of the 1991/92 sera reacted with this peptide (p 0.005). 20% of the 1991/92 sera, compared with only 7% of the 1986 sera, did not react with any of the peptides (p 0.05). There was no obvious correlation between the specificities of antibody binding and the V3 loop sequence of the corresponding virus isolate or provirus. Competitive inhibition and antibody adsorption experiments indicated that the MN peptide, the Uganda A consensus peptide, the Uganda D consensus peptide, and the U31 peptide were recognized by different sets of antibodies. 18% of the sera from AIDS patients and 26% of the sera from asymptomatic persons were monospecific for one of the MN, Uganda A, or Uganda D peptides. Whereas all except one of the singly reactive AIDS sera were specific for MN, 39% of the singly reactive asymptomatic sera were specific for MN, 39% for the Uganda A peptide, and 21% for the Uganda D peptides. The analysis of the specificities of antibodies against the V3 loop epitopes in sera from asymptomatic persons could provide useful epidemiological data about the prevalence of viral subtypes within a population.

Sequence analysis of the V3 loop regions of the env genes of Ugandan human immunodeficiency proviruses.

Ugandan strains of human immunodeficiency virus type 1 (HIV-1) were isolated by cocultivation of peripheral blood lymphocytes from infected individuals with cord blood lymphocytes. Sequences from the V3 region of the env gene were amplified by the polymerase chain reaction (PCR) from chromosomal DNA obtained from low passage virus cultures. The PCR products from 13 Ugandan isolates were cloned into a phagemid vector and sequenced. Many isolates contained divergent V3 loop sequences and adjacent regions: diversity was associated with codon deletions or duplications and with nucleotide substitutions, especially G----A transitions. Proviruses from some of the cultures showed extensive diversity within the V3 loop sequences but others were more homogeneous. The V3 loop apices were conserved in 6 of the Ugandan proviruses and these were very similar to the equivalent regions of several Zairean proviruses. The V3 loop apices of African isolates of HIV-1 are divergent from those of North American isolates. The possible biological consequences of this divergence are discussed.

Nucleotide sequence of the S RNA of Lassa virus (Nigerian strain) and comparative analysis of arenavirus gene products.

The nucleotide sequence of the small (S) genomic RNA of Lassa virus (strain GA391, of Nigerian origin) has been determined. The RNA has features which conform to those seen in most other arenavirus S RNAs which have been characterised, including conserved terminal sequences, an ambisense arrangement of the coding regions for the precursor glycoprotein (GPC) and nucleocapsid (N) proteins and an intergenic region capable of forming a base-paired "hairpin" structure. Comparison of the nucleotide sequence with that of the Josiah strain of Lassa virus (from Sierra Leone) reveals considerable nucleotide divergence in the third base of codons in the reading frames of all three proteins, although the resulting protein sequences are highly conserved, with 92, 94 and 91% identical residues for the mature glycoproteins G1 and G2 and the N protein, respectively. Sequence alignments of the available arenavirus structural proteins and dendrograms summarising the relationships between the viral proteins are presented.

Nucleotide sequence of a Ugandan HIV-1 provirus reveals genetic diversity from other HIV-1 isolates.

A Ugandan isolate of human immunodeficiency virus type 1 (HIV-1), designated U455, was adapted to growth in U937 cells, the provirus cloned into the lambda L47.1 vector, and its DNA sequence determined. The sequences of some of the U455 genes showed a marked divergence from those of North American and other African isolates. The sequenced clone was defective with single in-phase stop codons in the vpr and env genes and frame shift, resulting in a stop codon, within the vpu gene.

Complete nucleotide sequence of the Rhodosporidium toruloides gene coding for phenylalanine ammonia-lyase.

The complete nucleotide sequence of the Rhodosporidium toruloides gene coding for the enzyme phenylalanine ammonia-lyase (PAL) has been determined. The primary structure of PAL was deduced from the nucleotide sequence of the two cDNA clones, pPAL1 and pPAL2, which covered the entire amino acid-coding sequence. Comparison of cDNA and genomic sequences of pal revealed the presence of six introns. The nucleotide sequences of these introns were compared to those from other fungi. The primary amino acid sequence of the enzyme exhibits only 30.8% identity with the determined primary sequence of PAL from Phaseolus vulgaris. Upstream from the structural gene there is a stretch of C + T-rich DNA similar to that found upstream from a number of Neurospora and Saccharomyces cerevisiae genes. In the case of S. cerevisiae, these C + T-rich sequences are thought to be involved in the transcription of highly expressed genes.

Molecular cloning of Lassa virus RNA: nucleotide sequence and expression of the nucleocapsid protein gene.

Lassa virus RNA isolated from purified virus particles was polyadenylated and reverse transcripts were cloned into the PstI site of plasmid pUC9. Clones containing sequences of the smaller (S) segment of the Lassa virus genome were identified by hybridization with purified S RNA. They were characterized by their ability to hybridize with fragments of 3'-labeled Lassa virus S RNA and with each other, and by restriction mapping. The largest insert was 1830 bp long and began with the 3'-terminal 19-base sequence characteristic of all arenavirus S RNAs so far analyzed. The virus complementary strand contained a single large open reading frame, beginning at the ATG nearest its 5' end (nucleotides 103-5) and terminating with a TGA triplet at position 1813-15, that encodes a protein of 570 amino acids. A recombinant was constructed which expressed the gene as a fusion protein in Escherichia coli. The product was a 60-kDa polypeptide which reacted with monoclonal antibodies specific for the nucleocapsid protein. Comparison of the predicted amino acid sequence with the corresponding sequences deduced from the nucleotide sequences of the other arenavirus S RNAs, lymphocytic choriomeningitis (LCM) and Pichinde, reveals a considerable degree of similarity between Old and New World arenaviruses.

The structure of the major immediate early gene of human cytomegalovirus strain AD169.

The nucleotide sequence of the major immediate early (IE) gene of human cytomegalovirus strain AD169 was determined. The structure of the gene was examined by nuclease mapping and by sequence analysis of a cDNA clone made from IE mRNA. The gene encodes a spliced molecule of 1736 nucleotides, made up of four exon sequences of 121, 88, 185 and 1342 nucleotides. Three introns (827, 114 and 170n) were located near the 5' end of the gene. A single open reading frame starting in the second exon extends for 491 amino acids, corresponding to a protein of molecular weight 64000. The putative promoter region contains several short direct and inverted repeat sequences of 16, 18, 19 and 21 nucleotides, which extend 509n upstream from the transcription start site. The structure of the major IE gene and its protein product are discussed and compared with the corresponding IE gene from the Towne strain of HCMV.

Characterization of the human cytomegalovirus envelope glycoproteins.

Virions of human cytomegalovirus were shown to contain two discrete membranes. An outer, loose fitting, membrane was sensitive to osmotic shock and could be partially removed by diluting buffered preparations of virions with water. Purified virions were shown, by SDS-polyacrylamide gel electrophoresis of virion membranes labelled by carbohydrate-specific procedures, to contain five glycoproteins with molecular weights of 52, 67, 95, 130 and 250, all X 10(3). Digestion of virions with endoglycosidases revealed that there were structural differences between the carbohydrate portions of the glycoproteins. All five glycoproteins were recognized by antibodies present in pools of human convalescent sera.

Human cytomegalovirus DNA: BamHI, EcoRI and PstI restriction endonuclease cleavage maps.

The cloned HindIII fragments of human cytomegalovirus (HCMV) strain AD169 DNA were mapped with respect to the BamHI, EcoRI and PstI restriction endonuclease cleavage sites. Composite restriction endonuclease cleavage maps for the entire virus genome were constructed using the previously established linkages between the HindIII fragments.

Use of recombinant plasmids to investigate the structure of the human cytomegalovirus genome.

Human cytomegalovirus (HCMV) DNA was digested with restriction endonucleases and the fragments characterized with respect to molecular weight and relative mole proportions. The terminal fragments were identified by digesting HCMV DNA with exonucleases before restriction endonuclease treatment and subsequent gel analysis. The HindIII fragments of HCMV DNA were cloned in Escherichia coli and recombinant plasmids were characterized by digestion with restriction endonucleases and by molecular hybridization with HindIII, Bg/II and XbaI fragments of the virus genome. Data from these experiments were used to construct physical maps of HCMV DNA for the HindIII, Bg/II and XbaI restriction endonucleases. The terminal regions of the genome and the region containing fragment HindIII M were shown to be heterogeneous.

A comparison of labelled antibody methods for the detection of virus antigens in cell monolayers.

A number of labelled antibody methods have been applied to the detection of Semliki Forest virus antigens after replication of the virus in monolayers of host cells in multi-wellpolystyrene plates. The importance of several reaction variables has been investigated and the sensitivity of the methods compared for different periods of virus replication. Direct assays with radio-labelled antibody (RLA) and indirect assays using peroxidase-antiperoxidase complexes (PAP) were equally sensitive. Direct and indirect assays using enzyme-linked antibodies (ELA) were slightly less sensitive than direct RLA and PAP methods but were more sensitive than the indirect RLA or fluorescent antibody (FLA) methods. Direct assays using ELA were more rapid and easier to perform than the other assay methods.

The inhibition of streptococci by lactoperoxidase, thiocyanate and hydrogen peroxide. The effect of the inhibitory system on susceptible and resistant strains of group N streptococci.

1. The growth of the lactoperoxidase-sensitive Streptococcus cremoris 972 in a synthetic medium was inhibited by lactoperoxidase and thiocyanate. The glycolysis and oxygen uptake of suspensions of Strep. cremoris 972 in glucose or lactose were also inhibited. The lactoperoxidase-resistant Strep. cremoris 803 was not inhibited under these conditions but was inhibited in the absence of a source of energy. 2. Lactoperoxidase (EC 1.11.1.7), thiocyanate and hydrogen peroxide completely inhibited the hexokinases of non-metabolizing suspensions of both strains. The inhibition was reversible, hexokinase and glycolytic activities of Strep. cremoris 972 being restored by washing the cells free from inhibitor. The aldolase and 6-phosphogluconate-dehydrogenase activities of Strep. cremoris 972 were partially inhibited but several other enzymes were unaffected. 3. The resistance of Strep. cremoris 803 to inhibition was not due to the lack of hydrogen peroxide formation, to the destruction of peroxide, to the inactivation of lactoperoxidase or to the operation of alternative pathways of carbohydrate metabolism. 4. A ;reversal factor', which was partially purified from extracts of Strep. cremoris 803, reversed the inhibition of glycolysis of Strep. cremoris 972. The ;reversal factor' also catalysed the oxidation of NADH(2) in the presence of an intermediate oxidation product of thiocyanate and was therefore termed the NADH(2)-oxidizing enzyme. 5. The NADH(2)-oxidizing enzyme was present in lactoperoxidase-resistant streptococci but was absent from lactoperoxidase-sensitive streptococci.