Striking similarities in amino acid sequence among nonstructural proteins encoded by RNA viruses that have dissimilar genomic organization.

The plant viruses alfalfa mosaic virus (AMV) and brome mosaic virus (BMV) each divide their genetic information among three RNAs while tobacco mosaic virus (TMV) contains a single genomic RNA. Amino acid sequence comparisons suggest that the single proteins encoded by AMV RNA 1 and BMV RNA 1 and by AMV RNA 2 and BMV RNA 2 are related to the NH2-terminal two-thirds and the COOH-terminal one-third, respectively, of the largest protein encoded by TMV. Separating these two domains in the TMV RNA sequence is an amber termination codon, whose partial suppression allows translation of the downstream domain. Many of the residues that the TMV read-through domain and the segmented plant viruses have in common are also conserved in a read-through domain found in the nonstructural polyprotein of the animal alphaviruses Sindbis and Middelburg. We suggest that, despite substantial differences in gene organization and expression, all of these viruses use related proteins for common functions in RNA replication. Reassortment of functional modules of coding and regulatory sequence from preexisting viral or cellular sources, perhaps via RNA recombination, may be an important mechanism in RNA virus evolution.     

HB Coimbra or α2β299(Gl)ASP→GLU,A Newly Discovered High Oxygen Affinity Variant

We have identified a new high oxygen affinity hemoglobin variant in members of a Portuguese family; it is characterized by an Asp→Glu replacement at codon 99 of the β chain which is in the α1β2 interface. The altered functional properties of Hb Coimbra likely result from the inability to form a hydrogen bond between β99Glu and α42Tyr; such a bond is formed in deoxy Hb A between the normally occurring β99Asp and α42Tyr. The two affected members of the family have a distinct erythrocytosis with hemoglobin levels of 18 to 20 g/dl. The mutation in the β-globin gene (GAT→GAA at codon 99) resulting in the Asp→Glu replacement is the seventh type at this specific location. A review of the many variants of the α and β chains identifies primarily aspartic acid and glutatnic acid residues as being most frequently replaced; it is speculated that codons GAC and GAT (for Asp), and GAGand GAA (for Glu) are most susceptible to mutational events.     

OK10, an avian acute leukemia virus of the MC29 subgroup with a unique genetic structure

The RNA of defective avian acute leukemia virus OK10 was isolated from a defective virus particle, released by OK10-transformed nonproducer avian fibroblasts, as a 60S complex consisting of 8.6-kilobase subunits. Oligonucleotide fingerprinting and RNA.cDNA hybridization identified two sets of sequences in OK10 RNA: group-specific sequences, which are related to all nondefective members of the avian tumor virus group, and a sequence closely related to the subgroup-specific sequences (mcv) of the myelocytomatosis virus (MC29) subgroup of avian acute leukemia viruses. Hence, OK10 is classified as a member of the MC29 subgroup of avian tumor viruses, in agreement with classification based on its oncogenic spectrum. The group-specific sequences of OK10 RNA include partial (Delta) pol and env genes, a c-region, and, unlike those of all other members of the MC29 subgroup, a complete gag gene. Oligonucleotide mapping revealed 5'-gag-Deltapol-mcv-Deltaenv-c-3' as the order of the subgroup-specific and group-specific elements of OK10 RNA. The genetic unit gag-Deltapol-mcv, measuring approximately 6.4 kilobases, codes for the nonstructural, presumably transforming, 200,000-dalton OK10-specific protein and also includes the gag gene coding for the internal virion proteins. Because gag is the only intact virion gene shared in addition to regulatory RNA sequences between OK10 and nondefective avian tumor viruses, it is concluded that the gag gene is sufficient for the formation of a defective virus particle. Comparisons among the RNAs and gene products of different viruses of the MC29 subgroup show that they share 5'-terminal gag-related and internal mcv sequences but differ from each other in intervening gag-, pol-, and mcv-related sequences. It follows that the probable transforming genes and their protein products have two essential domains, one consisting of conserved 5' gag-related and the other of 3' mcv-related sequence elements. In the light of this and previous knowledge we can now distinguish two designs among five different transforming onc genes of avian tumor viruses: onc genes with coding sequences unrelated to virion genes, like those of Rous sarcoma virus and avian myeloblastosis virus, and onc genes with coding sequences that are hybrids of virion genes and specific sequences, like those of the MC29 subgroup viruses, of avian erythroblastosis virus, and of Fujinami sarcoma virus.     

Making of viral replication organelles by remodeling interior membranes

Positive-stranded RNA (+RNA) viruses exploit host cell machinery by subverting host proteins and membranes and altering cellular pathways during infection. To achieve robust replication, some +RNA viruses, such as poliovirus (PV), build special intracellular compartments, called viral replication organelles. A recent work from the Altan-Bonnett laboratory [1] gave new insights into the formation of poliovirus replication organelles, which are unique subcellular structures containing many individual replication complexes as a result of dynamic cellular membrane remodeling.     

Poliovirus chimeras replicating under the translational control of genetic elements of hepatitis C virus reveal unusual properties of the internal ribosomal entry site of hepatitis C virus.

Chimeric genomes of poliovirus (PV) have been constructed in which the cognate internal ribosomal entry site (IRES) element was replaced by genetic elements of hepatitis C virus (HCV). Replacement of PV IRES with nt 9-332 of the genotype Ib HCV genome, a sequence comprising all but the first eight residues of the 5' nontranslated region (5'NTR) of HCV, resulted in a lethal phenotype. Addition of 366 nt of the HCV core-encoding sequence downstream of the HCV 5'NTR yielded a viable PV/HCV chimera, which expressed a stable, small-plaque phenotype. This chimeric genome encoded a truncated HCV core protein that was fused to the N terminus of the PV polyprotein via an engineered cleavage site for PV proteinase 3CPpro. Manipulation of the HCV core-encoding sequence of this viable chimera by deletion and frameshift yielded results suggesting that the 5'-proximal sequences of the HCV open reading frame were essential for viability of the chimera and that the N-terminal basic region of the HCV core protein is required for efficient replication of the chimeric virus. These data suggest that the bona fide HCV IRES includes genetic information mapping to the 5'NTR and sequences of the HCV open reading frame. PV chimeras replicating under translational control of genetic elements of HCV can serve to study HCV IRES function in vivo and to search for anti-HCV chemotherapeutic agents.     

Relations immunologiques entre les hormones glycoprotéiques hypophysaires de poissons et de Mammifères ainsi qu'entre leurs sous-unités α et β

Immunological relationships among pituitary glycoproteic hormones and their subunits from fishes and mammals were studied with antisera (IS) raised in rabbits against carp gonadotropin (c-GTH), its α subunit (c-GTH α), and its β subunit (c-GTH β). Properties of the following preparations were compared: c-GTH and its subunits; gonadotropins (GTH) from two other Teleosts (indian catfish and eel), and a Chondrostean (sturgeon); and mammalian hormones (bovine LH and TSH, ovine FSH) and their subunits. In the α radioimmunoassays (RIAs) (IS c-GTH α, [¹²⁵I]c-GTH α) or (IS c-GTH, [¹²⁵I]c-GTH α) only the GTH from Cypriniform Teleosts (carp and indian catfish) cross-reacted: thus these α RIAs showed a high zoological specificity. However a weak immunological relatedness between c-GTH α and mammalian α subunits (b-LH α, b-TSH α, o-FSH α) could be shown in binding studies; in contrast, even at high concentration, the IS c-GTH α did not bind mammalian β subunits. The β RIA (IS c-GTH β, [¹²⁵I]c-GTH β) exhibited a much weaker zoological specificity: not only all fish GTHs studied but also b-LH and b-LH β were able to compete with [¹²⁵I]c-GTH β; furthermore, the degree of cross-reactivity (reflected by the slopes of the regressions) followed phylogenetic relationships. No cross-reaction was observed with the other mammalian preparations. The c-GTH RIA (IS c-GTH, [¹²⁵I]c-GTH) showed an intermediate zoological specificity. Binding studies carried out with ¹²⁵I-labeled mammalian preparations confirmed these data: b-LH and b-LH β were highly bound by IS c-GTH and IS c-GTH β whereas very low or no binding was observed with o-FSH or o-FSH β. A weak binding of b-TSH and b-TSH β by IS c-GTH and IS c-GTH β indicated a relationship between mammalian TSH β and TSH β or GTH β itself from the carp. Even at high concentration the IS c-GTH β did not bind mammalian α subunits. Thus the comparison of antigenic determinants gives additional evidence for homology among fish and mammal pituitary glycoproteins, indicating evolutionary relationship between the subunits of the same type (α or β). In the case of the β subunits, c-GTH β appears more closely related to mammalian LH β than to FSH β. Finally, due to the relative lack of immunological species specificity of the IS c-GTH β, the β RIA may be a useful tool to assay specifically GTHs from other species of fish.     

Sequence-motif analysis of 5'-untranslated region of GB virus-C in Japanese patients

BACKGROUND: GB Virus C (GBV-C) is considered to belong to the Flaviviridae; however, the structures of the N-terminal end of its putative polyprotein are not well known. The internal ribosomal entry site (IRES) at the 5'-untranslated region of GBV-C and an initiating codon at nucleotides (nt) 552-554 have been proposed. We investigated the validity of this proposal. METHODS: The 5'-untranslated region of GBV-C was amplified from serum samples of 17 Japanese patients. Polymerase chain reaction-amplified products were directly sequenced and the obtained sequences were analysed by comparing them with the IRES structure of other viruses. RESULTS: Fifteen of the 17 (88%) GBV-C strains in our patients were classified as being Asian type. The box-A-like sequence (UUUC) and box-B-like sequence (AUCAUGG) observed in the IRES of picornaviruses were highly conserved in all the strains. Based on pair-wise comparisons with the multiple alignment data, overall sequence divergence for the 5'-terminus was 2.9-12%. When compared with the proposed secondary structure of the IRES model, the sequence divergences of the Asian-type GBV-C were higher at the regions of loop structures and lower at the regions of double-stranded RNA. The AUG codons, except for the one located at nt 552-554, produced truncated polyproteins or were not in-frame with the putative protein. CONCLUSIONS: Our examination of the sequence motif of GBV-C supports the proposal that the GBV-C has common structural motifs for IRES at its 5'-untranslated region and the AUG codon at nt 552-554 may be an initiating codon.     

Selective incorporation of influenza virus RNA segments into virions

The genome of influenza A virus is comprised of eight viral RNA (vRNA) segments. Although the products of all eight vRNA segments must be present for viral replication, little is known about the mechanism(s) responsible for incorporation of these segments into virions. Two models have been proposed for the generation of infectious virions containing eight vRNA segments. The random-incorporation model assumes a common structural feature in all the vRNAs, enabling any combination of vRNAs to be incorporated randomly into virions. The selective-incorporation model predicts the presence of specific structures in each vRNA segment, leading to the incorporation of a set of eight vRNA segments into virions. Here we demonstrate that eight different vRNA segments must be present for efficient virion formation and that sequences within the coding region of (and thus unique to) the neuraminidase vRNA possess a signal that drives incorporation of this segment into virions. These findings indicate a unique contribution from individual vRNA segments and thus suggest a selective (rather than random) mechanism of vRNA recruitment into virions. The neuraminidase vRNA incorporation signal and others yet to be identified should provide attractive targets for the attenuation of influenza viruses in vaccine production and the design of new antiviral drugs.     

In vivo mapping of a sequence required for interference with the yeast killer virus.

The Saccharomyces cerevisiae viruses are noninfectious double-stranded RNA viruses whose segments are separately encapsidated. A large viral double-stranded RNA (L1; 4580 base pairs) encodes all required viral functions. M1, a double-stranded RNA of 1.9 kilobases, encodes an extracellular toxin (killer toxin) and cellular immunity to that toxin. Some strains contain smaller, S, double-stranded RNAs, derived from M1 by internal deletion. Particles containing these defective interfering RNAs can displace M1 particles by faster replication and thus convert the host strain to a nonkiller phenotype. In this work, we report the development of an assay in which the expression of S plus-strand from an inducible plasmid causes the loss of M1 particles. This assay provides a convenient method for identifying in vivo cis-acting sequences important in viral replication and packaging. We have mapped the sequence involved in interference to a region of 132 base pairs that includes two sequences similar to the viral binding site sequence previously identified in L1 by in vitro experiments.     

Molecular cloning and characterization of an isolate of hepatitis delta virus from Taiwan

The genomic RNA of an Asian isolate of hepatitis delta virus was cloned from a Chinese patient from Taiwan, using the polymerase chain reaction to amplify cDNA for cloning and sequencing. The sequence of this hepatitis delta virus isolate shares an 86% to 88% similarity with the three published hepatitis delta virus RNA sequences, suggesting heterogeneity of hepatitis delta viruses from different geographical areas. Four highly conserved, long stretches of sequence were found. These four regions corresponded to the sequences required for the autocatalytic cleavage activities of the genomic and antigenomic RNAs and the middle and the carboxyl terminal parts of the open reading frame for the delta antigen on the antigenomic strand. The conservation of nucleotide sequence in these four regions was further confirmed by sequencing additional hepatitis delta virus RNAs obtained from three patients with chronic delta hepatitis who lived in Los Angeles. These findings suggest that the conserved sequences are critical for viral replication. These conserved regions offer ideal sites for primer selection to carry out polymerase chain reactions to detect hepatitis delta virus RNA in patients with hepatitis delta virus infection.     

Compound heterozygosity for Hb S [β6(A3)Glu→Val] and Hb Kenya (Aγ81Leu-β86Ala) in a Ugandan woman

Hb Kenya is a hemoglobin (Hb) tetramer composed of two normal α- and two non α-globin chains. The latter are the product of a fusion gene in which the 5' end is (A)γ and the 3' end is β. The crossover point is between codon 81 of the (A)γ gene and codon 86 of the β gene. Like the other non α genes, the hybrid protein product ((A)γ81Leu-β86Ala) has 146 amino acids. The purpose of this report is to highlight the laboratory findings of Hb Kenya and to emphasize the pitfalls in misdiagnosis, particularly when associated with another variant such as Hb S [β6(A3)Glu→Val].     

Three-dimensional model of the active site of the self-splicing rRNA precursor of Tetrahymena.

The rRNA intervening sequence of Tetrahymena is a catalytic RNA molecule, or "ribozyme." A tertiary-structure model of the active site of this ribozyme has been constructed based on comparative sequence analysis of related group I intervening sequences, data on the accessibility of each nucleotide to chemical and enzymatic probes, and principles of RNA folding derived from a consideration of the structure of tRNA determined by x-ray crystallography. In the model, the catalytic center has a two-helix structural framework composed of the base-paired segments of the group I conserved sequence elements. The structural framework supports and orients the conserved nucleotides that are adjacent to the base-paired sequence elements; these conserved nucleotides are proposed to form the active site and to bind the 5' splice-site duplex and the guanine nucleotide substrate. Tests of the model are proposed.     

Genotype of subjects with borderline hemoglobin A2 levels: Implication for, β-thalassemia carrier screening

In this study, we have defined by molecular analysis, the α, β, and δ globin genotype in a group of individuals with normal or thal-like red cell indices but borderline hemoglobin (Hb)A2 levels, who were identified in a program for β-thal carrier screening. In 37 of 125 individuals with borderline HbA2 levels, we detected a molecular defect in the β, in both the δ and the β, or in the α globin gene. Specifically seven of these subjects were carriers of the ?101 C T mutation, ten of the IVSI nt6 T C mutation, 16 were double heterozygotes for δ and β thal, and two had the triple α globin gene and two the single α globin gene deletion. From these results, we may conclude that subjects with borderline HbA2, particularly when they marry a typical β-thal carrier, should be extensively investigated in order not to miss heterozygous β-thalassemia. © 1994 Wiley-Liss, Inc.     

Sequence analysis of the 5'' noncoding region of hepatitis C virus.

We have determined the nucleotide sequence of the 5' noncoding (NC) region of the hepatitis C virus (HCV) genome in 44 isolates from around the world. We have identified several HCV isolates with significantly greater sequence heterogeneity than reported previously within the 5' NC region. The most distantly related isolates were only 90.1% identical. Nucleotide insertions were seen in three isolates. Analysis of the nucleotide sequence from 44 HCV isolates in this study combined with that of 37 isolates reported in the literature reveals that the 5' NC region of HCV consists of highly conserved domains interspersed with variable domains. The consensus sequence was identical to the prototype HCV sequence. Nucleotide variations were found in 45 (16%) of the 282 nucleotide positions analyzed and were primarily located in three domains of significant heterogeneity (positions -239 to -222, -167 to -118, and -100 to -72). Conversely, there were three highly conserved domains consisting of 18, 22, and 63 completely invariant nucleotides (positions -263 to -246, -199 to -178, and -65 to -3, respectively). Two nucleotide domains within the 5' NC region, conserved among all HCV isolates studied to date, shared statistically significant similarity with pestivirus 5' NC sequences, providing further evidence for a close evolutionary relationship between these two groups of viruses. Additional analysis revealed the presence of short open reading frames in all HCV isolates. Our sequence analysis of the 5' NC region of the HCV genome provides additional information about conserved elements within this region and suggests a possible functional role for the region in viral replication or gene expression. These data also have implications for selection of optimal primer sequences for the detection of HCV RNA by the PCR assay.     

Compound Heterozygosity for Hb S [β6(A3)Glu→Val] and Hb Kenya (Aγ81Leu-β86Ala) in a Ugandan Woman

Hb Kenya is a hemoglobin (Hb) tetramer composed of two normal α- and two non α-globin chains. The latter are the product of a fusion gene in which the 5′ end is ^Aγ and the 3′ end is β. The crossover point is between codon 81 of the ^Aγ gene and codon 86 of the β gene. Like the other non α genes, the hybrid protein product (^Aγ81Leu-β86Ala) has 146 amino acids. The purpose of this report is to highlight the laboratory findings of Hb Kenya and to emphasize the pitfalls in misdiagnosis, particularly when associated with another variant such as Hb S [β6(A3)Glu→Val].     

Organization and sequences of the diversity, joining, and constant region genes of the human T-cell receptor beta chain.

The organization and sequences of the human beta-chain T-cell receptor diversity, joining, and constant region segments are described. The beta chain of the human T-cell receptor, analogous to the mouse counterpart, consists of two distinct constant region genes approximately equal to 10 kilobases apart. The two constant region genes, C beta 1 and C beta 2, are very similar not only in sequence but also in genomic organization. The coding sequences of each of these C beta constant region genes are divided into four exons. The first two exons encode most of the extracellular constant domain. The third exon encodes a major part of the presumed transmembrane portion, and the last exon contains the cytoplasmic coding sequence as well as 3' untranslated sequences. Except for a stretch of approximately equal to 95 highly conserved nucleotides extending 3' of the first exon of the C region genes, little homology can be found between the intron sequences of C beta 1 and C beta 2. A small cluster of joining region (J beta) gene segments is located approximately equal to 5 kilobases upstream of each of these two constant regions. The first cluster, J beta 1, contains six functional J gene segments while the second, J beta 2, contains seven functional J gene segments. In addition, diversity region (D beta) gene segments are located approximately equal to 600 base pairs upstream of each J beta. Recombinational signals containing highly conserved heptamer and nonamer sequences separated by 12 or 23 bases are found adjacent to all of these D beta and J beta gene segments. These signal sequences are thought to be involved in the somatic recombination processes. These results indicate that what appears to be a gene duplication event giving rise to these two distinct regions must have arisen a long time ago in the evolution of this gene locus.     

Complementation between Sindbis viral RNAs produces infectious particles with a bipartite genome.

Sindbis virus, the type member of the alpha-viruses, is an enveloped virus containing a nonsegmented positive-strand RNA genome. We show that the nonstructural and the structural genes can function to produce infectious virus particles when they are expressed on two different RNA segments. The nonstructural genes are translated from an RNA in which the structural genes have been replaced by the chloramphenicol acetyltransferase gene [Xiong, C., Levis, R., Shen, P., Schlesinger, S., Rice, C. M. & Huang, H. V. (1989) Science 243, 1188-1191]. The structural genes are encoded in a defective-interfering RNA but are translated from a subgenomic RNA. Both segments contain the cis-acting sequences required for replication and packaging and are copackaged. This type of genome provides a model for an ancestral intermediate between alphaviruses and the multipartite positive-strand RNA viruses of plants. These different viruses show sequence similarities in their replicative proteins and are thought to have evolved from a common ancestor.