Major Hydrophobic Region Research Articles

Genetic variation of hepatitis B surface antigen among acute and chronic hepatitis B virus infections in The Netherlands.

Genetic variation within hepatitis B surface antigen (HBsAg), in particular within the major hydrophobic region (MHR), is related to immune/vaccine and test failures and can have a significant impact on the vaccination and diagnosis of acute infection. This study shows, for the first time, variation among acute cases and compares the amino acid variation within the HBsAg between acute and chronic infections. We analyzed the virus isolated from 1231 acute and 585 chronic cases reported to an anonymized public health surveillance database between 2004 and 2014 in The Netherlands. HBsAg analysis revealed the circulation of 6 genotypes (Gt); GtA was the dominant genotype followed by GtD among both acute (68.2% and 17.4%, respectively) and chronic (34.9% and 34.2%, respectively) cases. Variation was the highest among chronic strains compared to that among acute strains. Both acute and chronic GtD showed the highest variation compared to that of other genotypes (P < .01). Substitutions within the MHR were found in 8.5% of the acute strains and 18.6% of the chronic strains. Specific MHR substitutions described to have an impact on vaccine/immune escape and/or HBsAg test failure were found among 4.1% of the acute strains and 7.0% of the chronic strains. In conclusion, we show a high variation of HBsAg among acute and chronic hepatitis B virus–infected cases in The Netherlands, in particular among those infected with GtD, and compare, for the first time, variation in frequencies between acute and chronic cases. Additional studies on the impact of these variations on vaccination and test failure need to be conducted, as well as whether HBsAg false–negative variants have been missed.

Antirhinoviral vinyl acetylene benzimidazoles

The nucleotide sequence of the hemagglutinin-neuraminidase (HN) gene of human parainfluenza type 2 virus (PIV2) was determined. The PIV-2 HN gene was 2112 nucleotides excluding poly(A) tail. There was a single large open reading frame in the mRNA which encoded a protein of 571 amino acids with a calculated molecular weight of 63,262. Analysis of the deduced amino acid sequence revealed that there were fourteen potential glycosylation sites and a major hydrophobic region near the N-terminus, which would anchor the protein in the viral membrane. Comparisons of the HN protein sequences of PIV-2 with those of Simian virus 5 (SV5), Sendai virus (SV, parainfluenza virus type 1), human parainfluenza virus type 3 (PIV-3), type 4 (PIV-4), bovine parainfluenza virus type 3 (BPIV-3), mumps virus (MuV), and Newcastle disease virus (NDV) showed definite amino acid sequence relatedness, indicating a common ancestor for these viruses. Furthermore, statistical analysis of the protein sequences suggested a possible evolutionary relatedness among the paramyxoviruses. This is the first time that a phylogenetic tree has been constructed for all the parainfluenza viruses and mumps virus which are infectious to humans. In addition, amino acid sequences involved in hemag-glutinating and neuraminidase activities of paramyxovirus were discussed.

Definition and functional analysis of the signal/anchor domain of the human respiratory syncytial virus glycoprotein G

The attachment protein G of human respiratory syncytial (RS) virus is a type II transmembrane glycoprotein. A secreted from of the G protein is also produced. To examine the two distinct hydrophobic regions in the N-terminal 63 amino acids of G protein for their role(s) in membrane insertion and anchoring, transport to the cell surface, and secretion, G proteins that contained point mutations or deletions were synthesized by cell-free transcription-translation and in cells by expression from recombinant vaccinia virus vectors. A mutant protein lacking the entire major hydrophobic region (amino acids 38-63) was not glycosylated, not expressed on the cell surface, and not secreted, because it was not inserted into membranes. In contrast, deletion of the minor hydrophobic region (amino acids 23-31) had no detectable effect on membrane insertion or anchoring. These data provided direct evidence that amino acids 38-63 were necessary for membrane insertion and contained the signal/anchor domain of RS virus G protein. Mutant proteins that lacked either the N-terminal or the C-terminal half of this 26 residue hydrophobic region were inserted into membranes and processed to maturity, showing that either half of this region was sufficient for membrane insertion. However, these two mutant proteins were secreted more abundantly than wild-type G protein. We propose that their truncated hydrophobic domains interacted with membranes in a way that mimicked the N-terminal signal sequence of naturally secreted proteins, allowing proteolytic cleavage of the mutant proteins.

The haemagglutinin-neuraminidase glycoprotein of the porcine paramyxovirus LPMV: comparison with other paramyxoviruses revealed the closest relationship to simian virus 5 and mumps virus

The complete nucleotide sequence of the haemagglutinin-neuraminidase (HN) gene of the porcine paramyxovirus LPMV, was determined from cDNA derived from viral genomic RNA. The gene was 1906 nucleotides long including a putative gene end and poly A signal. One long open reading frame was found encoding a protein of 576 amino acids with a calculated molecular weight of 63,324. The protein contains four potential N-glycosylation sites and a major hydrophobic region near the N-terminal, suggesting a membrane anchor domain. Comparison of the deduced amino acid sequence of the LPMV HN protein with that of other paramyxovirus HN proteins, revealed the highest amino acid identity to simian virus 5 of 43% and mumps virus of 41%.

The nucleotide and predicted amino acid sequence of the attachment protein of canine distemper virus

The nucleotide sequence of the gene coding for the attachment protein of the Convac strain of the canine distemper virus (CDV), corresponding to the haemagglutinin (H) gene of measles virus was determined using a mRNA-derived cDNA clone and genomic viral RNA. The mRNA transcribed from the CDV H gene is 1944 nucleotides long excluding the polyadenylated tail. Only one long open reading frame was found comprising nucleotides 21-1841. The predicted protein has a single hydrophobic region which can serve as a membrane anchoring domain. The deduced 607 amino acids would code for a protein of 68,247 Da, to be compared with an approximate protein molecular weight in SDS-PAGE of the glycosylated protein, which is 85,000 Da. The CDV H protein exhibited seven potential N-linked glycosylation sites. These were concentrated to the carboxyterminal part of the CDV H protein and differed markedly from measles virus (MV) and rinderpest virus (RPV) where the potential sites were mostly conserved and located in the amino-terminal half of the proteins. In spite of the differences in amino acid composition of these three H proteins their hydrophilicity/hydrophobicity plots were closely similar with the major hydrophobic region at an identical location. All the 12 cysteine residues found in the CDV H protein were conserved in MV and RPV. The amino acid homology between CDV and MV H protein was 37% and between CDV and RPV H protein 38%. The fact that the corresponding homology between the MV and RPV proteins is almost 60% shows that the evolutionary separation between CDV and RPV occurred at a much earlier time than the separation between RPV and MV.

Sequence of the hemagglutinin-neuraminidase gene of human parainfluenza virus type 1

The nucleotide sequence of the gene encoding the hemagglutinin-neuraminidase (HN) glycoprotein of human parainfluenza virus type 1 (PI1) was determined from cDNA clones derived from poly(A) + RNA extracted from infected cells. A single open reading frame in the sequence was found to encode a putative protein of 575 amino acids with a calculated molecular weight of 63,960. The predicted amino acid sequence contains ten potential sites for N-linked glycosylation and one major hydrophobic region located 35 amino acids from the amino terminus, which appears to be the signal-anchor domain of HN. Comparison of the amino acid sequence with the HN glycoproteins of other paramyxoviruses indicated that the PI1 HN protein is most closely related to the Sendai virus (SV) HN protein.

Sequence determination of the hemagglutinin-neuraminidase (HN) gene of human parainfluenza type 2 virus and the construction of a phylogenetic tree for HN proteins of all the paramyxoviruses that are infectious to humans

The nucleotide sequence of the hemagglutinin-neuraminidase (HN) gene of human parainfluenza type 2 virus (PIV2) was determined. The PIV-2 HN gene was 2112 nucleotides excluding poly(A) tail. There was a single large open reading frame in the mRNA which encoded a protein of 571 amino acids with a calculated molecular weight of 63,262. Analysis of the deduced amino acid sequence revealed that there were fourteen potential glycosylation sites and a major hydrophobic region near the N-terminus, which would anchor the protein in the viral membrane. Comparisons of the HN protein sequences of PIV-2 with those of Simian virus 5 (SV5), Sendai virus (SV, parainfluenza virus type 1), human parainfluenza virus type 3 (PIV-3), type 4 (PIV-4), bovine parainfluenza virus type 3 (BPIV-3), mumps virus (MuV), and Newcastle disease virus (NDV) showed definite amino acid sequence relatedness, indicating a common ancestor for these viruses. Furthermore, statistical analysis of the protein sequences suggested a possible evolutionary relatedness among the paramyxoviruses. This is the first time that a phylogenetic tree has been constructed for all the parainfluenza viruses and mumps virus which are infectious to humans. In addition, amino acid sequences involved in hemag-glutinating and neuraminidase activities of paramyxovirus were discussed.

Molecular cloning and sequence analysis of the rinderpest virus mRNA encoding the hemagglutinin protein

We cloned the full-length cDNAs corresponding to the mRNA for the hemagglutinin[dH) protein of rinderpest virus (RV) and determined the nucleotide sequence of RV-H. The gene of RV-H was composed of 1952 nucleotides and contained a single large open reading frame, which was capable of encoding a protein of 609 amino acids with a molecular weight of 68,330 Da. The nucleotide sequence and predicted amino acid sequence were compared with those of the measles virus (MV)-H. The 5′ end of the message(nucleotides 1 to 485) was largely conserved, with a homology of 75.1% of the nucleotides and 78.0% of the predicted amino acids. In the middle portion[dnucleotides 486–1310), where the potential glycosylation sites exist, 56.6% of the nucleotides and 49.5% of the amino acids were identical. In the 3′ end of the message[dnucleotides 1311–1850), 63.3% of the nucleotides and 58.1% of the amino acids were identical. Four potential glycosylation sites were found in RV-H protein and three of them were the same as those of MV-H protein. The positions of 13 cysteine residues of RV-H were absolutely identical to those of MV-H. The hydropathy profile of RV-H protein resembled that of MV-H. One major hydrophobic region long enough to be an anchor in the membrane was located near the N-terminus.

Nucleotide sequence of the Escherichia coli motB gene and site-limited incorporation of its product into the cytoplasmic membrane.

The motB gene product of Escherichia coli is an integral membrane protein required for rotation of the flagellar motor. We have determined the nucleotide sequence of the motB region and find that it contains an open reading frame of 924 nucleotides which we ascribe to the motB gene. The predicted amino acid sequence of the gene product is 308 residues long and indicates an amphipathic protein with one major hydrophobic region, about 22 residues long, near the N terminus. There is no consensus signal sequence. We postulate that the protein has a short N-terminal region in the cytoplasm, an anchoring region in the membrane consisting of two spanning segments, and a large cytoplasmic C-terminal domain. By placing motB under control of the tryptophan operon promoter of Serratia marcescens, we have succeeded in overproducing the MotB protein. Under these conditions, the majority of MotB was found in the cytoplasm, indicating that the membrane has a limited capacity to incorporate the protein. We conclude that insertion of MotB into the membrane requires the presence of other more hydrophobic components, possibly including the MotA protein or other components of the flagellar motor. The results further reinforce the concept that the total flagellar motor consists of more than just the basal body.

The predicted primary structure of the measles virus hemagglutinin

The complete nucleotide sequence of cloned cDNAs corresponding to the full length of the mRNA encoding the measles virus hemagglutinin (H) protein has been determined. the mRNA contains a single large open reading frame which is capable of encoding a protein of 617 amino acids with a molecular mass of 69,250 Da. The deduced amino acid structure of the protein indicates that the only major hydrophobic region of sufficient length to anchor the molecule in membranes is located near the amino terminus. Comparison of the amino acid structure of the measles virus H protein with that of the hemagglutinin-neuraminidase (HN) molecules of Sendai virus and simian virus 5 (SV5) reveals little homology. However, 11 of the 13 cysteine residues found in the measles H protein can be aligned with cysteines in the Sendai virus HN protein in similar positions relative to one another. Five potential N-linked glycosylation sites are present in the measles H protein sequence. These are relatively closely grouped between amino acids residues 168 and 240 in the amino terminal half of the molecule. No obvious structural features are present in the measles H protein amino acid sequence which might explain the reported absence of neuraminidase activity associated with the molecule.

Hemagglutinin-neuraminidase protein of the paramyxovirus simian virus 5: nucleotide sequence of the mRNA predicts an N-terminal membrane anchor.

The nucleotide sequence of a cloned cDNA copy of the mRNA coding for the hemagglutinin-neuraminidase of the paramyxovirus SV5 was determined. There was a single large open reading frame on the mRNA which encoded a protein of 565 amino acids with a molecular weight of 62,134. The deduced amino acid sequence indicated that the only major hydrophobic region in the protein sufficiently long to anchor the protein in the membrane is located near the N terminus (amino acids 18 to 36). It is suggested that, like the influenza virus neuraminidase, hemagglutinin-neuraminidase of paramyxoviruses is oriented with its N terminus inserted into the membrane.

Gating of ion channels made by a diphtheria toxin fragment in phospholipid bilayer membranes.

B45, a fragment containing the major hydrophobic region of diphtheria toxin, increases the conductance of thin lipid membranes by forming ion-conducting channels that are gated by transmembrane voltage, Vm, and the bath pH. Single-channel currents show "bursting" behavior in the form of rapid transitions between a closed and an open conductance level. The average duration of a current "burst," as well as the total time a channel is actually open within a burst, decreases with increasing Vm. Analysis of these data suggests that, over a range of Vm, increases in the rate constants for transitions from the open to the closed states largely account for the decline in macroscopic conductance with increasing Vm. Increases in rate constants for transitions from a closed to an open conductance state are more likely to account for the increase in macroscopic conductance with increasing bath pH. Since several diphtheria toxin fragments and mutants are currently available, each containing various portions of the B45 region, it may be possible to study the relationship of the structure of these complex proteins to the detailed gating properties of the ion channels that they form.

The influence of primary structure changes on the higher order structure of histone H2B

Structural changes of histones H2B in aqueous media were studied under conditions of varying pH and ionic strength. The techniques involved intrinsic fluorescence of tyrosine residues, extrinsic fluorescence using the hydrophobic probe 8-anilinonaphthalene sulfonate and fluorescence polarization. Secondary structure predictive methods were also used to study potential effects of primary mutations. In spite of many primary structure changes, the predicted effect on a globular structure of H2B is minimal. The results indicate the formation of a major hydrophobic region, the formation of helices and the burial of tyrosine residue 83 (calf), the structural changes of which appear to be identical in all H2B's as ionic strength is increased. The effect of primary structure changes on protein-protein interaction where H2B′s are concerned is likely to be negligible, whereas the major effect is likely to be the protein-DNA interaction where H2B could be at least partially responsible for diffferentiation.