Polypeptide 2A of human rhinovirus type 2: identification as a protease and characterization by mutational analysis

Evidence is presented that the protein 2A of human rhinovirus serotype 2 (HRV2) is a protease. On expression of the VP1-2A region of HRV2 in bacteria, protein 2A was capable of acting on its own N-terminus; derived extracts specifically cleaved a 16 amino acid oligopeptide corresponding to the sequence at the cleavage site. Cleavage of the oligopeptide substrate provides a convenient in vitro assay system. Deletion experiments showed that removal of 10 amino acids from the carboxy terminus inactivated the enzyme. Site-directed mutagenesis identified an essential arginine close to the C-terminus and showed that the enzyme was sensitive to changes in the putative active site. This analysis supports the hypothesis that 2A belongs to the group of sulfhydryl proteases, although sequence comparisons indicate that the putative active site of HRV2 2A is closely related to that of the serine proteases.     

Functional conservation of the hydrophobic domain of polypeptide 3AB between human rhinovirus and poliovirus

In this study we exchanged portions of the poliovirus type 1 (PV1) hydrophobic domain within the membrane-associated polypeptide 3AB for the analogous sequences from human rhinovirus 14 (HRV14). The sequence exchanges were based upon a previous report in which the 22 amino acid hydrophobic region was subdivided into two domains, I and II, the latter of which was shown to be required for membrane association (J. Biol. Chem. 271 (1996), 26810). Using these divisions, the HRV14 sequences were cloned into the complete poliovirus type 1 cDNA sequence. RNAs transcribed from these cDNAs were transfected into HeLa cell monolayers and used in HeLa cell-free translation/replication assays. The data indicated that 3AB sequences from PV1 and HRV14 are interchangeable; however, the substitutions cause a range of significant RNA replication defects, and in some cases, protein processing defects. Following transfection of RNAs encoding the domain substitutions into HeLa cell monolayers, virus isolates were harvested, and the corresponding viral RNAs were sequenced. The sequence data revealed that for the carboxy-terminal domain substitutions (domain II), multiple nucleotide changes were identified in the first, second, and third positions of different codons. In addition, the data indicated that for one of the PV1/HRV14 chimeras to replicate, compensatory mutations within poliovirus protein 2B may be required.     

Trypsin sensitivity of several human rhinovirus serotypes in their low pH-induced conformation

Five serotypes of human rhinovirus (HRV) were examined for sensitivity to trypsin at physiological pH, HRV1A, HRV2, and HRV14 were found to be resistant whereas in serotypes HRV49 and HRV89 degradation of VP2 was observed. However, exposure to low pH followed by neutralization, a treatment which causes irreversible conformational changes in the capsid, led to rapid cleavage by trypsin of VP1 in HRV1A, HRV2, and HRV49 at defined sites followed by degradation of VP2. In the case of HRV2, the cleavage site in VP1 was determined by direct protein sequencing and was shown to occur between Arg260 and Thr261, close to the C-terminus. HRV49 behaves similarly to HRV2 as expected from extensive sequence similarity in this region, whereas VP1 in HRV1A is most probably cleaved at a site closer to the C-terminus than that in HRV2. Although HRV14 contains the same amino acid pair present in HRV2 and HRV49, it was not cleaved under these conditions. HRV89, which lacks a basic residue at the corresponding position, was also insensitive. Examination of the cleavage site on the three-dimensional structural map of native HRV2 reveals that it is most probably buried inside the capsid and thus not accessible. Structural rearrangements of the viral capsid are thus necessary to account for the cleavage observed after low pH treatment.     

Isolation of a peptide inhibitor of human rhinovirus

Cell culture-based transdominant genetic techniques provide new methods for discovering peptide/RNA modulators of cellular pathways. We applied this technology to isolate a peptide inhibitor of human rhinovirus. A green fluorescent protein (GFP)-scaffolded library of cDNA fragments was expressed in HeLa cells from a retroviral vector and screened for inhibitors of rhinovirus-mediated cell killing. A DNA clone, I421, increased cell survival in an HRV14 challenge assay from less than 0.5% to greater than 60%. It encodes a 53-amino-acid C-terminal extension of the GFP scaffold. Particular subclones of Hela cells expressing I421 (exemplified by I421dp3) show a delay in virus production and a 50-fold decrease in viral RNA levels at 6-8 h postinfection. HRV2, HRV14, and HRV16 show a dramatic decrease in plaque-forming ability on I421dp3 while Coxsackievirus B3 showed a small reduction. Levels of ICAM-1, the receptor for the main rhinovirus serotype, are not altered in I421dp3.     

Substrate requirements of a human rhinoviral 2A proteinase.

The genetic information contained within the RNA genome of picornaviruses is expressed as a single large open reading frame; processing of the primary translation product begins while translation is still in progress. In rhinoviruses and enteroviruses, two picornavirus genera, the virally encoded proteinase 2A begins the processing cascade, cleaving between the C-terminus of VP1 and its own N-terminus. The natural variation in the amino acid sequences amongst rhinoviruses and enteroviruses at the cleavage site of the viral proteinase 2A served as the basis for a mutational analysis of the substrate specificity of the 2A proteinase of human rhinovirus 2. This enzyme was shown to have an unusual preference at the P1 site; out of eight amino acid substitutions made, only the branched amino acids Val and Ile were not readily accepted. The HRV2 2A was shown to process poorly the HRV89 2A cleavage site and to be unable to cleave at sites which included the P' region of poliovirus or HRV14. Furthermore, the 2A of HRV89 preferred the cleavage site of HRV2 to its own.     

Human rhinovirus mutants resistant to low pH.

Mutants of human rhinovirus serotype 14 (HRV14) with increased resistance to treatment at low pH were obtained by repeated cycles of exposure to pH 4.5 and propagation in HeLa cells. Whereas wild-type virus lost more than 5 logs of infectivity upon incubation at pH 4.3, the three isolates examined were essentially unaffected. Conformational change of the viral capsid upon exposure to low pH was assessed as an increase of hydrophobicity by partition between an aqueous phase and a Triton X-114 phase; the mutants required exposure to a much lower pH to accumulate in the Triton phase than wild-type HRV14. The sequence of the capsid region was determined for three isolates; two isolates were found to have the changes Thr17 to lie in VP2 and Asn 100 to lie in VP1. The third isolate also had the change Thr17 to Ile in VP2; however, in VP1, Asp101 was replaced by Glu. Separate introduction of the mutations into full length cDNA clones of the wild-type sequence of HRV14 showed that only the changes in VP1 were necessary for the increased stability at pH 4.5. The implications of the mutations for the three-dimensional structure of the viral capsid are discussed.     

Complete nucleotide sequence of the chiba virus genome and functional expression of the 3C-like protease in Escherichia coli

We cloned the genome RNA of the Chiba virus (ChV; Hu/NLV/Chiba 407/1987/JP) and determined its complete nucleotide sequence. The genome is predicted to be a positive-sense, single-stranded RNA of 7697 bases, excluding a poly(A) tract. Comparison of the nucleotide and amino acid sequences with those of other members of the species Norwalk virus (NV) revealed that ChV belongs to genogroup I NV. The ChV genome contains three open reading frames (ORFs). A large 5'-terminal ORF (ORF1) encodes a polyprotein with 1785 amino acids that are likely processed into functional proteins, including RNA helicase, VPg, protease, and RNA-dependent RNA polymerase. ORF2 encodes the capsid protein with 544 amino acids, and a small 3'-terminal ORF (ORF3) encodes a basic protein with 208 amino acids. The amino acid sequences of five cleavage sites in ORF1 are highly conserved compared with those of other members of NV. When expressed in Escherichia coli, the glutathione-S-transferase (GST) fusion protein of the ChV protease connected via a short peptide containing a human rhinovirus 3C protease cleavage site was cleaved into GST and the protease; however, this cleavage did not occur when the Cys mutation was introduced into the putative active site of the protease. Moreover, the ChV protease recognized and cleaved the predicted proteolytic sites between VPg and protease and between protease and RNA polymerase. Therefore, the ChV protease expressed in E. coli retained an enzymatic activity and a substrate specificity similar to that of the human rhinovirus 3C protease.     

Identification of rhinoviruses by cDNA probes

We have used nucleic acid hybridization for the detection and grouping of human rhinoviruses (HRV) according to their genetic relationships. Fifteen rhinovirus reference strains, seventy-one clinical isolates and four enteroviruses were propagated in cell cultures, spotted onto membrane filters and hybridized with radioactively labelled cDNA probes covering different parts of the genomes of HRV-1B, HRV-2, HRV-14, HRV-85 and HRV-89. When the rhinovirus and enterovirus reference strains were tested, the 5' probe of HRV-2 hybridized with thirteen of the fifteen HRV reference strains, with poliovirus type 3 and with ECHO virus 11. The HRV-14 5' probe reacted with eleven HRV reference strains and with all the enteroviruses studied. Sixty-nine of the 71 clinical isolates were recognised by the HRV-2 5' probe, whereas the HRV-14 probe from the same part of the genome hybridized with 54 field isolates. One of the two isolates that remained negative with the HRV-2 5' probe was detected with the HRV-2 probe that derived from the P2 region of the genome, and the other isolate was not detected by any of the probes. Probes from other parts than the 5' end of the genome were generally more specific, and clusters could be formed based on the reactivity of the HRV strains with these probes.     

Human rhinovirus protein synthesis and polyprotein cleavage in infected HeLa-RH cells

Summary Precursor and mature polypeptides of four human rhinovirus types (HRV 30, 63, 81 and 88) were compared. The SDS-PAGE profiles of the HRV-specific polypeptides differed significantly from each other, as well as from those of the HRV types studied previously (HRV 1A, 2 and 14). Our results provide further evidence for considerable heterogeneity within the rhinovirus genus.With 2 Figures     

Construction and characterization of a poliovirus/rhinovirus antigenic hybrid.

In order to study the properties of foreign antigenic sites expressed on poliovirus a hybrid was constructed in which neutralization antigenic site IA of poliovirus type 1 strain Mahoney [PV1(M)] was replaced by neutralization immunogenic site IA (NImIA) of human rhinovirus 14 (HRV14). The resulting hybrid was viable, but growth was impaired in comparison to PV1(M). The hybrid expressed both PV1(M) and HRV14 antigenic determinants. When inoculated into rabbits it elicited neutralizing antibodies against both PV1(M) and HRV14. Furthermore, the hybrid was efficiently neutralized by polyclonal antisera specific for either PV1(M) or HRV14 and by three out of five monoclonal antibodies directed to NImIA. The monoclonal antibodies also blocked binding of the hybrid to the cellular receptor for poliovirus. One of them is thought to neutralize rhinovirus in this manner, and it appears that NImIA is expressed in a sufficiently favorable context on the hybrid for the same mechanism to be effective. This can be interpreted to mean that the interactions between the parental viruses and their respective cellular receptors are very similar.     

我国新分离乙脑病毒02-76株的全基因序列特征

目的对我国新分离乙脑病毒02-76株进行全基因序列测定和分析，了解乙脑病毒基因组结构及毒力特性。方法设计乙脑病毒全基因组扩增引物，RT-PCR扩增片段，PCR产物直接测序，拼接后获得全基因序列。通过Clustal X（1．8）、DNASTAR、GENEDOC（3．2）等生物学软件进行核苷酸序列及氨基酸序列分析和病毒的系统进化分析。结果新分离乙脑病毒02．76株全基因组全长10977个核苷酸，从96位到10391位，共10296个核苷酸编码一个开放阅读框，编码3432个氨基酸。与我国1949年分离的Beijing-1株相比较共存在248个核苷酸差异，16个氨基酸差异。与GenBank中选择的29株乙脑病毒全基因序列比较发现，其核苷酸总体差异率为0．6％-15．1％，氨基酸总体差异率为0．2％-4．6％。通过PrM／C区段、E区段、3’NTR区段及全基因序列进行系统进化分析均显示该毒株属于基因3型乙脑病毒。结论新分离的乙脑病毒02-76株属于基因3型，与中国分离株SA-14进化关系最接近。     

Complete nucleotide sequence of polyprotein gene 1 and genome organization of turkey coronavirus

The complete nucleotide sequence of polyprotein gene 1 and the assembled full-length genome sequence are presented for turkey coronavirus (TCoV) isolates 540 and ATCC. The TCoV polyprotein gene encoded two open reading frames (ORFs), which are translated into two products, pp1a and pp1ab, the latter being produced via -1 frameshift translation. TCoV polyprotein pp1a and pp1ab were predicted to be processed to 15 non-structure proteins (nsp2-nsp16), with nsp1 missing. ClustalW analysis revealed 88.99% identity and 96.99% similarity for pp1ab between TCoV and avian infectious bronchitis virus (IBV) at the amino acid level. The whole genome consists of 27,749 nucleotides for 540 and 27,816 nucleotides for ATCC, excluding the poly(A) tail. A total of 13 ORFs were predicted for TCoV. Five subgenomic RNAs were detected from ATCC-infected turkey small intestines by Northern blotting. The whole genome sequence had 86.9% identity between TCoV and IBV, supporting that TCoV is a group 3 coronavirus.     

Complete Nucleotide Sequence of Bean Pod Mottle Virus RNA1: Sequence Comparisons and Evolutionary Relationships to Other Comoviruses

The complete nucleotide sequence of bean pod mottle comovirus (BPMV) RNA1 was determined. It is 5983 nucleotides long, excluding the poly(A) tail, and encodes a polyprotein of 1850 amino acid (aa) residues. Multiple alignments of the deduced aa sequence of BPMV polyprotein with those of cowpea mosaic virus (CPMV), red clover mottle virus (RCMV) and cowpea severe mosaic virus (CPSMV) indicated that BPMV RNA1 encodes the predicted set of five mature proteins: the equivalent of CPMV 32K protease cofactor, 58K putative helicase, VPg, 24K protease and 87K putative RNA-dependent RNA polymerase. Of the four proposed cleavage sites in BPMV RNA1 polyprotein, the one at the 32K/58K site (Q/A) is distinct for BPMV polyprotein and those at the 58K/VPg and VPg/24K junctions (Q/S and Q/M, respectively) are identical in all four comovirus polyproteins. Sequence comparison and phylogenetic analysis revealed that BPMV RNA1 is more closely related to CPSMV than to CPMV or to RCMV.     

Complete Nucleotide Sequence Analysis of a Western Pacific Dengue-1 Virus Strain

We have determined the complete nucleotide sequence and the deduced amino acid polypeptide sequence of the genome of a dengue-1 (DEN-1) virus strain isolated from a patient on Nauru in the Western Pacific in 1974 (West Pac 74). The complete genome is 10,735 nucleotides in length and contains a single long open reading frame of 10,176 nucleotides encoding a polyprotein of 3392 amino acids. When compared to DEN-1 Singapore S275/90, the nucleotide and amino acid sequence homology are 94% and 97.8%, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Entry of a heparan sulphate-binding HRV8 variant strictly depends on dynamin but not on clathrin, caveolin, and flotillin

The major group human rhinovirus type 8 can enter cells via heparan sulphate. When internalized into ICAM-1 negative rhabdomyosarcoma (RD) cells, HRV8 accumulated in the cells but caused CPE only after 3 days when used at high MOI. Adaptation by three blind passages alternating between RD and HeLa cells resulted in variant HRV8v with decreased stability at acidic pH allowing for productive infection in the absence of ICAM-1. HRV8v produced CPE at 10 times lower MOI within 1 day. Confocal fluorescence microscopy colocalization and the use of pharmacological and dominant negative inhibitors revealed that viral uptake is clathrin, caveolin, and flotillin independent. However, it is blocked by dynasore, amiloride, and EIPA. Furthermore, HRV8v induced FITC-dextran uptake and colocalized with this fluid phase marker. Except for the complete inhibition by dynasore, the entry pathway of HRV8v via HS is similar to that of HRV14 in RD cells that overexpress ICAM-1.     

Sequence and phylogenetic analyses of highly virulent infectious bursal disease virus

Summary.  The nucleotide sequences of the genome segment A and B encoding the precursor polyprotein (NH₂-VP2-VP4-VP3-COOH) and VP1 were determined for a highly virulent strain of infectious bursal disease virus (IBDV). The precursor polyprotein and VP1 coding regions of highly virulent OKYM strain consisted of 3 039 nucleotides (1 012 deduced amino acids) and 2 640 nucleotides (879 deduced amino acids), respectively. Comparison of the deduced amino acid sequences of the highly virulent IBDV (HV-IBDV) with other serotype 1 and 2 sequences revealed 17 amino acid residues which were conserved only in the HV-IBDV. Among the 17 unique amino acid differences, 8 were in VP1, 4 were in VP2, 3 were in VP3 and 2 were in VP4. Although it is impossible to predict the effect of the unique amino acid residues without detailed knowledge of the three-dimensional structure and function of the proteins, they could affect the virulence of HV-IBDV. Alignment of the nucleic acid sequences of precursor polyprotein, VP1, VP2, VP3 and VP4 coding regions followed by distance analysis allowed the generation of phylogenetic trees. The same tree topology was obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. On the other hand, the tree topology of VP1 was quite different from that obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. These findings indicate that not a genetic recombination but a genetic reassortment may play an important role in the emergence of HV-IBDV. Accepted January 16, 1997; Received October 25, 1996     

Complete nucleotide sequence of the genome of coxsackievirus B1

The complete nucleotide sequence of the genome of the coxsackievirus B1, a human enterovirus that belongs to the Picornaviridae, was determined by using molecular cloning and rapid sequence analysis techniques. Sequence analysis of the cloned cDNAs revealed that the virion RNA was 7389 nucleotides long and polyadenylylated at the 3' terminus. Similar to other picornavirus genomes, a single large open reading frame was identified. The translated sequence starts at nucleotide position 742 and ends at 7287 of the genome. Thus, the viral polyprotein should consist of 2182 amino acids. When the predicted amino acid sequence of the viral polyprotein was compared with those of other human enteroviruses such as polioviruses, a striking sequence homology was observed, especially in viral proteins 1B, 2C, and 3D. This allowed us to predict precise map locations of the viral structural and nonstructural proteins on the genome, although two proteolytic processing sites, between 1D and 2A and between 2B and 2C, were obscure. The result presented here implied important information with respect to the genetical variation of human enteroviruses.     

The complete nucleotide sequence of plum pox potyvirus RNA

The complete nucleotide sequence of the plum pox virus (PPV) RNA genome has been determined. The RNA sequence is 9786 nucleotides in length, excluding the 3'-terminal poly(A) tail. An AUG triplet at position 147-149 was assigned as the initiation codon for the translation of the genome size viral polyprotein which would consist of 3140 amino acid residues. The nucleotide sequence of the non-coding regions and the predicted amino acid sequence of the polyprotein of PPV were compared with those previously reported for two other potyviruses (tobacco etch virus, TEV, and tobacco vein mottling virus, TVMV), with nucleotide and amino acid sequences of other viruses, as well as with sequences from data banks. The potyvirus genomic expression is discussed in relation to the homologies observed, in particular the predicted protease recognition sequences in related viruses.     

Localization of rhinovirus replication in vitro with in situ hybridization.

To facilitate understanding of human rhinovirus (HRV) pathogenesis, methods were developed for detection of HRV infection in vitro using in situ hybridization (ISH). HRV-14 RNA probes and oligonucleotide probes representing conserved sequences in the 5'-non-translated region were labeled with 35S and used to detect infected HeLa or WI-38 strain human embryonic lung cells in cytological preparations. ISH was shown to be specific for detection of HRV on a single-cell basis. Subsequently, in human nasal polyps infected in vitro, both oligonucleotide- and ribo-probes produced a strong signal in association with ciliated epithelial cells. In human adenoids infected in vitro, a signal was observed in non-ciliated epithelial cells. This study shows that HRV replicates in ciliated cells in the epithelium of human nasal polyps infected in vitro, and the presence of viral RNA in non-ciliated cells of the human adenoid infected in vitro suggests that other cell types may also support rhinovirus replication.