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PubMed1999

Genomic and phylogenetic characterization of coxsackievirus B2 prototype strain Ohio-1.

The human picornavirus coxsackievirus B2 (CVB2) is often linked to several infections, from mild respiratory diseases to more severe illnesses such as myocarditis. In this study, we report the complete genome sequence of CVB2 prototype strain Ohio-1. The genome sequence was determined from reverse transcribed viral RNA, amplified with long distance PCR and used for non-radioactive sequencing. The full length PCR amplicons were used for in vitro transcription and the obtained cRNA was lipofected onto green monkey kidney cells, in order to confirm that the PCR generated sequence reflects a viable virus RNA. The CVB2 genome sequence shows a typical enterovirus genome organization with a total length of 7411 nucleotides. Phylogenetic analysis, using the CVB2 polyprotein in comparison with other enterovirus polyproteins, clearly shows that CVB2 clusters with the coxsackievirus B-like enteroviruses and is more related to coxsackievirus B4 (CVB4) than any other published CVB serotype. The grouping of CVB2 and CVB4 as one subgroup has earlier been reported in connection with receptor usage and ability to replicate in different cell lines. The exposed viral capsid proteins of CVB2 (VP1-VP3) show high similarity to other CVB proteins, except in regions that are likely to be surface epitopes.

Amino Acid Sequence↗
PubMed1999

Identification and analysis of truncated and elongated species of the flavivirus NS1 protein.

The flavivirus non-structural glycoprotein NS1 is often detected in Western blots as a heterogeneous cluster of bands due to glycosylation variations, precursor-product relationships and/or alternative cleavage sites in the viral polyprotein. In this study, we determined the basis of structural heterogeneity of the NS1 protein of Murray Valley encephalitis virus (MVE) by glycosylation analysis, pulse-chase experiments and terminal amino acid sequencing. Inhibition of N-linked glycosylation by tunicamycin revealed that NS1 synthesised in MVE-infected C6/36 cells was derived from two polypeptide backbones of 39 kDa (NS1(o)) and 47 kDa (NS1'). Pulse-chase experiments established that no precursor-product relationship existed between NS1(o) and NS1' and that both were stable end products. Terminal sequencing revealed that the N- and C-termini of NS1(o) were located at amino acid positions 714 and 1145 in the polyprotein respectively, consistent with the predicted sites based upon sequence homology with other flaviviruses. Expression of the NS1 gene alone or in conjunction with NS2A by recombinant baculoviruses demonstrated that the production of NS1' was dependent on the presence of NS2A, indicating that the C-terminus of the larger protein was generated within NS2A. A smaller form (31 kDa) of NS1 (deltaNS1) was also identified in MVE-infected Vero cultures, and amino acid sequencing revealed a 120-residue truncation at the N-terminus of this protein. This corresponds closely with the in-frame 121-codon deletion at the 5' end of the NS1 gene of defective MVE viral RNA (described by Lancaster et al. in 1998), suggesting that deltaNS1 may be a translation product of defective viral RNA.

Aedes↗
PubMed1999

Sequencing of prototype viruses in the Venezuelan equine encephalitis antigenic complex.

The 5' nontranslated region (5'NTR) and nonstructural region nucleotide sequences of nine enzootic Venezuelan equine encephalitis (VEE) virus strains were determined, thus completing the genomic RNA sequences of all prototype strains. The full-length genomes, representing VEE virus antigenic subtypes I-VI, range in size from 11.3 to 11.5 kilobases, with 48-53% overall G+C contents. Size disparities result from subtype-related differences in the number and length of direct repeats in the C-terminal nonstructural protein 3 (nsP3) domain coding sequence and the 3'NTR, while G+C content disparities are attributable to strain-specific variations in base composition at the wobble position of the polyprotein codons. Highly-conserved protein components and one nonconserved protein domain constitute the VEE virus replicase polyproteins. Approximately 80% of deduced nsP1 and nsP4 amino acid residues are invariant, compared to less than 20% of C-terminal nsP3 domain residues. In two enzootic strains, C-terminal nsP3 domain sequences degenerate into little more than repetitive serine-rich blocks. Nonstructural region sequence information drawn from a cross-section of VEE virus subtypes clarifies features of alphavirus conserved sequence elements and proteinase recognition signals. As well, whole-genome comparative analysis supports the reclassification of VEE subtype-variety IF and subtype II viruses.

5' Untranslated Regions↗
PubMed2000

TROMB, a new retrotransposon of the gypsy-Ty3 group from the fly Megaselia scalaris.

We describe TROMB, a new LTR retrotransposon, from the phorid fly Megaselia scalaris. Three full-length copies (4226, 4160 and 4129bp) and a truncated one (319bp) have been isolated. The target site consensus is TATAT, with a 4bp target site duplication TATA. The LTRs are short (142bp) and contain a TATA-box and a polyadenylation signal. The isolated copies are degenerate to different degrees and presumably inactive. The polyprotein coding sequence contains scattered stop codons and deletions/insertions at non-homologous positions. The consensus sequence among the three full-length copies, however, has an uninterrupted open reading frame and, presumably, represents the original sequence of the active element. Southern hybridization experiments showed TROMB to be present at a low copy number in two wild-type strains of M. scalaris and absent in a related species, M. abdita. The order of domains in the polyprotein coding region, the target site specificity for AT-rich sequences, and the protein sequence similarity to blastopia, mdg3 and micropia place TROMB in the gypsy-Ty3 group of LTR retrotransposons.

Amino Acid Sequence↗
PubMed2000

A genomic approach of the hepatitis C virus generates a protein interaction map.

The hepatitis C virus (HCV) causes severe liver disease, including liver cancer. A vaccine preventing HCV infection has not yet been developed, and, given the increasing number of infected people, this virus is now considered a major public-health problem. The HCV genome is a plus-stranded RNA that encodes a single polyprotein processed into at least 10 mature polypeptides. So far, only the interaction between the protease NS3 and its cofactor, NS4A, which is involved in the processing of the non-structural region, has been extensively studied. Our work was aimed at constructing a protein interaction map of HCV. A classical two-hybrid system failed to detect any interactions between mature HCV polypeptides, suggesting incorrect folding, expression or targetting of these proteins. We therefore developed a two-hybrid strategy, based on exhaustive screens of a random genomic HCV library. Using this method, we found known interactions, such as the capsid homodimer and the protease dimer, NS3-NS4A, as well as several novel interactions such as NS4A-NS2. Thus, our results are consistent with the idea that the use of a random genomic HCV library allows the selection of correctly folded viral protein fragments. Interacting domains of the viral polyprotein are identified, opening the possibility of developing specific anti-viral agents, based on their ability to modulate these interactions.

Genome, Viral↗
PubMed1997

In vitro and ex vivo inhibition of hepatitis A virus 3C proteinase by a peptidyl monofluoromethyl ketone.

Hepatitis A virus (HAV) 3C proteinase is the enzyme responsible for the processing of the viral polyprotein. Although a cysteine proteinase, it displays an active site configuration like those of the mammalian serine proteinases (Malcolm, B. A. Protein Science 1995, 4, 1439). A peptidyl monofluoromethyl ketone (peptidyl-FMK) based on the preferred peptide substrates for HAV 3C proteinase was generated by first coupling the precursor, N,N-dimethylglutamine fluoromethylalcohol, to the tripeptide, Ac-Leu-Ala-Ala-OH, and then oxidizing the product to the corresponding peptidyl-FMK (Ac-LAAQ'-FMK). This molecule was found to be an irreversible inactivator of HAV 3C with a second-order rate constant of 3.3 x 10(2) M-1 s-1. 19F NMR spectroscopy indicates the displacement of fluoride on inactivation of the enzyme by the fluoromethyl ketone. NMR spectroscopy of the complex between the 13C-labeled inhibitor and the HAV 3C proteinase indicates that an (alkylthio)methyl ketone is formed. Studies of polyprotein processing, using various substrates generated by in vitro transcription/translation, demonstrated efficient blocking of even the most rapid proteolytic events such as cleavage of the 2A-2B and 2C-3A junctions. Subsequent ex vivo studies, to test for antiviral activity, show a 25-fold reduction in progeny virus production as the result of treatment with 5 microM inhibitor 24 h post-infection.

3C Viral Proteases↗
PubMed2001

Hepatitis C virus (HCV) genotype 1b sequences from fifteen patients with hepatocellular carcinoma: the 'progression score' revisited.

The genome of hepatitis C virus (HCV) associated with hepatocellular carcinoma (HCC) may have some characteristics which would barely be found in those of HCV from asymptomatic carriers (ASC). We analyzed 15 HCC patients who were infected with HCV genotype 1b (HCV-1b) for complete nucleotide sequences of the viral genomes. Of the 15 isolates, three were sequenced up to the first nucleotide of the 5'UTR, and six were sequenced to encompass the X-tail at the 3' end: sequencing of at least three-quarters of the 5'UTR and entire polyprotein-ORF was accomplished in all 15 isolates. Analyses of these sequences together with those reported previously by Nagayama et al. [Hepatology; 31 (2000) 745] suggested that nine residues (nt 119 of 5'UTR and aa 90, 434, 938, 962, 1176, 1412, 2143, and 2774 of polyprotein) might be useful to discriminate HCC-type sequences from ASC-type ones. The 'progression score' was 1.4+/-0.9 in ASC versus 3.7+/-1.5 in HCC (P=3.87E-07) when calculated with the Nagayama et al.'s seven residues, but was 1.4+/-0.6 versus 4.6+/-1.9 (P=1.33E-09) with our nine residues: a greater difference between HCC and ASC was achieved in the latter system. Further analyses, by increasing the sample size and/or by extending the comparison to include entire 5'UTR and 3'UTR/X-tail, may thus contribute to define the 'progression score' more appropriately.

Journal Article↗
PubMed1998

The polypyrimidine tract binding protein (PTB) requirement for internal initiation of translation of cardiovirus RNAs is conditional rather than absolute.

Picornavirus RNAs are translated by an unusual mechanism of internal ribosome entry that requires a substantial segment of the viral 5'-untranslated region, generally known as the internal ribosome entry segment (IRES), and in some circumstances may require cellular trans-acting proteins, particularly polypyrimidine tract binding protein (PTB). It is shown here that for encephalomyocarditis virus (EMCV), the PTB dependence of IRES function in vitro is determined partly by the nature of the reporter cistron, and more especially by the size of an A-rich bulge in the IRES. With a wild-type EMCV IRES (which has a bulge of 6 As), translation is effectively independent of PTB provided the IRES is driving the synthesis of EMCV viral polyprotein. With an enlarged (7A) bulge and heterologous reporters, translation is highly dependent on PTB. Intermediate levels of PTB dependence are seen with a 7A bulge IRES driving viral polyprotein synthesis or a wild-type (6A) bulge IRES linked to a heterologous reporter. None of these parameters influenced the binding of PTB to the high-affinity site in the IRES. These results argue that PTB is not an essential and universal internal initiation factor, but, rather, that when it is required, its binding to the IRES helps to maintain the appropriate higher-order structure and to reverse distortions caused, for example, by an enlarged A-rich bulge.

Adenine Nucleotides↗
PubMed1993

Studies on the substrate specificity of the proteinase of equine infectious anemia virus using oligopeptide substrates.

The proteinase of the equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus (HIV), was purified from concentrated virus. The specificity of the enzyme was characterized using oligopeptides representing naturally occurring cleavage sites in the Gag and Gag-Pol polyproteins. The length of the substrate binding pocket was found to be 1-2 residues longer than that of HIV proteinases. Although the EIAV and HIV proteinases cleaved most of the peptides at the same bond, some were hydrolyzed by only the EIAV enzyme. Oligopeptides representing cleavage sites in the nucleocapsid protein were also found to be substrates of the EIAV proteinase. However, these peptides were not hydrolyzed by the HIV proteinases. While peptides representing the corresponding sequences in the first cysteine arrays of the nucleocapsid proteins of HIV-1 and HIV-2 were substrates of the proteinases, peptides representing the homologous sequences in the second Cys arrays were resistant against the proteolytic attack. A three-dimensional model of the EIAV proteinase built on the basis of homology with HIV-1 proteinase was used to interpret the differences. In addition to the oligopeptides representing cleavage sites in the Gag and Gag-Pol polyproteins, the EIAV proteinase was also able to cleave an oligopeptide mimicking a cleavage site in the transmembrane protein. Our results suggest that the specificity of lentiviral proteinases share common characteristics, although substantial differences may exist in hydrolysis of some peptides.

Amino Acid Sequence↗
PubMed1994

Proteolytic processing mechanisms of a miniprecursor of the aspartic protease of human immunodeficiency virus type 1.

The infectivity of the human immunodeficiency virus (HIV) depends upon correct proteolytic processing of viral polyprotein precursors, the Pr55gag and Pr160gag-pol polyproteins. The processing is mediated spontaneously by the viral protease unit (PR) contained within the Pr160gag-pol precursor. However, little is known about the mechanism of this process. The expression in Escherichia coli and the isolation of a 14-kDa HIV-1 PR "miniprecursor" with Ala28 mutated to serine has permitted study of the mechanism for cleavage at the N-terminus of the protease. The miniprecursor is active against a synthetic peptide substrate, and its specific activity is near that of the mutant mature protease. The rate of conversion of radiolabeled precursor to mature protease is quantitated by measuring the amounts of the two radiolabeled proteins separated by SDS-PAGE. The apparent first-order conversion rate constant, kapp, is dependent on miniprecursor concentration indicating a second-order reaction and suggesting an interdimeric processing mechanism. A significant first-order rate constant is observed when the plot of kapp versus initial precursor concentration is extrapolated to zero. This observation suggests the presence of an alternative processing mechanism involving a single active precursor dimer. The presence of both mechanisms is an advantage for the virus to ensure processing under various conditions.

Amino Acid Sequence↗
PubMed1996

Influence of flanking sequences on the dimer stability of human immunodeficiency virus type 1 protease.

The maturation of the human immunodeficiency virus type 1 protease (PR) from the Gag-Pol polyprotein is dependent on the intrinsic proteolytic activity of the dimeric Gag-Pol. Herein, we report the kinetics and conformational stabilities of two unique fusion proteins of the protease. In one, X28-PR, a random sequences of 28 amino acids (X28) was linked to the N terminus of the mature protease. In the second construct, X28-delta TF*PR*delta Pol, X28 is fused to the protease which is flanked at both its termini by short sequences (delta) which correspond to the native sequences of the Gag-Pol precursor. Autoprocessing of the latter protein was prevented by inserting an Ala at the native protease cleavage sites. The measured kinetic parameters and the pH-rate profile of both enzymes are nearly identical to those of the mature protease. However, these fusion proteins are more sensitive to acid and urea denaturation than the mature protease. The decrease in the conformational stability of X28-PR and X28-delta TF*PR*delta Pol is reflected by increases in their apparent dissociation constants (Kd) from < 5 nM to approximately 180 and 25 nM, respectively. These results suggest that subunit interactions and hence the dimer stability of the protease domain in the Gag-Pol polyprotein differ from those of the mature protease. The high Kd of X28-PR further suggests that addition of non-native sequences to the N terminus of the protease destablizes the dimer.

Amino Acid Sequence↗
PubMed2000

Alpha-ketoacids are potent slow binding inhibitors of the hepatitis C virus NS3 protease.

The replication of the hepatitis C virus (HCV), an important human pathogen, crucially depends on the proteolytic maturation of a large viral polyprotein precursor. The viral nonstructural protein 3 (NS3) harbors a serine protease domain that plays a pivotal role in this process, being responsible for four out of the five cleavage events that occur in the nonstructural region of the HCV polyprotein. We here show that hexapeptide, tetrapeptide, and tripeptide alpha-ketoacids are potent, slow binding inhibitors of this enzyme. Their mechanism of inhibition involves the rapid formation of a noncovalent collision complex in a diffusion-limited, electrostatically driven association reaction followed by a slow isomerization step resulting in a very tight complex. pH dependence experiments point to the protonated catalytic His 57 as an important determinant for formation of the collision complex. K(i) values of the collision complexes vary between 3 nM and 18.5 microM and largely depend on contacts made by the peptide moiety of the inhibitors. Site-directed mutagenesis indicates that Lys 136 selectively participates in stabilization of the tight complex but not of the collision complex. A significant solvent isotope effect on the isomerization rate constant is suggestive of a chemical step being rate limiting for tight complex formation. The potency of these compounds is dominated by their slow dissociation rate constants, leading to complex half-lives of 11-48 h and overall K(i) values between 10 pM and 67 nM. The rate constants describing the formation and the dissociation of the tight complex are relatively independent of the peptide moiety and appear to predominantly reflect the intrinsic chemical reactivity of the ketoacid function.

Alanine↗
PubMed1994

Haloperidol-based irreversible inhibitors of the HIV-1 and HIV-2 proteases.

The proteases expressed by the HIV-1 and HIV-2 viruses process the polyproteins encoded by the viral genomes into the mature proteins required for virion replication and assembly. Eight analogs of haloperidol have been synthesized that cause time-dependent inactivation of the HIV-1 protease and, in six cases, HIV-2 protease. The IC50 values for the analogues are comparable to that of haloperidol itself. Enzyme inactivation is due to the presence of an epoxide in two of the analogues and carbonyl-conjugated double or triple bonds in the others. Irreversible inactivation is confirmed by the failure to recover activity when one of the inhibitors is removed from the medium. At pH 8.0, the agents inactivate the HIV-1 protease 4-80 times more rapidly than the HIV-2 protease. Faster inactivation of the HIV-1 protease is consistent with alkylation of cysteine residues because the HIV-1 protease has four such residues whereas the HIV-2 protease has none. Inactivation of the HIV-2 protease requires modification of non-cysteine residues. The similarities in the rates of inactivation of the HIV-2 protease by six agents that have intrinsically different reactivities toward nucleophiles suggest that the rate-limiting step in the inactivation process is not the alkylation reaction itself. At least five of the agents inhibit polyprotein processing in an ex vivo cell assay system, but they are also toxic to the cells.

Acetates↗
PubMed1998

Simultaneous accumulation of multiple viral coat proteins from a TEV-NIa based expression vector.

We previously described an expression cassette that relies on the tobacco etch virus (TEV) nuclear inclusion a (NIa) protease and leads to the coordinated accumulation of multiple proteins through self processing of a polyprotein [21]. However, low levels of proteins accumulated when the full-length protease was encoded within the polyprotein [22]. Studies were conducted to evaluate whether the disruption of NIa nuclear localization would affect the levels of proteins produced via the cassette. Modifications comprised either removal of its nuclear localization signals (NLSs), removal of the VPg domain (which includes the NLSs), and fusion to the 6 kDa protein, previously demonstrated to be a viral cytoplasmic anchor [28]. In in vitro translation reactions and in vivo protoplast experiments the modified NIa retained sequence-specific proteolysis. Moreover, the removal of the NLSs correlated with an increase in GUS reporter accumulation. The modified cassette, pPRO10, led to the synthesis of up to three viral coat protein (CPs) in addition to NIa. However, the accumulation of proteins in protoplasts depended upon the position of the CP coding sequence within the cassette as well as on the stability of the protein.

Base Sequence↗
PubMed1997

A cDNA clone to clover yellow vein potyvirus genome is highly infectious.

We obtained a highly infectious cDNA clone of clover yellow vein virus (CIYVV). The cDNA fragments, from which a full-length cDNA clone was constructed, were sequenced, and the complete nucleotide sequence of C1YVV RNA was determined. The viral genome is 9584 nucleotides (nt) in length excluding the poly(A) tail and contains one open reading frame (ORF) encoding a large polyprotein of 3072 amino acids. The non-coding region preceding the ORF is 190 nt long. The termination codon is followed by a 175-nt sequence. Seven potential protease NIa, one HC-pro and one P1 protease recognition sites were found in the C1YVV polyprotein by searching for cleavage consensus sequences among the potyvirus group. The cleavage dipeptides of C1YVV NIa protease are Q(E)/S(A,G). The F is conserved at the -2 position from the cleavage site except for at the P3/6K1 junction, and the V conserved at the -4 position among many potyviruses is not present at all. The genome organization of C1YVV was determined, and the amino acid sequence was compared with that of other potyviruses. The full-length cDNA clone of C1YVV was constructed by combining cDNA fragments and placed it under the control of the cauliflower mosaic virus 35S promoter. The full-length cDNA was constructed so that no extra nucleotide was present at the transcription initiation site and only 10 adenine residues were present at the 3' end of the C1YVV cDNA clone. Mechanical inoculation of a circular-formed plasmid DNA onto broad bean seedlings led to systemic infection, and the symptoms were similar to those caused by the wild-type virus but rather mild. Plasmid diluted as low as 500 pg/microl was able to induce symptoms, demonstrating that this full-length C1YVV cDNA is more infectious than any other infectious cDNAs so far reported. Filamentous particles reacting with the antiserum to C1YVV were observed in the crude sap of infected plants by immunoelectron microscopy, and genome replication was demonstrated by RT-PCR of 3' non-coding regions of C1YVV genome in total plant RNAs.

Amino Acid Sequence↗
PubMed1999

Sequence analysis of the medium (M) segment of Cache Valley virus, with comparison to other Bunyaviridae.

The complete sequence of the medium (M) segment of Cache Valley virus (CVV), a human neuropathogen, has been determined using a series of overlapping cDNA clones. The viral complementary-sense RNA is comprised of 4463 nucleotides which encodes a polyprotein precursor of 1435 amino acids, starting at AUG at bases 49-51 to a UGA stop codon at bases 4351-4353. This polyprotein-encoding sequence is arranged as G2-NSm-G1. The base composition of the segment is 34.9% A, 17.0% C, 19.4% G and 28.7% U. Comparison of the nucleotide sequence to the prototype Bunyamwera virus sequence shows an identity of 63%, indicating several differences exist within the individual coding regions, most notably within the NSm and G1 coding regions. Based on two presumed cleavage points within the precursor, the G2 glycoprotein, encoded from nt 94-951, is 286 amino acids long, and has two sites of potential glycosylation. NSm, encoded from nt 952-1476, is 175 amino acids, while the largest glycoprotein, G1, encoded from nt 1477-4350, consists of 958 amino acids, and has five potential glycosylation sites, two of which appear to be unique to CVV. The subsequent study of these glycosylation sites and potential differences between the sequence of this prototype CVV strain and other geographic isolates may suggest the means for improving detection of human infections as well as mapping differences in neurovirulence, neuroinvasiveness and other aspects of pathogenicity.

Amino Acid Sequence↗
PubMed2002

Complete nucleotide sequence, genome organization and phylogenic analysis of the canine calicivirus.

The complete genomic sequence of canine calicivirus (CaCV) isolated from feces of a dog with diarrhea was determined. The CaCV genome, a positive-sense single-stranded RNA, contained 8513 nucleotides excluding the poly(A) tail and was longer than that of any other calicivirus strain with a completely known sequence. There were three open reading frames (ORF1, nt 12-5801; ORF2, nt 5805-7880; and ORF3, nt 7877-8278). ORF1 encoded a polyprotein (calculated Mr of 214,802) which had the conserved motifs of non-structural proteins of other caliciviruses and picornaviruses. Regions containing characteristic motifs in the non-structural polyprotein of CaCV showed highest similarity with those of the species Feline calicivirus and Vesicular exanthema of swine virus in the genus Vesivirus. Phylogenic analysis indicated that CaCV formed a distinct branch within the genus. Our results strongly suggested that CaCV is a new species in the genus Vesivirus.

Animals↗
PubMed2003

Evolution of North American PVY(NTN) strain Tu 660 from local PVY(N) by mutation rather than recombination.

A North American (NA) isolate of tobacco veinal necrotic strain of Potato virus Y (PVY(N)) (N-Jg) and a NA isolate of potato tuber necrotic strain of Potato virus Y (PVY(NTN) (Tu 660) were tested for their phenotypes by inoculation to potato plants of three potato cultivars. Upon inoculation with Tu 660, tubers of the cultivars 'Norchip' and 'Ranger Russet' developed potato tuber necrotic ringspot disease (PTNRD) but not the tubers of 'Russet Burbank'. N-Jg failed to induce PTNRD in the tested cultivars. The genomic RNAs of both strains were completely sequenced and analysed. High homology (98% and 99% identity on nucleotide and polyprotein, respectively) was found between Tu 660 and N-Jg. When polyproteins were compared with other isolates, high identity was observed between Tu 660 and an European (Eu) PVY(N)-605 (98%) and with an Eu-PVY(NTN)-H (96%). However, when individual mature proteins were compared, much lower identities (86.5-94%) were found between Tu 660 and PVY9(NTN))-H compared to 98-99.5% between Tu 660 and PVY(N)-605 in the P3, 6K1 and CI regions. Further sequence analysis indicated that the PVY(NTN)-H is a hybrid molecule of the genomic RNA recombination of PVY(O) and Eu-PVY(N) as shown by Glais et al. (Arch Virol 147, 363-378), whereas NA-PVY(NTN) Tu 660 is free of recombination points. Phylogenetic analysis confirmed this observation, and suggested that, in light of high homology, the Tu 660 might have evolved from NA-PVY(N) by mutations rather than the genome recombinations. The non-recombinant nature of NA-PVY(NTN) Tu 660 strongly suggests that the recombinant structure of genome is not a necessary prerequisite for the PTNRD phenotype.

Cloning, Molecular↗