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W K Joklik

Publications and source records attributed to W K Joklik.

At least 19 recordsLinked to original sources
PubMed1995

Identification of signals required for the insertion of heterologous genome segments into the reovirus genome.

In cells simultaneously infected with any two of the three reovirus serotypes ST1, ST2, and ST3, up to 15% of the yields are intertypic reassortants that contain all possible combinations of parental genome segments. We have now found that not all genome segments in reassortants are wild type. In reassortants that possess more ST1 than ST3 genome segments, all ST1 genome segments appear to be wild type, but the incoming ST3 genome segments possess mutations that make them more similar to the ST1 genome segments that they replace. In reassortants resulting from crosses of the more distantly related ST3 and ST2 viruses that possess a majority of ST3 genome segments, all incoming ST2 genome segments are wild type, but the ST3 S4 genome segment possesses two mutations, G74 to A and G624 to A, that function as acceptance signals. Recognition of these signals has far-reaching implications for the construction of reoviruses with novel properties and functions.

Animals↗
PubMed1994

Reovirus exists in the form of 13 particle species that differ in their content of protein sigma 1.

When electrophoresed in 0.7% agarose gels, populations of reovirus particles can be resolved into 13 well-defined bands that possess from 0 to 12 projection/spike-associated trimers of protein sigma 1. This state of affairs is not an artifact of purification, of the techniques used to demonstrate it, of aggregation, or of virus particle instability. Complexes of monoclonal antibody against protein sigma 1 with virus particles that possess only 1, or, to a lesser extent, 2 sigma 1 trimers are less stable (that is, more readily dissociated by sonication) than complexes of antibody and virus particles that possess 3 or more sigma 1 trimers. The specific infectivity of virus particles that possess 3 or more sigma 1 trimers is essentially the same; virus particles that possess only 2 sigma 1 trimers are about two-thirds as infectious; and particles that possess only 1 sigma 1 trimer still possess very significant infectivity (about one-third of maximum). Reovirus particles that possess no sigma 1 trimers (about 1 in 30) are essentially noninfectious. The reason reovirus particles do not possess a full complement of sigma 1 trimers is presumably the fact that only very small amounts of protein sigma 1 are synthesized in infected cells; and since possession of 3 such trimers is sufficient for maximal infectivity, and since the average number of sigma 1 trimers per reovirus particle is 7.1, there is presumably no selection for variants that synthesize larger amounts of sigma 1. On the contrary, such variants may well be at a selective disadvantage.

Capsid Proteins↗
PubMed1993

Translation of reovirus RNA species m1 can initiate at either of the first two in-frame initiation codons.

The m1 species of reovirus RNA, which encodes the minor protein component mu 2, possesses two initiation codons, one "strong" according to Kozak rules and preceded by 13 residues (IC1), the other "weak" and located 49 codons downstream of the first (IC2). In reovirus-infected cells only IC2 is used, but initiation from IC1 can be activated, and efficiency of initiation from either initiation codon modulated over a wide range, by coupling unrelated sequences to either or both ends of m1 RNA. For example, when the M1 genome segment is cloned into the thymidine kinase gene of vaccinia virus in such a way that various "irrelevant" stretches of nucleotides comprising restriction endonuclease cleavage sites or promoter remnants are coupled to the 5' end of m1 RNA, translation of the resultant transcripts is also initiated at IC2, with frequencies controlled by the nature of the attached sequences. However, in rabbit reticulocyte lysates these same transcripts are translated from IC1 as well as from IC2, and transcripts in which m1 RNA is preceded by long sequences of encephalomyocarditis virus RNA (from the T7 polymerase-controlled pTM1 vector) are translated exclusively from IC1. By contrast, m1 RNA itself is translated only from IC2. It appears that the most important factor that controls the extent to which translation is initiated from IC1 and IC2 is their "availability," which is likely to be a function of the extent to which the regions on either side of them interact with each other (and also, to a lesser extent, with the 3' untranslated region) either directly or via interaction with host cell proteins. The effects described here are of considerable potential significance when genetic material is rearranged as a result of translocations, insertions, deletions, and amplifications--that is, when sequences that are normally separated are brought into apposition.

Animals↗
PubMed1993

Reovirus protein lambda 3 is a poly(C)-dependent poly(G) polymerase.

Reovirus protein lambda 3 has been isolated from cells infected with two recombinant vaccinia viruses into the TK gene of which the reovirus serotype3 strain Dearing L1 genome segment under the control of the bacteriophage T7 RNA polymerase promoter, or the T7 polymerase gene itself, had been cloned. Highly purified protein lambda 3 does not transcribe double-stranded reovirus RNA into single-stranded RNA, or plus-stranded reovirus RNA into minus-stranded RNA, but it does transcribe poly(C) into poly(G). It prefers Mn2+ to Mg2+. A polymer consisting of poly(C) linked linearly to poly(U) provided template activity only for its poly(C) moiety. Protein lambda 3 forms complexes with protein lambda 1, as well as with protein lambda 2, and with both lambda 1 and lambda 2, which are sufficiently stable to be precipitated by monospecific antisera. None of these complexes are capable of transcribing either ds- or ssRNA.

Base Sequence↗
PubMed1993

Generation of reovirus core-like particles in cells infected with hybrid vaccinia viruses that express genome segments L1, L2, L3, and S2.

When mouse L fibroblasts are infected with various combinations of recombinant vaccinia viruses possessing thymidine kinase (TK) genes with inserted reovirus genes that encode core components, particles are formed that closely resemble reovirus cores. In cells infected with vaccinia viruses that express reovirus proteins lambda 1 and sigma 2, particles are formed that are very similar to reovirus core shells; if, in addition, the cells are also infected with vaccinia virus that expresses protein lambda 2, particles are formed that also possess the characteristic icosahedrally located projections/spikes that are present on reovirus cores. If, in either case, the cells are also infected with vaccinia virus that expresses the reovirus RNA polymerase, protein lambda 3, the resultant particles are morphologically identical with those formed in its absence, but also contain protein lambda 3.

Animals↗
PubMed1992

Reovirus genome segment assortment into progeny genomes studied by the use of monoclonal antibodies directed against reovirus proteins.

Using a panel of monoclonal antibodies (MABs) against reovirus proteins, we have identified proteins that associate with reovirus messenger RNA molecules prior to the generation of progeny double-stranded (ds) genome segments and proteins that are components of the structures within which progeny ds genome segments are generated. The following conclusions can be drawn from the results obtained. (1) Three proteins rapidly become associated with mRNA molecules to form single-stranded RNA-containing complexes (ssRCCs): the nonstructural protein microNS, the nonstructural protein sigma NS, and protein sigma 3. (2) Analysis of populations of ssRCCs in density gradients and by sequential exposure to various MABs indicates that some ssRCCs contain only microNS, others microNS and sigma NS or sigma 3, and others all three proteins. Each ssRCC contains one RNA molecule and, depending on the size of the RNA, 10-30 protein molecules. (3) The relative proportions of the individual RNA species in the ssRCC populations reflect the composition of the total mRNA population present in infected cells (which differs substantially from equimolarity). (4) RCCs that contain dsRNA, which become detectable as early as 4 hr after infection, contain not only microNS, sigma NS, and sigma 3, but also lambda 2. (5) The relative proportions of the 10 genome segments in dsRCCs are equimolar. This suggests that genome segment assortment into progeny genomes is linked to the transcription of plus strands into minus strands.

Animals↗
PubMed1991

Isolation and enzymatic characterization of protein lambda 2, the reovirus guanylyltransferase.

Protein lambda 2 of reovirus serotype 3 has been purified to homogeneity from extracts of cells infected with hybrid vaccinia virus strain WR into whose TK gene of the reovirus L2 genome segment under the control of the CPV ATI protein gene promoter had been inserted. Protein lambda 2 is formed in large amounts (final purification factor about 180) as a monomer that shows no tendency to pentamerize into the reovirus core projections/spikes. Isolated protein lambda 2 is reversibly guanylylated by GTP (that is, it carries out the GTP-PPi exchange reaction) and can transfer the -GMP moiety to GTP to form GppppG, to GDP to form GpppG, and to 5'-pp-terminated RNA to form GpppG- caps. These studies confirm previous studies on reovirus cores that indicated that protein lambda 2 is the reovirus guanylyltransferase. Protein lambda 2 possesses neither nucleoside nor RNA triphosphatase activities, nor methyltransferase activities; thus it is the reovirus capping enzyme, but provides neither the required 5'-ppG-terminated substrate nor does it methylate the cap structure. These must be functions of lambda 2 pentamers or of other individual or complexed components of reovirus cores.

Animals↗
PubMed1990

Identification of conserved domains in the cell attachment proteins of the three serotypes of reovirus.

Sequence analysis of reovirus serotype 1 (ST1) and 2 (ST2) S1 genome segment cDNAs identified several differences from previously reported versions of their sequences. The sequences reported here comprise 1463 and 1440 base pairs, respectively; for comparison, the ST3 S1 genome segment is 1416 nucleotides long. The serotype 1 and 2 sigma 1 proteins are predicted to contain 470 and 462 amino acids, respectively; the ST3 sigma 1 protein is 455 amino acids long. As previously observed, the ST1 and ST2 sigma 1 proteins are much more closely related to each other than to that of ST3 (about 48 and 25% similarity, respectively, using a computer program that finds about 14% similarity among unrelated proteins). The sequences of the three S1 genome segments have diverged very extensively in all three codon positions, in some cases almost to the extent of randomness. Despite this, not only function but also shape and configuration have been retained (since the three sigma 1 proteins can be incorporated efficiently into completely heterologous capsids). Seventy-nine amino acid residues are conserved among all three serotypes, many of them clustered into five regions in which one-third or more of the residues are triply conserved. These regions may represent functionally conserved domains involved in oligomerization, cell attachment, and hemagglutination.

Amino Acid Sequence↗
PubMed1990

Reovirus RNA is infectious.

Conditions under which reovirus RNA is infectious have been worked out. In brief, single-stranded (plus-stranded, ss) and/or double-stranded (ds) RNA of reovirus serotype 3 (ST3 virus) is lipofected into L929 mouse fibroblasts together with a rabbit reticulocyte lysate in which ss or melted dsRNA has been translated. After 8 hr the cells are then infected with a helper virus, ST2 reovirus. Virus yields are harvested 24 or 48 hr later. Under these conditions virus that forms plaques by 5 days is produced, all of which is ST3 virus; ST2 virus forms plaques only after 12 days. No reassortants are present among the progeny. The virus yields are about 0.2 PFU/cell; immunofluorescence assays show that this progeny is derived from about 4% of the cells. Double-stranded RNA is 20 times as infectious as ssRNA; ds and ssRNA together yield 10 times as much infectious virus as dsRNA alone, the reason being that dsRNA greatly increases the infectiousness of ssRNA. All species of both ss and dsRNA are required for the operation of this additive effect. The primed rabbit reticulocyte lysate is not essential, but increases virus yields by 100-fold. Its activity is proportional to the time for which translation has proceeded; however, this activity is not due solely to newly synthesized proteins because destruction of the RNA following translation abolishes activity which cannot be restored by simple addition of more RNA. Translation of all species of RNA is essential. Whereas no reassortants are formed when ss and dsRNA of different genotypes are lipofected together, mixtures of dsRNAs of different genotypes do yield reassortants. The same is true for such mixtures of ssRNA. These findings will permit the introduction of new or altered genome segments into the reovirus genome. They open the way to the identification of encapsidation and assortment signals on reovirus genome segments, the characterization of functional domains on reovirus proteins, and the development of reovirus as an expression vector.

Animals↗
PubMed1990

Reovirus protein sigma 1 translated in vitro, as well as truncated derivatives of it that lack up to two-thirds of its C-terminal portion, exists as two major tetrameric molecular species that differ in electrophoretic mobility.

Reovirus protein sigma 1 is the cell attachment protein that modulates tissue tropism and the nature of the antiviral immune response. This protein is present in reovirus particles in the form of 12 tetramers that are associated with the projections or spikes. We have analyzed a series of deletion mutants of protein sigma 1 in order to localize its oligomerization domain and found that progressive deletion from the C-terminus fails to affect ability to oligomerize, even when the deletion extends into the N-terminal heptapeptide repeat region. It was also found that native tetrameric protein sigma 1 synthesized in vitro, as well as its truncated derivatives, exists in two forms that differ in electrophoretic mobility. Possible reasons for this are discussed.

Capsid Proteins↗
PubMed1989

The sequences of the reovirus serotype 1, 2, and 3 L1 genome segments and analysis of the mode of divergence of the reovirus serotypes.

We report the sequence of the L1 genome segment of reovirus serotype 3 strain Dearing, which encodes the minor core component protein lambda 3. It is 3854 bp long, with a long open reading frame starting at position 19 that is 1267 codons long. Protein lambda 3 is not detectably related to any other protein, nor does it appear to possess motifs indicative of recognized specialized functions. We have also sequenced the L1 genome segments of reovirus serotypes 1 and 2. The serotype 1 and 3 L1 genome segments are extremely closely related; there are only 154 mismatches (4.1%), 80% of which are in third base codon positions, so that these two lambda 3 proteins are 98.3% related (only 22 mismatches out of 1267). The serotype 2 L1 genome segment is only 75% related to the serotype 1 and 3 genome segments, and the serotype 2 lambda 3 protein is 92% related to the serotype 1 and 3 lambda 3 proteins. We have also analyzed the divergence patterns by which the various reovirus genome segments evolved into the three serotype forms. It appears that serotype 2 separated from the serotype 1/3 precursor long before serotypes 1 and 3 themselves diverged. In all cases the third base codon positions in the various genome segments have diverged about 80% toward randomness. The first and second base codon positions have diverged much less and to varying degree, depending, presumably, on each protein's ability to accept changes without significant loss of function. For the separation into the serotype 1 and 3 forms, the extent of divergence of the various genome varies over a very wide range. The S1 genome segments have again diverged most extensively, the extent of divergence in the first, second, and third base codon positions being about 50, 35 and 75%, respectively. For seven other genome segments that we examined the extent of third base codon position divergence is 56, 53, 48, 29, 22, 13, and 6%, whereas first and second base codon position divergence ranges from no more than 6 to 2 and 3 to less than 1%, respectively. The most likely explanation of these patterns is that the separation of the various genome segments into the present-day serotype 1 and 3 associated forms occurred at different times during evolution, from progenitors that were genome segment reassortants with survival rates as high as or higher than those of homologous genome segment sets.

Amino Acid Sequence↗
PubMed1989

The sequences of reovirus serotype 3 genome segments M1 and M3 encoding the minor protein mu 2 and the major nonstructural protein mu NS, respectively.

The sequences of the M1 and M3 genome segments of reovirus serotype 3 strain Dearing, which encode protein mu 2, a minor capsid, component, and protein mu NS, one of the two nonstructural proteins, are reported. They are 2304 and 2235 base pairs long, respectively, and proteins mu 2 and mu NS comprise 736 and 719 amino acids, respectively. This completes the sequencing of the reovirus serotype 3 genome: it comprises 23,549 basepairs. Neither protein mu 2 nor protein mu NS possesses any sequence similarity to any protein sequence in gene banks, nor any of the commonly recognized motifs indicative of specialized function. Protein mu 2 has a higher alpha-helix content (36%) than other capsid proteins; for it, the ratio of amino acids in alpha-helix/beta-sheet configuration is 1.2, whereas that of typical reovirus capsid proteins ranges from 0.5 to 0.9. Thus it is not a typical capsid protein. Protein mu NS has a very high alpha-helix content (about 50%; alpha-helix/beta-sheet ratio 2.5), which is very similar to that of the other nonstructural reovirus protein, protein sigma NS. The C-terminal regions of mu NS and various myosins exhibit periodic sequence similarity elements indicative of helical structure. Protein mu NS exists in two forms in infected cells: protein mu NS and a protein, mu NSC, which lacks a region of about 5 kDa at its N-terminus. Pulse-chase analysis in vivo suggests that protein mu NSC is not a cleavage product of protein mu NS; further, protein mu NSC is formed along with protein mu NS in in vitro protein synthesizing systems, whereas protein mu 1C, the cleavage product of protein mu 1, is not. It is likely, therefore, that protein mu NSC is a primary translation product, formed either by ribosomes reading through the first initiation codon of m1 messenger RNA at position 14 and initiating at codon 42, or by de novo internal initiation at this codon.

Amino Acid Sequence↗
PubMed1989

Control of reovirus messenger RNA translation efficiency by the regions upstream of initiation codons.

The 10 species of reovirus messenger RNA are translated in vivo with efficiencies/frequencies that differ by as much as 100-fold. The s1 mRNA, which is translated 10 times less efficiently than the s4 mRNA but 10 times more efficiently than the/1 and m1 mRNAs, has a unique BamH1 cleavage site located immediately downstream of its initiation codon. Because the reovirus mRNAs have been cloned, this provides the opportunity for placing modified and altered sequences upstream of its coding sequence. The translation efficiencies of the variant mRNAs, transcribed via the SP6 in vitro transcription system, can then be measured in the rabbit reticulocyte lysate in vitro translation system. Using this system it was found that replacing the 5'-upstream sequence of the s1 mRNA with that of the s4 mRNA increases its in vitro translation efficiency by 4-fold; that the trinucleotide immediately upstream of the s1 initiation codon renders it very weak, and that it is only slightly superior to the weakest Kozak consensus sequence; that the nature of the nucleotides further upstream than position -3 can profoundly affect translation efficiency; that the nature of this effect is in turn markedly modified by the nature of nucleotides in positions -1 to -3; and that there is a minimum optimal 5'-upstream sequence length of about 14 nucleotides. We also investigated the effect of secondary structure involvement on the ability of 5'-upstream sequences to promote translation. Two effects were noted. First, being part of moderately stable stem loops (delta G, -18 kcal/mol) decreased translation efficiency about 3-fold; second, mRNA in which only three 5'-terminal nucleotides were unpaired were translated five times less efficiently than mRNA in which six nucleotides were unpaired. Accessibility of the 5'-cap as well as secondary structure of the 5'-upstream sequences are therefore factors that affect translation efficiency. Finally, we showed that the m1 mRNA, which is transcribed very poorly in vivo, is translated very efficiently in vitro; and that its 5'-upstream sequence is as effective in increasing protein sigma 1 formation as that of s4 mRNA. Since both m1 mRNA and protein mu 2 are stable in infected cells, the reason why m1 mRNA is translated so inefficiently in vivo therefore remains unexplained.

Codon↗
PubMed1989

The sequences of the S2 genome segments of reovirus serotype 3 and of the dsRNA-negative mutant ts447.

The most temperature-sensitive dsRNA-negative mutant of reovirus serotype 3 is ts447; the amount of dsRNA formed in cells infected with it at 39 degrees is less than 0.1% of that formed in cells with wt virus at 37 degrees. The genome segment in which this mutation is located is S2. We compare here the sequence of the S2 genome segment of wt reovirus serotype 3 with that of mutant ts447. The two sequences differ in three locations, at two of which there are C to U transitions, while at the third there is an A to G transition. All cause amino acid changes (Ala to Val, Ala to Val, and Asn to Asp, respectively). One mutation (at nucleotide position 581, which causes an Ala to Val change) causes the length of an alpha-helix to be significantly reduced and may be that which is responsible for the ts phenotype.

Amino Acid Sequence↗
PubMed1989

Studies on the mechanism of the antiviral activity of ribavirin against reovirus.

We have examined the mechanism by which ribavirin inhibits the multiplication of reovirus. At a concentration of 12.5 microM (3 micrograms/ml) ribavirin inhibits viral multiplication, ssRNA formation, dsRNA formation, and protein synthesis by about 90%; when much higher concentrations are used for brief periods of time, the primary target of ribavirin is seen to be viral ssRNA synthesis. When the effect of ribavirin triphosphate (RTP) was tested on the in vitro transcription by cores of the dsRNA genome segments into plus-stranded RNA, elongation, that is, the formation of intact mRNA molecules, was found to be inhibited to the greatest extent; initiation was at least 2.5 times less sensitive, and cap formation and methylation were almost unaffected. The inhibition of elongation and initiation was not competitive with respect to any of the four nucleoside triphosphates. Remarkably, the transcription of plus strands into minus strands by immature reovirus particles (the replicase reaction) was insensitive to RTP. A model is proposed that envisages RTP binding to a site close to the catalytic site of the transcriptase. This binding is postulated to inhibit the helicase function of the transcriptase and lower its affinity for template RNA so that the likelihood of premature termination is greatly increased. The helicase activity is not, of course, necessary for the transcription of plus strands into minus strands, which would account for the differential sensitivity of the transcriptase and the replicase to RTP.

Animals↗
PubMed1989

The function of reovirus proteins during the reovirus multiplication cycle: analysis using monoreassortants.

When cultured cells are injected with mixtures of cores of two reovirus strains, a high proportion of reassortants are monoreassortants, that is, virus particles that contain one genome segment of 1 parent and 9 genome segments of the other. We have isolated two complete sets of monoreassortants, those that contain a single serotype 2 genome segment and 9 serotype 3 genome segments, and those that contain 1 serotype 3 genome segment and 9 serotype 1 genome segments. We have used the former set of monoreassortants (because reovirus serotypes 2 and 3 are less closely related than serotypes 1 and 3) to assess the effect of all 10 genome segments, or rather of the proteins that they encode, in controlling parameters of the reovirus multiplication cycle such as yield size, extent of viral ssRNA, dsRNA and protein synthesis, plaque size, and cytopathogenicity. Among the major findings are: proteins lambda 2, mu 1/mu 1C, and sigma 3 control yield size and extent of RNA and protein synthesis; proteins mu 2 and sigma 1 control severity of cytopathic effects; and proteins sigma 1, mu 1/mu 1C, and mu 2 control plaque size. Identification of monoreassortant phenotypes is useful for identifying which viral proteins are functionally involved at the various stages of the reovirus multiplication cycle.

Animals↗