Search PubMed⌕ Search

Biomedical subjects

J T Patton

Publications and source records attributed to J T Patton.

At least 55 records · Page 3Linked to original sources
PubMed1994

The rotavirus RNA-binding protein NS35 (NSP2) forms 10S multimers and interacts with the viral RNA polymerase.

The gene 8 product of SA11 rotavirus, NS35 (NSP2), is a nonspecific RNA-binding protein that accumulates in cytoplasmic inclusions (viroplasms) and is required for genome replication. To gain additional information on the role of NS35 in virus replication, lysates of simian rotavirus SA11-infected cells were treated with the thio-cleavable crosslinking agent, dithiobis(succinimidyl propionate) (DSP). Gel electrophoresis of NS35-specific immunoprecipitates recovered from the crosslinked lysates indicated that infected cells contained NS35 multimers, the largest consisting of four or more molecules of the protein. Sedimentation analysis of NS35 expressed in rabbit reticulocyte lysates by cell-free translation and in vTF7-3-infected cells by transfection with a gene 8-containing transcription vector showed that NS35 assembles into multimers of approximately 10S and that the formation of the multimers does not require other viral proteins. The 10S multimers were also detected in rotavirus-infected cells, providing evidence that they function in virus replication. The lack of RNase sensitivity indicates that the 10S multimers probably lack an RNA component. However, by an NS35-specific RNA capture assay, the multimers were shown to possess the RNA-binding activity previously demonstrated for NS35. Despite its ability to multimerize and bind RNA, indirect immunofluorescence assays showed that when transiently expressed in cells, NS35 alone is not sufficient to induce the formation of viroplasms. DSP-crosslinking of infected cell lysates and immunoprecipitation also revealed that NS35 interacts with the putative viral RNA polymerase VP1. Analysis of cytoplasmic extracts resolved by sedimentation on glycerol gradients suggested that the VP1-NS35 complexes are soluble and RNA-free. Complexes formed from NS35 multimers, VP1, and viral messenger RNA may function to coordinate RNA packaging and the assembly of viral cores.

Cytoplasm↗
PubMed1994

The carboxyl-half of the rotavirus nonstructural protein NS53 (NSP1) is not required for virus replication.

The rotavirus nonstructural protein NS53 (NSVP1), the product of genome segment 5, possesses RNA-binding activity and contains a highly conserved cysteine-rich motif located in the amino-terminal half of the protein. The genome of the bovine rotavirus variant, brvA, lacks a normal segment 5 but includes a novel dsRNA (gene A) of approximately 2600 basepairs (bp) that contains segment 5-specific sequences (F. Hundley, B. Biryahwaho, M. Gow, and U. Desselberger, Virology 143, 88-103, 1985). To gain information about the nature of the rearrangement in gene A and its capacity to encode a protein product, we prepared and sequenced complementary (c)DNA of the gene A RNA. The results showed that gene A is 2693 bp in size and contains a head-to-tail duplication of 1112 bp that originates from the open reading frame (ORF) of gene 5. The duplication begins at nucleotide (nt) 1454, which is 53 nt upstream from the end of the ORF for NS53. Gene A contains a point mutation at nt 808 which results in the presence of a nonsense codon near the middle of the ORF for NS53. Thus the predicted product of gene A is a truncated NS53 of 258 amino acids (aa) (31 kDa), approximately one-half the size of the authentic 491-aa NS53 (58 kDa). Examination of lysates from brvA-infected cells by Western blot assay using an NS53-specific antibody confirmed that the variant encodes only a truncated gene 5 product. Despite the truncation, analysis of the gene A product suggested that it, like full-length NS53, accumulated in association with the cytoskeleton of the infected cell, thus providing evidence that the subcellular localization signal in NS53 resides in the amino terminal half of the protein. Given that brvA is a viable, nondefective mutant, these results demonstrate that the carboxyl-terminal 233 aa of NS53 are not required for rotavirus replication in vitro.

Amino Acid Sequence↗
PubMed1994

Temperature-sensitive lesions in the capsid proteins of the rotavirus mutants tsF and tsG that affect virion assembly.

The SA11 rotavirus mutants tsF and tsG contain temperature sensitive (ts) lesions in the capsid proteins VP2 and VP6, respectively, that interfere with their ability to assemble. To understand the nature of their lesions, full-length cDNAs of tsF gene 2 and tsG gene 6 were prepared from viral mRNA by reverse transcription and polymerase chain reaction. Comparative sequence analysis indicated that the ts phenotype of tsF VP2 is due to an Ala-->Asp substitution at position 387. The mutation falls outside of those regions of VP2 previously suggested to be of functional significance and therefore points to a previously unidentified site in VP2 that is important for the assembly of viral cores. Comparative sequence analysis showed that tsG VP6 contains two mutant amino acids, i.e., Thr-10 and His-13, and therefore one or both of these mutations are responsible for the ts phenotype of the mutant VP6. In the case of other group A and group C VP6 sequences, these residues are Ser and Asp, respectively. Characterization of tsG-infected cells by indirect immunofluorescence staining showed that while viroplasmic inclusions are formed at the nonpermissive temperature, the mutant VP6 accumulates in these structures only at the permissive temperature. While influencing intracellular accumulation, the Thr-10-->Ser and His-13-->Asp mutations in tsG VP6 are probably not directly involved in the interaction of VP6 with VP2, as VP6 deletion mutants lacking residues 10 and 13 retain the ability to bind VP2 in vitro. Analysis of VP6 failed to confirm previous reports that the protein was myristylated and thus excludes the possibility that this cotranslational modification is temperature-dependent for tsG VP6. Together, these data suggest that the amino terminus of VP6 plays an essential role in virus assembly in vivo, perhaps by being necessary for the movement of the protein to viroplasmic inclusions, the site of core and single-shelled particle formation.

Amino Acid Sequence↗
PubMed1994

Deletion mapping of the rotavirus metalloprotein NS53 (NSP1): the conserved cysteine-rich region is essential for virus-specific RNA binding.

NS53 (NSP1), the gene 5 product of the group A rotaviruses, is a minor nonstructural protein of 486 to 495 amino acids which binds zinc and contains an amino-terminal highly conserved cysteine-rich region that may form one or two zinc fingers. To study the structure-function of the gene 5 product, wild-type and mutant forms of NS53 were produced by using a recombinant baculovirus expression system and a recombinant vaccinia virus/T7 (vTF7-3) expression system. Analysis of the RNA-binding activity of the wild-type NS53 immobilized onto protein A-Sepharose beads with NS53-specific antiserum showed that the protein exhibited specific affinity for all 11 rotavirus mRNAs. The use of short virus-specific RNA probes indicated that NS53 specifically recognizes an element located near the 5' ends of viral mRNAs. Analysis of the RNA-binding activity of deletion mutants of NS53 showed that the RNA-binding domain resides within the first 81 amino acids of the protein and that the highly conserved cysteine-rich region within this region of the protein is essential for the activity. Gel electrophoresis and Western immunoblot analyses of intracellular fractions derived from infected cells revealed that large amounts of NS53 were present in the cytosol and in association with the cytoskeletal matrix. Indirect immunofluorescence analysis of cells programmed to transiently express mutant forms of NS53 using vTF7-3 indicated that the intracellular localization domain resides between amino acids 84 and 176 of NS53. Together, these data show that the RNA-binding domain and the intracellular localization domain lie upstream from the region of NS53 previously determined not to be essential for replication of rotaviruses in cell culture (J. Hua and J. T. Patton, Virology 198:567-576, 1994).

Amino Acid Sequence↗
PubMed1993

Computerized analysis of tumor cells flowing in a parallel plate chamber to determine their adhesion stabilization lag time.

The importance of cell adhesion in a variety of physiological phenomena requires development of an understanding of the factors and molecular mechanisms underlying these behaviors. Cell adhesion is a multistep process involving primary receptor-ligand interactions followed by secondary events that may lead to the formation of focal contacts. Due to the lack of well-defined assays to study adhesion stabilization, little is known about this process, except that it may involve signaling events, receptor recruitment, and, as we have demonstrated, covalent peptide cross-linking by cell membrane-associated transglutaminase [Menter et al.: Cell Biophys. 18:123-143, 1992). To study the stabilization process we have developed a dynamic assay employing a parallel plate flow chamber coupled with video microscopy and digital image processing. Our studies utilize wheat germ agglutinin-selected human metastatic melanoma cell variants that exhibit differences in their experimental metastatic potential and expression of transglutaminase. Using this assay, quantifying cell-substrate stabilization was found to be quick, reliable, reproducible, and useful in evaluating agents that block this process.

Aged↗
PubMed1993

Nucleotide and amino acid sequence analysis of the rotavirus nonstructural RNA-binding protein NS35.

NS35, a basic protein encoded by gene 8 of SA11 rotavirus, possesses RNA-binding activity and is essential for genome replication. To identify conserved regions in the NS35 gene and its protein product, we determined the nucleotide sequences of the NS35 gene for the mammalian and avian rotaviruses Wa, DS1, SA11 (Patton and Ramig strains), NCDV, and Ty-1 and compared them and their deduced amino acid sequences to those reported for SA11 (Both strain), OSU, and UK. The results indicated that the NS35 genes of the mammalian rotaviruses are 1058-1059 bases in length and encode proteins of 317 amino acids that exhibit high levels of sequence conservation (> or = 83%). The NS35 gene of the turkey rotavirus Ty-1 differed from those of the mammalian rotaviruses with respect to size of the predicted protein (315 amino acids) and of the gene (1042 bases). NS35 of Ty-1 exhibited a relatively low degree of amino acid homology (52-57%) with NS35 of the mammalian viruses. Phylogenetic analysis of the NS35 gene indicated that avian (TY-1) and mammalian rotaviruses are distantly related. Comparison of the predicted sequences of NS35 showed that all possessed a conserved basic domain of 37 amino acids at residues 205-241 that may serve as the RNA-binding domain. Electrophoretic examination showed that NS35 contains a disulfide bond probably located in the amino-terminal half of the protein. Comparison of NS35 genes at the nucleotide level revealed two regions of extensive conservation, (i) a 75-base (b) sequence that includes the 35-base 5'-noncoding region and the first 30 bases of the open reading frame for NS35, and (ii) a 28-b sequence in the 3'-noncoding region of the gene. Secondary structure predictions for the NS35 mRNA suggest that the 75-base sequence can fold to produce a stem double-loop structure. Such a structure may serve as a packaging signal for the assortment of NS35 mRNA into replicase particles.

Amino Acid Sequence↗
PubMed1993

Comparative analysis of the rotavirus NS53 gene: conservation of basic and cysteine-rich regions in the protein and possible stem-loop structures in the RNA.

NS53, the product of rotavirus gene 5, is an RNA-binding protein that contains a cysteine-rich region and is a component of early replication intermediates. To gain information about the structure of NS53 and its RNA, we determined the nucleotide sequence of gene 5 for the human viruses Wa (serotype 1) and DS1 (2) and the simian virus SA11 (3) (Patton strain) and compared them and their deduced amino acid sequences to those reported for the bovine viruses UK (6) and RF (6), SA11 (3) (Both strain), the human virus Rohivg803, and the group C porcine virus PRV. The results showed that gene 5 for human, simian, and bovine strains have lengths of 1564-1567, 1611, and 1579-1581 nucleotides (nt) and encode proteins of 486, 495, and 491 amino acids, respectively. Comparison of the protein sequences for NS53 among different serotypes showed that they are extremely divergent with many sharing amino acid homologies of only 36-38%. Even NS53 from viruses isolated from the same species possessed relatively poor homology, e.g., DS1 versus Wa was 68%. The first 150 amino acids of NS53 exhibited a greater degree of conservation than the rest of the protein. Near the amino terminus, NS53 contains three basic regions and a cysteine-rich domain, suggesting that this area is responsible for the RNA-binding activity of the protein. Present in the cysteine-rich domain of all group A and C viruses was the motif C-X2-C-X8-C-X2-C-X3-H-X-C-X2-C-X5-C. Although this motif may form one or two zinc fingers, the fact that it is highly conserved indicates that it plays a critical role in the function of protein. Comparison of the nucleotide sequences for gene 5 showed that the entire 5'-noncoding region and the first 24 nt of the NS53 ORF are conserved. RNA-folding predictions suggest that this region of the NS53 mRNA can interact with itself, producing a stem-loop structure similar to that found near the 5'-terminus of the NS35 mRNA. Thus, such structures may be common to all rotavirus mRNAs, perhaps functioning as signals for packaging of RNAs into replication intermediates or regulating mRNA translation.

Amino Acid Sequence↗
PubMed1993

Location of intrachain disulfide bonds in the VP5* and VP8* trypsin cleavage fragments of the rhesus rotavirus spike protein VP4.

Because the rotavirus spike protein VP4 contains conserved Cys residues at positions 216, 318, 380, and 774 and, for many animal rotaviruses, also at position 203, we sought to determine whether disulfide bonds were structural elements of VP4. Electrophoretic analysis of untreated and trypsin-treated rhesus rotavirus (RRV) and simain rotavirus SA11 in the presence and absence of the reducing agent dithioerythritol revealed that VP4 and its cleavage fragments VP5* and VP8* possessed intrachain disulfide bonds. Given that the VP8* fragments of RRV and SA11 contain only two Cys residues, those at positions 203 and 216, these data indicated that these two residues were covalently linked. Electrophoretic examination of truncated species of VP4 and VP4 containing Cys-->Ser mutations synthesized in reticulocyte lysates provided additional evidence that Cys-203 and Cys-216 in VP8* of RRV were linked by a disulfide bridge. VP5* expressed in vitro was able to form a disulfide bond analogous to that in the VP5* fragment of trypsin-treated RRV. Analysis of a Cys-774-->Ser mutant of VP5* showed that, while it was able to form a disulfide bond, a Cys-318-->Ser mutant of VP5* was not. These results indicated that the VP4 component of all rotaviruses, except B223, contains a disulfide bond that links Cys-318 and Cys-380 in the VP5* region of the protein. This bond is located between the trypsin cleavage site and the putative fusion domain of VP4. Because human rotaviruses lack Cys-203 and, hence, unlike many animal rotaviruses cannot possess a disulfide bond in VP8*, it is apparent that VP4 is structurally variable in nature, with human rotaviruses generally containing one disulfide linkage and animal rotaviruses generally containing two such linkages. Considered with the results of anti-VP4 antibody mapping studies, the data suggest that the disulfide bond in VP5* exists within the 2G4 epitope and may be located at the distal end of the VP4 spike on rotavirus particles.

Amino Acid Sequence↗
PubMed1992

The rotavirus nonstructural protein, NS35, possesses RNA-binding activity in vitro and in vivo.

Toward the goal of identifying and characterizing rotavirus RNA-binding proteins, we have used a gel retardation assay and protein-RNA cross-linking by ultraviolet (uv) light to examine cytoplasmic lysates prepared from SA11-infected cells for the presence of RNA-binding proteins. Analysis of band shifts produced in the gel retardation assay indicated that infected cells contained significant amounts of a viral protein which had affinity for both single-stranded and double-stranded RNA but lacked sequence specificity. Cross-linking of this protein to radiolabeled RNA in vitro followed by RNase treatment and immunoprecipitation with an anti-NS35 monoclonal antibody revealed that the RNA-binding activity was associated with NS35. Moreover, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein-RNA complex isolated from native gels revealed that NS35 was the only viral protein component of the complex. Since NS35 expressed by translation in rabbit reticulocyte lysates exhibited affinity for poly(U)-Sepharose, NS35 must possess intrinsic RNA-binding activity that is able to function in the absence of other viral proteins. Immunoprecipitation of RNase-treated cross-links formed in intact cells following exposure to uv light confirmed that NS35 was intimately associated with ssRNA in the infected cell. On the basis of its ability to bind RNA and given that previous studies have shown that NS35 localizes to the viroplasm in infected cells, is essential for RNA replication, and is a component of replicase particles, we propose that NS35 functions to concentrate viral mRNAs in the viroplasm and that NS35-mRNA complexes serve as substrates for genome assortment and replication.

Animals↗
PubMed1991

Transglutaminase stabilizes melanoma adhesion under laminar flow.

To resist substantial wall shear stress (WSS) exerted by flowing blood, metastatic melanoma cells can form adhesive contacts with subendothelial extracellular matrix proteins, such as fibronectin (FN). Such contacts may be stabilized by transglutaminase catalyzed-cross-linkage of cell focal adhesion proteins. We analyzed human melanoma cell adhesion under flow by decreasing the flow (WSS) of melanoma cell suspensions and allowing them to adhere to immobilized wheat germ agglutinin or FN. At the wall shear adhesion threshold (WSAT), cell adherence was rapid with no rolling. Following cell adherence, we increased the flow and determined the wall shear detachment threshold (WSDeT). Cells spread and remained adherent on immobilized FN at high WSDeTs (greater than or equal to 32.5 dynes/cm2). The high resistance of adherent cells to shear forces suggested that transglutaminase-mediated crosslinking might be involved. Transglutaminase inhibitors monodansylcadaverine and INO-3178 decreased WSAT, and at low concentrations completely inhibited tumor cell spreading and promoted detachment at low WSDeTs (0.67 dynes/cm2). In static adhesion assays, transglutaminase inhibitors decreased cell adhesion to immobilized-FN in a dose-dependent manner and prevented the formation of crosslinked 125I-FN complex that failed to enter a SDS-polyacrylamide gradient gel. The data suggest that transglutaminase-catalyzed crosslinking, particularly in the presence of WSS, may be important in stabilizing cellular adhesive contacts during adhesion to immobilized-FN.

Aged↗
PubMed1991

Rotavirus morphogenesis: domains in the major inner capsid protein essential for binding to single-shelled particles and for trimerization.

A cell-free system containing rotavirus subviral particles (SVPs), rabbit reticulocyte lysate, and [35S]methionine was programmed to synthesize viral protein by the addition of messenger RNA (mRNA). Electrophoretic analysis of single-shelled particles recovered from the system by CsCl centrifugation showed that newly made VP6 assembled into the particles in vitro. Electrophoretic analysis also showed that the newly made VP6 which bound to single-shelled particles in vitro was arranged in trimeric units. To identify the domain within VP6 essential for assembly into single-shelled particles, amino- and carboxyl-truncated species of VP6 were assayed for the ability to associate with single-shelled particles in the cell-free system. The truncated proteins were introduced into the system by adding VP6 mRNAs containing 5'- and 3'-terminal deletions. The terminally deleted mRNAs were prepared using SP6 RNA polymerase to transcribe portions of cDNAs of the rotavirus SA11 gene for VP6 (gene 6). Analysis of the ability of truncated VP6 to associate with single-shelled particles showed that a domain essential for assembly resides at the carboxyl-end of VP6 located between amino acid residues 251 and 397. To contrast the domain for assembly with that for trimerization, amino- and carboxyl-truncated species of VP6 were also examined by electrophoretic assay for the ability to trimerize in vitro. The results showed that the domain for trimerization resides near the center of VP6 located between amino acid residues 105 and 328. Comparison of the domains for assembly and trimerization showed that they are unique but may overlap. The fact that some truncated species of VP6, although able to bind to single-shelled particles were unable to form trimers in vitro, suggests that trimerization of VP6 is not prerequisite for the assembly of single-shelled particles.

Animals↗
PubMed1991

Imaging of bone and joint infections.

In recent years, world interest in bone infection has centered on investigating the potential of the newer imaging methods for detecting early osteomyelitis and on their sensitivity and specificity in evaluating both the acute disease and recurrence of chronic disease and the development of infection in prostheses. No revolutionary new techniques were forthcoming since the advent of MR imaging, and the past 12 months have seen a decline in new analyses, presumably because most researchers have now made up their minds about the relative imaging modalities. Interest lately has been mainly in the imaging of chronic (granulomatous) disease and patterns in children and in drug abuse patients. Because of the importance of differentiating neuropathy from infection in the diabetic foot, research is also active in this field, particularly in relation to dynamic scintigraphy. Interesting observations continue to emerge on infection resulting from the depressing spread of drug abuse and immunosuppression.

Bacterial Infections↗
PubMed1990

Evidence for equimolar synthesis of double-strand RNA and minus-strand RNA in rotavirus-infected cells.

The genome of the rotaviruses consists of eleven segments of double-strand RNA (dsRNA). Each segment is replicated asymmetrically with viral plus-strand RNA, i.e. messenger (m)RNA, serving as the template for the synthesis of minus-strand RNA to produce dsRNA. To examine the relative frequency of replication of each of the eleven genome segments, MA104 cells were infected with low (3rd) and high (12th) passage stocks of simian rotavirus SA11. The total cytoplasmic RNA of the infected cell was radiolabeled either by maintaining the infected cells in the presence [3H]uridine prior to harvest or by 3'-endlabeling the purified RNA with [32P]pCp and T4 RNA ligase. The RNA was then analyzed for the presence of 3H- and 32P-labeled dsRNA by electrophoresis on 10% polyacrylamide gels. Total cytoplasmic RNA from infected cells was also 3'-end-labeled with [32P]pCp and T4 RNA ligase and examined for the presence of minus-strand RNA by electrophoresis on low pH agarose-urea gels. Bands representing dsRNAs and minus-strand RNAs on autoradiographs of the gels were analyzed for intensity by densitometry. The results showed that the eleven segments of viral dsRNA were present in equimolar concentrations in cells either infected with low or high passage stocks of virus. Like intracellular dsRNAs, full-length minus-strand RNAs were also present in equimolar concentration in cells either infected with low or high passage rotavirus. These data indicate that, despite the non-equimolar levels of viral RNAs in the cell, the eleven genome segments of rotavirus are replicated with equal frequencies in vivo.

Animals↗
PubMed1990

Rotavirus RNA replication: single-stranded RNA extends from the replicase particle.

The rotavirus genome consists of 11 segments of dsRNA that are replicated asymmetrically with plus strand RNA serving as the template for minus strand RNA synthesis. In this study, we have used non-denaturing gel electrophoresis to examine subviral particles that synthesize dsRNA (replicase particles), for possible changes in structure during RNA replication. Analysis of SVPs purified from simian rotavirus SA11-infected MA104 cells and resolved on 0.6% agarose gels containing 50 mM-Tris-glycine pH 8.8 showed that the overall size of particles able to synthesize dsRNA in a cell-free system was 100 nm or more. Electrophoretic analysis of the size of replicase particles as a function of length of incubation in the cell-free system demonstrated that replicase particles decreased in size with increasing length of incubation. However, after 60 to 90 min of incubation, replicase particles no longer changed in size but were similar in size to the rotavirus single-shelled (75 nm), core (60 nm) and precore (45 nm) replicative intermediates which have been described previously. As the size of replicase particles decreased with increasing length of incubation, the number of newly made genome-length dsRNAs in the particles increased. Analysis of the RNA products detected in replicase particles showed that RNA replication is regulated such that the synthesis of full-length dsRNAs in the replicase particle proceeds from the smallest to the largest genome segments. Treatment of replicase particles with single-strand-specific RNase reduced their size to that of replicative intermediates and interfered with their ability to synthesize dsRNA, thus indicating that the plus strand RNA template for replication extends from the replicase particle. This study showed that replicase particles undergo a continuous change in size during RNA replication due apparently to plus strand RNA templates moving into the replicase particle during the synthesis of dsRNA.

Animals↗
PubMed1990

Rotavirus RNA replication: VP2, but not VP6, is necessary for viral replicase activity.

Temperature-sensitive mutants of simian rotavirus SA11 were previously developed and organized into 10 of a possible 11 recombination groups on the basis of genome reassortment studies. Two of these mutants, tsF and tsG, map to genes encoding VP2 (segment 2) and VP6 (segment 6), respectively. To gain insight into the role of these proteins in genome replication, MA104 cells were infected with tsF or tsG and then maintained at permissive temperature (31 degrees C) until 9 h postinfection, when some cells were shifted to nonpermissive temperature (39 degrees C). Subviral particles (SVPs) were recovered from the infected cells at 10.5 and 12 h postinfection and assayed for associated replicase activity in a cell-free system shown previously to support rotavirus genome replication in vitro. The results showed that the level of replicase activity associated with tsF SVPs from cells shifted to nonpermissive temperature was ca. 20-fold less than that associated with tsF SVPs from cells maintained at permissive temperature. In contrast, the level of replicase activity associated with tsG SVPs from cells maintained at nonpermissive temperature was only slightly less (twofold or less) than that associated with tsG SVPs from cells maintained at permissive temperature. Analysis of the structure of replicase particles from tsG-infected cells shifted to nonpermissive temperature showed that they were similar in size and density to virion-derived core particles and contained the major core protein VP2 but lacked the major inner shell protein VP6. Taken together, these data indicate that VP2, but not VP6, is an essential component of enzymatically active replicase particles.

Animals↗
PubMed1989

Characterization of rotavirus replication intermediates: a model for the assembly of single-shelled particles.

The segmented double-stranded (ds)RNA genome of the rotaviruses is replicated asymmetrically with viral mRNA serving as the template for minus-strand RNA synthesis. To identify intermediate structures in rotavirus replication, subviral particles (SVPs) purified from the cytoplasm of simian rotavirus SA11-infected cells were assayed for RNA polymerase activity in a cell-free system that supports viral RNA replication. Intact SVPs containing newly made RNA were resolved by electrophoresis under nondenaturing conditions on 0.6% agarose gels (50 mM Tris-glycine, pH 8.8). This gel system was found to separate without disrupting SA11 single- and double-shelled virions and virion-derived core particles. SVPs from the cell-free system that contained newly made dsRNA migrated in the agarose gels at positions between virion-derived cores and intermediate of single- and double-shelled virions. SVPs containing newly made dsRNA were eluted from the gel and analyzed for protein content by electrophoresis on polyacrylamide gels. The results showed that three distinct types of replication intermediates (RIs) were present in SA11-infected cells. The smallest intermediate (precore RI, 45 nm, 220 S) contained the structural proteins VP1, VP3, and VP9 and the nonstructural proteins NS53, NS35, and NS34. A second intermediate (core RI, 60 nm, 310 S) contained the core proteins VP1, VP2, and VP3 and the proteins VP9, NS35 and NS34. The largest RI (single-shelled RI, 75 nm, 420 S) contained the inner shell proteins VP1, VP2, VP3, and VP6 and the proteins VP9, NS35 and NS34. Analysis of the formation and turnover of RIs in infected cells pulse-labeled with 35S-amino acids supports a hypothesis that rotavirus single-shelled particles are assembled in vivo by the sequential addition of VP2 and VP6 to precore RIs consisting of VP1, VP3, VP9, NS35, and NS34.

Animals↗