Purification and characterization of a strain of coxsackievirus B4 of human origin that induces diabetes in mice

A diabetogenic strain of coxsackievirus B4 of human origin has been purified to study its biochemistry and diabetogenicity. Tissue culture cells infected with the virus contain two distinct types of particles--virions and membrane-bound virions (MBV). MBVs are lighter (p = 1.29) than virions (p = 1.34), and they contain relatively more protein than RNA. Virons contain four capsid proteins, VPI-4, of various molecular weights: VP1, 37,500; VP2, 36,000; VP3, 26,000; and VP4, 5,500. MBVs contain three of these proteins and several additional proteins of molecular weights 45,000 to greater than 92,500, possibly of host or viral origin. The RNA in each type of particle is a 35S molecule; T1 oligonucleotide fingerprint profiles suggest minor differences in the two RNAs. Hybridization experiments show a great deal of sequence homology between the RNA of the diabetogenic strain and the RNA of prototype CB4, which does not induce overt diabetes. MBVs are 10-70 times less infective than virions, yet they are more pathogenic in mice and induce significantly higher glucose intolerance (hyperglycemia). The hyperglycemic response appears to be lower in mice infected with both types of particles than in mice infected with MBVs alone. Thus, the two subpopulations of virions present in the diabetogenic strain differ biochemically and in their ability to induce diabetes.     

Formation of RNA and protein in cells infected with standard and defective Sindbis virus

Our studies on the formation of Sindbis virus proteins have established that: 1. one of the two envelope proteins (E2) accumulates in infected cells as a higher molecular weight precursor that is slowly converted to the virion protein; 2. the large protein (mol. wt 130000 daltons), that accumulates in cells infected with a temperature-sensitive mutant of Sindbis virus contains sequences of the three virion proteins; and 3. the protein (mol. wt. 100000 daltons) isolated from BHK cells infected with Sindbis virus is related in sequence to the two envelope proteins.We have investigated the formation of defective-interfering (DI) particles of Sindbis virus and their ability to inhibit the replication of standard virus. BHK cells infected with passages of Sindbis virus containing DI particles accumulate a species of RNA (20S) that is about half the molecular weight of the 26S RNA. We have demonstrated by competitive hybridization experiments that 20S RNA contains half the sequences of 26S RNA. We also present evidence that in contrast to 26S RNA, 20S does not bind to polysomesin vivo and is not translatedin vitro.Presented on the Meeting on Studies on Virus Replication of the Commission of the European Communities in Brüssel, May 1974.     

The inhibition of influenza virus RNA synthesis by actinomycin D and cycloheximide

Chick embryo fibroblast monolayers infected with influenza virus (WSN strain) were treated with either actinomycin D or cycloheximide at various times after infection. Analysis of cell homogenates indicated that in the presence of actinomycin D, synthesis of RNA complementary to virion type RNA was preferentially inhibited. Consequently, no labeled polysomes are found in cells so treated, although virion-type RNA, isolated as the RNP, was found. Cycloheximide, on the other hand, while suppressing the synthesis of complementary type RNA, completely inhibited the synthesis of virion-type RNA.These results confirm and extend the work of Scholtissek and Rott (1970) that showed the same differential effect of these two drugs on RNA synthesis and are added evidence that in influenza virus-infected cells the messenger RNA is that RNA which is complementary to the RNA found in virions.     

Electron microscopic observations onAedes albopictus cells infected with dengue viruses

Summary In dengue virus infectedAedes albopictus cells, electron-dense particles, larger than single ribosomes, were arranged on the cytoplasmic sides of rough endoplasmic reticulum (RER) membranes. Mature virions 40–45 nm in diameter as well as vesiculotubular structures 50–120 nm in diameter appeared in enlarged cisternae of RER filled with fine granular substance. Many of the mature virions and somewhat degenerated vesiculotubular structures remained to be enclosed in membranous structures presumably derived from RER, even after degeneration of infected cells. The findings suggest that development of dengue viruses in culturedA. albopictus cells takes place in close relationship with the activated membranes of RER.Other morphological changes observed in dengue infectedA. albopictus cells were 1. electron-dense double-track structures in areas of virion morphogenesis, 2. fine crystalline structures in type-2 dengue infected cells, and 3. aggregates of nucleoid structures, in cells persistently infected with type 2 dengue virus. The implication and nature of these structures in relation to virion morphogenesis remain to be investigated.With 6 Figures     

Host-induced influences on the syntheses of defective vaccinia particles

Virion subpopulations of the WR strain of vaccinia virus which plaque well on mouse L cells but not at all on rabbit kidney cells (L⁺RK^?) were isolated by clonal selection techniques. Initial passage of these cloned subpopulations in nonpermissive rabbit kidney cells at an input multiplicity of 100 virions per cell resulted in a low yield of virus which could be detected with the aid of the electron microscope but which lacked rabbit kidney plaquing capability. Further passage of these particles in rabbit kidney cells resulted in the production of defective particles along with standard virions which were fully infectious for both mouse L and rabbit kidney cells (L⁺RK⁺). Passage of these standard L⁺RK⁺ virion populations and their associated defective particle subpopulations back into mouse L cells did not rever L⁺RK⁺ variants to L⁺RK^? but did eliminate defective particle synthesis. Defective particles produced in rabbit kidney cells sedimented faster in sucrose and had a lower buoyant density than standard virions. However, no difference between defective and standard virion sizes were observed in the electron microscope. In contrast, cores produced from virion populations rich in defective particles sedimented at the same rate and had the same buoyant density as cores produced from standard virion populations. Defective particle synthesis was a host-induced phenomenon associated with rabbit kidney but not mouse L cells.     

Defective-interfering particles of murine coronavirus: mechanism of synthesis of defective viral RNAs

The mechanism of synthesis of the defective viral RNAs in cells infected with defective-interfering (DI) particles of mouse hepatitis virus was studied. Two DI-specific RNA species, DIssA of genomic size and DIssE of subgenomic size, were detected in DI-infected cells. Purified DI particles, however, were found to contain predominantly DIssA and only a trace amount of DIssE RNA. Despite its negligible amount, the DIssE RNA in virions appears to serve as the template for the synthesis of DIssE RNA in infected cells. This conclusion was supported by two studies. First, the uv target size for DIssE RNA synthesis is significantly smaller than that for DIssA. Second, when purified DIssE RNA was transfected into cells which had been infected with a helper virus, DIssE RNA could replicate itself and became a predominant RNA species in the infected cells. Thus, DIssE RNA was not synthesized from the genomic RNA of DI particles. By studying the relationship between virus dilution and the amount of intracellular viral RNA synthesis, we have further shown that DIssE RNA synthesis requires a helper function, but it does not utilize the leader sequence of the helper virus. In contrast, DIssA synthesis appears to be helper-independent and can replicate itself. Thus DIssA codes for a functional RNA polymerase.     

Identification of putative polymerase gene product in cells infected with murine coronavirus A59

The virion RNA of mouse hepatitis virus, strain A59 (MHV-A59) is believed to be the mRNA for the viral RNA-dependent RNA polymerase. The cell-free translation of virion RNA results in the synthesis of two predominant products p220 and p28 (M. R. Denison and S. Perlman, 1986, J. Virol. 60, 12-18). p28 is a basic protein and is readily detected by two-dimensional gel electrophoresis. When infected cells and isolated virions were assayed for this protein by two-dimensional gel electrophoresis, p28 could be detected in infected cells labeled at late times after infection, but not at early times or in purified virions. p28 represents the first protein product of the putative coronavirus polymerase gene to be identified in infected cells.     

Structural maturation of rubella virus in the Golgi complex

Rubella virus is a small enveloped virus that assembles in association with Golgi membranes. Freeze-substitution electron microscopy of rubella virus-infected cells revealed a previously unrecognized virion polymorphism inside the Golgi stacks: homogeneously dense particles without a defined core coexisting with less dense, mature virions that contained assembled cores. The homogeneous particles appear to be a precursor form during the virion morphogenesis process as the forms with mature morphology were the only ones detected inside secretory vesicles and on the exterior of cells. In mature virions potential remnants of C protein membrane insertion were visualized as dense strips connecting the envelope with the internal core. In infected cells Golgi stacks were frequently seen close to cytopathic vacuoles, structures identified as the sites for viral RNA replication, along with the rough endoplasmic reticulum and mitochondria. These associations could facilitate the transfer of viral genomes from the cytopathic vacuoles to the areas of rubella assembly in Golgi membranes.     

Identification of a satellite RNA associated with turnip crinkle virus

Turnip plants infected with turnip crinkle virus (TCV) contain four major RNA species which are not found in uninfected plants (A = 1.3 x 10(6) MW, B = 0.28 x 10(6) MW, C = 0.17 x 10(6) MW, and D = 0.13 x 10(6) MW). At least two of these RNAs, RNA A and RNA C, are packaged in the mature virion, but only the large RNA A is required for infection. Plants infected with RNA A alone produce neither the small virion RNA C nor the small nonvirion RNAs B and D. The small virion RNA C is not infective by itself, but requires coinfection with RNA A to replicate in plants. RNA C increases the severity of symptoms in plants infected with RNA A and restores the production of the nonvirion RNAs B and D. T1 RNase oligonucleotide mapping and copy DNA hybridization analysis indicate that the virion RNAs A and C do not have extensive homology. These data suggest that the large virion RNA A contains the full TCV genome and that the smaller virion RNA C is a dispensible satellite, designated S-TCV.     

Molecular weight estimates of vesicular stomatitis virus ribonucleic acids from virions,defective particles,and infected cells

Summary Ribonucleic acid from vesicular stomatitis virus (VSV) virions (B particles), defective particles (T), and infected cells was examined by polyacrylamide gel electrophoresis, and apparent molecular weights were estimated. B particle RNA of 4.0×10⁶ daltons from Indiana strain L and from Cocal virus was distinguishable from RNA of 4.5×10⁶ daltons from Indiana strain BT-78 and several strains of New Jersey VSV. Abundant T particle RNA of 1.0×10⁶ daltons, was present in one Indiana L stock and was absent in a plaque purified stock. Cocal virus had T particle RNA of 0.7×10⁶ daltons, and one stock of Ogden strain New Jersey VSV had T particle RNA of 1.2×10⁶ daltons. Little or no T particle RNA was detectable in several other VSV stocks. Major RNA components with apparent molecular weights, 2.4, 0.66, and 0.31×10⁶ daltons in cells infected with Indiana strain L were complementary to virion RNA. The size relationships of B particle RNA from Indiana L and New Jersey strains were examined by sucrose density gradient sedimentation of undenatured and of formaldehyde denatured RNA. Sedimentation of undenatured RNA led to a conflict with gel electrophoresis results, inferring New Jersey had a lower molecular weight than Indiana L, but upon denaturation the relation was consistent with electrophoresis results.     

Long-term persistent vesicular stomatitis virus and rabies virus infection of cells in vitro.

BHK 21 carrier cells persistently infected with VSV Indiana for over 2 years have been shedding generally very low levels of mature infectious virus or mature T particles (averaging less than one-hundredth p.f.u./cell/day) yet most cells are producing virus antigens and are resistant to homologous superinfection. However, large amounts of biologically active T particle RNP can be recovered from cytoplasmic extracts of these carrier cells even at times when they are shedding no detectable infectious virus. This recovered cytoplasmic RNP replicates (with helper B virions) to produce mature T particles, interferes strongly after DEAE dextran-facilitated uptake and, together with B virions, allows the establishment of a persistent carrier state in exposed cells. No 'provirus' DNA copies of the VSV RNA genome are detectable (less than 1/40 copy/cell or I copy per 40 cells) in carrier cells after more than 2 years of persistent infection, and all transfection attempts have failed using DNA from these VSV carriers or DNA from carrier cells persistently infected with some other negative strand RNA viruses (measles, mumps, LCM, influenza, rabies). Infectious viruses shed after more than I year from carrier cells originally infected with wild-type B virions are small plaque mutants showing a slight temperature sensitivity. Cured cell populations can be obtained from the long term VSV carrier culture by cloning in the presence or absence of antiviral antibody.     

Fractionation of biologically active and inactive populations of human rhinovirus type 2.

Infectious virions of human rhinovirus type 2 (HRV-2) migrate during electrophoresis to pH 6.4 in a sucrose-stabilized pH gradient. As already reported, acidification of HRV-2 produces two kinds of noninfectious components both of which have lost the smallest virion polypeptide, VP4: One has lost RNA in addition to VP4 and sediments at about 80 S, while the other retains RNA and sediments at 135 S. The 80 S particles migrate to pH 4.5 upon electrophoresis while the 135 S particles migrate to pH 4.2. Natural top component of HRV-2 is also separable into two subpopulations that are isoelectric at pH 6.3 and pH 4.5. Only the particles isoelectric at pH 6.3 react with virion (D) specific serum and attach to HeLa cells. The polypeptide compositions of both the attaching and nonattaching fractions of natural top component are the same, and therefore the differences in activity and isoelectric point may reflect the arrangement of the polypeptides in the capsids. After a preparation of purified infectious virus is focused to its isoelectric point some particles are found at pH 4.5. These particles are relatively noninfectious, yet contain RNA and a full complement of polypeptides, and they sediment at 140 S. They are unable to attach to host cells. These results argue against a direct role of VP4 or RNA in the attachment process and support the view that protein conformation changes occur during the inactivation of HRV-2 virions.     

A host range restriction in infectious pancreatic necrosis virus maps to the large RNA segment and involves virus attachment to the cell surface

Infectious pancreatic necrosis virus (IPNV) strain JV can replicate in both chinook salmon embryo (CHSE-214) cells and fathead minnow (FHM) cells; but another strain (OV) cannot replicate in FHM cells. In addition, a spontaneous host range mutant of JV has been isolated which has the same host range restriction as OV. By genetic reassortment of the two RNA segments in IPNV, we have determined that the gene responsible for the host range phenotype in both OV and the mutant maps to the large RNA segment which encodes the major capsid protein, the internal virion protein, and a nonstructural protein. Attachment of virions to the surface of FHM cells was impaired in OV and in the mutant. We have succeeded in transcapsidating OV RNA into the capsid of JV, thereby facilitating the entry of OV RNA into FHM cells. FHM cells infected with this transcapsidated virus showed abundant OV-specific antigen synthesis, suggesting that the host range block was solely at the level of virus attachment to the cell surface.     

Japanese encephalitis virus replication: A procedure for the selective isolation and characterization of viral RNA species

Summary The viral RNA species synthesized in a porcine kidney cell line, PS(Y-15), by Japanese encephalitis virus (JEV) are described. Virus titers on these cells ranged between 10⁶ to 10⁷ p.f.u./ml at the end of 2 to 3 days incubation at 35° C. Actinomycin D (AD) could not be used to unmask JEV RNA synthesis since it inhibited virus replication at concentrations necessary to substantially reduce host cell RNA synthesis. Treatment of cells with 1 g AD/ml and removal prior to infection permitted good JEV replication, and at the same time strongly suppressed synthesis of 28 S and 18 S cellular ribosomal RNA. The problem of separating viral RNA from non-ribosomal RNA that was still being synthesized by AD pretreated cells was resolved by the isolation of the cytoplasmic membrane fraction of infected cells. RNA extracted from the membranes of infected AD pretreated cells and analyzed for sedimentation characteristics on sucrose gradients has four RNA species not found in uninfected cells.They are: (1) 45 S single stranded RNA believed to be the infectious RNA found in the virion; (2) a 27 S RNA single stranded RNA; (3) a 20 S ribonuclease resistant RNA believed to be double stranded and (4) an 8 S RNA species. The RNA species found in JEV infected cells, except for the 8 S form, have been found in group A arboviruses. The procedure described utilizing AD pretreatment of host cells and the separation of the cellular cytoplasmic fraction may well have value for the study of the biosynthetic events involved in the replication of other animal viruses that are inhibited by AD.This work was supported by the Bureau of Medicine and Surgery, Department of the Navy, Project MR 041.05.01-0006B3GJ.The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the Naval Service at large.Postdoctoral Research Associate, National Research Council, supported by the Bureau of Medicine and Surgery, Department of the Navy.     

Ribonucleic acids of Machupo and Lassa viruses

Summary Sucrose gradient velocity centrifugation, polyacrylamide gel electrophoresis and RNA-RNA hybridization were used to characterize Lassa and Machupo virion RNAs as well as virus-specific RNAs from cells infected with Pichinde and Machupo viruses. Five RNA species: 30–31S, 28S, 22–24S, 18S and 4–6S have been detected in Lassa, Machupo, and Pichinde virion RNAs. Among them 28S, 18S and 4–6S RNAs cosediment and comigrate with respectivelv cell RNAs. RNase resistance analyses suggest the presence of extensive secondary structures and complementary RNAs in Lassa, Machupo, and Pichinde virion RNAs. Annealing with poly(A)-containing RNA from infected cells has revealed that the bulk of minus strands of Machupo virion RNA is located in 22–24S and 28–31S fractions of sucrose gradient. Thus Machupo and Lassa viruses as well as Pichinde virus contain two genomic RNA fragments: large (molecular weight of about 2.2 × 10⁶) and small (molecular weight of about 1.3 × 10⁶).In the cells infected with Pichinde virus and treated with actinomycin D (1.0 µg/ml) synthesis of 18S,22–24S and 30–31S RNAs has been registered. At least 22–24S and 30–31S classes comprise plus and minus strands. In cells infected with Machupo virus in the presence of actinomycin D the synthesis of similar sedimentation classes of RNAs and certain amounts of 28S RNA have been detected.With 4 Figures