Analysis of the Ribonucleic Acid of Murine Leukemia Virus

Cells producing the Rauscher strain of murine leukemia virus (MLV) were exposed to (3)H-uridine, and labeled virus was collected at hourly intervals. Ribonucleic acid (RNA) extracted from virions (vRNA) had a characteristic single peak when analyzed by electrophoresis in polyacrylamide-agarose composite gels. Exposure of vRNA to dimethyl sulfoxide, urea, formaldehyde, or heat altered the mobility to a faster moving form (vRNA'). This vRNA' sedimented more slowly than native vRNA in sucrose gradients. Incubation of labeled virions at 37 C resulted in fragmentation of viral RNA which was detectable only after denaturation. Also, large differences in the temperature required for the change from vRNA to vRNA' were seen with alterations in NaCl concentration. These experiments demonstrate that the vRNA of MLV is held in a specific conformation by hydrogen bonds distributed over a large part of the molecule. The possibility that an undefined factor is associated with viral RNA is discussed.     

Electron Microscopic Characterization of the Defectiveness of a Temperature-Sensitive Mutant of Moloney Murine Leukemia Virus Restricted in Assembly

The effect of temperature shiftdown on the assembly of ts3 virions was investigated by both scanning (SEM) and transmission (TEM) electron microscopy. Ts3 is a spontaneous temperature-sensitive mutant of Moloney murine leukemia virus (Mo-MuLV) which previous studies indicated to be defective in assembly or release of the virions. In the present study, both SEM and TEM revealed the following: (i) there were more cell-associated virions in ts3-infected cells grown at the nonpermissive temperature (39 degrees C) than either in cells grown at the permissive temperature (34 degrees C) or in wild-type MuLV-infected cells grown at 39 degrees C; (ii) there were more normal single particles than multiploids (virions with two or more pieces of genomic RNA) in ts3-infected cells grown at the nonpermissive temperature; (iii) there were more multiploids in ts3-infected cells grown at the nonpermissive temperature than either in cells grown at the permissive temperature or in wild-type MuLV-infected cells grown at the nonpermissive temperature; (iv) upon temperature shift from 39 to 34 degrees C, about 90% of the cell-associated virions dissociated from the cell surface. TEM studies also indicated that upon temperature shiftdown, virion assembly rapidly occurred. The above observations suggest that faulty assembly, which results in the production of multiploids, may not be the reason why ts3 virions accumulate on the cell surface at the nonpermissive temperature. The relatively higher proportion of multiploids found in ts3-infected cells grown at 39 degrees C compared with those grown at 34 degrees C may be due to the higher density of budding virions at the cell surface at the nonpermissive temperature, which increases the possibility of two or more particles assembling close to one another. The accumulation of ts3 virions in all stages of assembly at the nonpermissive temperature, together with the fact that rapid assembly and release of ts3 virions occurred on temperature shiftdown, indicates that virion assembly is restricted after it has been initiated. The probable role of altered glycoprotein(s) in restricting virion assembly is discussed.     

Electron Microscopic Studies of Tumor Viruses I. Entry of Murine Leukemia Virus into Mouse Embryo Fibroblasts

Entry of Rauscher leukemia virus into mouse embryo fibroblasts was studied by electron microscopy. The polycation diethylaminoethyl-dextran enhanced viral attachment and subsequent entry. At the site of viral attachment to the cell membrane, three distinct interactions occurred between the viral envelope and cell membrane, namely, (i) dissolution of viral envelopes on the cell membrane, which itself remained unaltered; (ii) simultaneous dissolution of both the envelope and cell membrane, resulting in passage of viral nucleoids directly into the cytoplasm; and (iii) dissolution of the cell membrane with direct penetration of intact enveloped particles into the cytoplasm, followed by intracytoplasmic disruption of the envelope, resulting in release of nucleoids into the cytoplasm. These interactions occurred with both mature and immature C-type particles. At no time was fusion of viral envelopes with the cell membrane observed. The mechanism of these interactions is discussed.     

Characterization of Intracellular Ribonucleic Acid Specific for the Murine Sarcoma-Leukemia Virus Complex

Virus particles were continuously produced by a cell line (78A1) of rat embryo fibroblasts that had been transformed by the murine sarcoma-leukemia virus complex. Since most of the mature virions were found in the extracellular fluid and were not cell-associated, a measurable quantity of viral ribonucleic acid (RNA) could not be extracted from these cells. Cycloheximide, a protein inhibitor, was successfully used to accumulate viral RNA within the cells. This ribonuclease-sensitive RNA, with a sedimentation coefficient of 71S, had the same base composition as the high molecular weight RNA (S(20,w) = 71) isolated from purified virions released by the transformed cells.     

Electron Microscopy of Friend Murine Leukemia Virus in the Mid-Gut of Experimentally Infected Mosquitoes

After 30 and 78 hr, Friend murine leukemia virus (FLV) particles were detected by electron microscopy in the mid-gut lumen of the mosquitoes Aedes aegypti (Linnaeus) and Anopheles stephensi Liston which had fed on leukemia BALB/c mice infected with FLV. Various developmental stages of the virions were observed within and on the surface of ingested blood cells, particularly young erythroblasts, as well as free in the lumen after budding. These preliminary findings indicate that FLV continues to multiply in the mid-gut of these species for at least 3 days despite the action of digestive enzymes. Detailed studies are in progress to determine the fate of FLV in these mosquito species.     

Characterization of Bluetongue Virus Ribonucleic Acid

An improved purification procedure yielded bluetongue virus free from any single-stranded ribonucleic acid (RNA) component. Double-stranded RNA obtained from purified virus or isolated from infected cells was fractionated into 5 components by means of sucrose gradient sedimentation analysis, and into 10 components by electrophoresis on polyacrylamide gels. The size of these components vary from 0.5 x 10(6) to 2.8 x 10(6) daltons, with a total molecular weight estimate of about 1.5 x 10(7) for the viral nucleic acid. The denaturation of the genome and separation of the resulting fragments are also discussed.     

Electron Microscopic Observations of Rabbit Antibodies

Electron micrographs were obtained showing the individual, shadow-cast macromolecules from solutions of purified anti-p-azobenzoate rabbit antibody and of normal γ-globulin. The two materials look alike and consist mainly of asymmetrical rod-like particles about 30 to 40 A in diameter. Lengths are not constant but the weight average is about 250 A for the antibodies and about 200 A for the γ-globulin. The average observed dimensions are reasonably consistent with values deduced from physical-chemical methods, although the shape is more nearly that of a cylindrical rod rather than the ellipsoid employed in hydrodynamical theory. Mixtures of antibody and specific dihaptenic dye were examined in attempts to establish the mode of the specific aggregation. At the high dilutions necessary for electron microscopy (0.1 mg./ml.), the effect of the dye was small and tended to be masked by non-specific aggregation on drying. The evidence suggests that under these conditions the specific reaction involves an end-to-end aggregation of the elementary particles to produce a weight average length about twice that of the pure antibody.     

粘质沙雷氏菌的电镜观察

用扫描电镜和透射电镜对粘质沙雷氏菌(Serratia marcescens)的临界点干燥标本和负染标本作了观察,均可见到细胞表面常有一至二个直径为0.12～0.24μm的颗粒。在超薄切片中颗粒则有两种不同的结构:一种是外膜泡(Outer membrane vesicle);另一种是致密体(Dense body)。致密体可能是一种分泌性颗粒,它既不是内部的贮存物,也不似外部进来的异物。它们看来形成于细胞质内后分泌到细胞外。在细菌中这是一种罕见现象。有关致密体的化学性质和功能尚不清楚。文中指出粘质沙雷氏菌的纲胞表面也具有茂密的菌毛(Pili)。     

Electron Microscopic Observations on the Taste Buds of the Rabbit

An examination of the fine structure of the taste buds in the rabbit was undertaken. Gustatory epithelium was fixed in OsO₄ or 1 per cent KMnO₄ solution, containing polyvinylpyrrolidone (PVP). Thick sections were examined in the phase microscope and contiguous sections prepared for the electron microscope. The bud contains two types of cells, gustatory receptors and sustentacular cells. The receptors are characterized by a dark nucleus and densely granular cytoplasm. The apical processes bear numerous microvilli which extend into the taste pore. Imbedded between the microvilli there is a dense substance, which is also present in the apical cytoplasm of the receptors. The sustentacular cells contain a large pale nucleus and less dense cytoplasm. Their basal surfaces rest upon a basement membrane. The subepithelial nerve plexuses comprise the fibers which innervate the gustatory receptors. The nerve fibers vary in diameter from 500 A to 0.3 µ, and are ensheathed by Schwann cells. The intragemmal fibers enter the taste bud between adjacent cells, and are ensheathed by the plasma membranes of the supporting cell until they synapse upon the gustatory cell. The synaptic terminals contain synaptic vesicles, which at this junction reside in the postsynaptic element. This observation is discussed with reference to synapses described elsewhere in the nervous system.     

Characterization of Ribonucleic Acid from Visna Virus

A single-stranded ribonucleic acid(s) has been isolated from purified virions of visna virus. It consists of two major components, namely 63S and "4S," under the conditions employed for ribonucleic acid (RNA) extraction. The 63S component can be converted to subunits by heat and dimethylsulfoxide treatments. Analyses by base composition indicate that the "4S" RNA isolated from visna virus is not a random breakdown product of the 63S component as a result of extraction, nor is it randomly derived from cellular RNA.     

Suppression of Murine Retrovirus Polypeptide Termination: Effect of Amber Suppressor tRNA on the Cell-Free Translation of Rauscher Murine Leukemia Virus, Moloney Murine Leukemia Virus, and Moloney Murine Sarcoma Virus 124 RNA

The effect of suppressor tRNA's on the cell-free translation of several leukemia and sarcoma virus RNAs was examined. Yeast amber suppressor tRNA (amber tRNA) enhanced the synthesis of the Rauscher murine leukemia virus and clone 1 Moloney murine leukemia virus Pr200^gag-pol polypeptides by 10- to 45-fold, but at the same time depressed the synthesis of Rauscher murine leukemia virus Pr65^gag and Moloney murine leukemia virus Pr63^gag. Under suppressor-minus conditions, Moloney murine leukemia virus Pr70^gag was present as a closely spaced doublet. Amber tRNA stimulated the synthesis of the “upper” Moloney murine leukemia virus Pr70^gag polypeptide. Yeast ochre suppressor tRNA appeared to be ineffective. Quantitative analyses of the kinetics of viral precursor polypeptide accumulation in the presence of amber tRNA showed that during linear protein synthesis, the increase in accumulated Moloney murine leukemia virus Pr200^gag-pol coincided closely with the molar loss of Pr63^gag. Enhancement of Pr200^gag-pol and Pr70^gag by amber tRNA persisted in the presence of pactamycin, a drug which blocks the initiation of protein synthesis, thus arguing for the addition of amino acids to the C terminus of Pr63^gag as the mechanism behind the amber tRNA effect. Moloney murine sarcoma virus 124 30S RNA was translated into four major polypeptides, Pr63^gag, P42, P38, and P23. In the presence of amber tRNA, a new polypeptide, Pr67^gag, appeared, whereas Pr63^gag synthesis was decreased. Quantitative estimates indicated that for every 1 mol of Pr67^gag which appeared, 1 mol of Pr63^gag was lost.     

Virus Interference by Cellular Double-Stranded Ribonucleic Acid

Ribonuclease-resistant ribonucleic acid (RNA) was isolated from uridine-labeled cultures of rabbit kidney, chicken embryo, and HeLa cells. This RNA, regardless of its source, was found to induce interference with virus growth in either rabbit kidney or chicken embryo cultures. Nuclease-treated cellular nucleic acids exhibited interference-inducing activity which eluted with a small fraction of RNA in the exclusion volume of a 6% agarose gel column. Besides resistance to ribonucleases, the interference inducer and RNA isolated from partially digested nucleic acids have in common two properties of double-stranded RNA: (i) similar sharp melting profiles were obtained for inducer and ribonuclease-resistant RNA, with T(m) dependent on NaCl concentration; (ii) ribonuclease-resistant inducer and RNA banded together in Cs(2)SO(4) density gradients at a density characteristic of known double-stranded RNA. After melting at low ionic strength, the labeled RNA shifted to a higher density and its capacity to inhibit virus replication was lost. Velocity sedimentation analysis of the cellular ribonuclease-resistant RNA indicated that the majority sedimented between 7 and 11S, but only RNA sedimenting at >==8 to 20S had a high specific activity of interference induction. Without prior ribonuclease treatment, the ribonuclease-resistant RNA can be precipitated with 2 m LiCl and thus appears to exist in purified cellular nucleic acids as part of molecular complexes with both single- and double-stranded regions of RNA. The biosynthesis of cellular double-stranded RNA is inhibited by actinomycin D.     

Nucleotide Composition of the Ribonucleic Acid of Rabies Virus

The nucleotide composition of the ribonucleic acid of three strains of rabies virus was determined and found to be similar to that of vesicular stomatitis virus.     

Cellular Origin of a Mouse Leukemia Viral Ribonucleic Acid

Mouse erythroblastosis virus, a member of the mouse leukemia virus group, was obtained from chronically infected C(3)H mouse embryo cells and purified on sucrose gradients. The ribonucleic acid (RNA) extracted from ribonuclease-treated virus consisted of a rapidly sedimenting (72S) species and a more slowly sedimenting component (4 to 30S). The 72S RNA did not contain base sequences homologous to deoxyribonucleic acid (DNA) from infected cells as determined by hybridization studies. In contrast, the slowly sedimenting RNA enclosed within the virus had base sequences homologous to DNA from infected and uninfected C(3)H mouse embryo cells.     

Electron Microscope Study of Ribonucleic Acid of Myxoviruses

Intact ribonucleic acid (RNA) molecules in an extended form were extracted from purified influenza virus and observed in the electron microscope. For this study, the RNA extraction procedure and the Kleinschmidt protein monolayer technique were modified. The mean lengths of RNA from X7, X7-F1, and WSN strains of influenza virus were found to be 2.69, 2.55, and 2.37 mum, respectively. From these measurements, the corresponding estimated molecular weights would be 2.9, 2.8, and 2.5 x 10(6) daltons. X7 and WSN RNA preparations were exposed to pH 3 to disrupt intact molecules. Histograms of length measurements showed five peaks, which were interpreted to represent the five pieces of RNA reported to exist in the influenza virion. X7 RNA appeared to be more stable than WSN RNA when stored at 4 C. The profiles of histograms of incomplete virus RNA suggest that the high molecular-weight component is missing. In preliminary experiments on Newcastle disease virus RNA, molecules of various lengths were observed.     

Single Amino Acid Insertion in Loop 4 Confers Amphotropic Murine Leukemia Virus Receptor Function upon Murine Pit1

Pit1 is the human receptor for gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B), while the related human protein Pit2 is a receptor for amphotropic murine leukemia virus (A-MuLV). The A-MuLV-related isolate 10A1 can utilize both Pit1 and Pit2 as receptors. A stretch of amino acids named region A was identified in Pit1 (residues 550 to 558 in loop 4) as critical for GALV and FeLV-B receptor function. We have here investigated the role of region A in A-MuLV and 10A1 entry. Insertion of a single amino acid in region A of mouse Pit1 resulted in a functional A-MuLV receptor, showing that region A plays a role in A-MuLV infection. Moreover, the downregulation of 10A1 receptor function by changes in region A of human Pit1 indicates that this region is also involved in 10A1 entry. Therefore, region A seems to play a role in infection by all viruses utilizing Pit1 and/or Pit2 as receptors.     

Interferon Action on Parental Semliki Forest Virus Ribonucleic Acid

Actinomycin D-treated chick fibroblasts were infected with purified (32)P-labeled Semliki forest virus, and ribonucleic acid (RNA) was extracted after 1 or 2 hr. Within 1 hr, viral RNA forms sedimenting in sucrose gradients at 42S, 30S, and 16S were present. The 42S form corresponded to the RNA of the virion. The 16S form appeared to be a double-stranded template for the formation of new viral RNA, since nascent RNA was associated with it and the molecule could be heat-denatured and subsequently reannealed by slow cooling. Interferon treatment before infection, or puromycin (50 mug/ml) or cycloheximide (200 mug/ml) added at the time of virus infection, had no effect on the formation of the 30S RNA but inhibited the production of the 16S form. Several findings made it unlikely that these results were due to breakdown of parental RNA and reincorporation of (32)P into progeny structures. The results suggested that the mechanism of interferon action involves inhibition of protein synthesis by parental viral RNA, since a specific viral RNA polymerase had previously been demonstrated to be necessary for production of 16S RNA. No protein synthesis appears necessary for formation of 30S RNA from parental virus RNA.     

Formation of Viral Ribonucleic Acid and Virus in Cells that are Permissive or Nonpermissive for Murine Encephalomyelitis Virus (GDVII)

GDVII virus growth in BHK-21 cells, a permissive host for the virus, resembled productive infections with other picornaviruses. Virus yields ranged from 100 to 600 plaque-forming units (PFU)/cell. Virus replication in HeLa cells, a nonpermissive host for GDVII virus, was characterized by virus yields of only 0.1 to 5 PFU/cell. Similar low yields of virus have been obtained from HeLa cells at all multiplicities of input up to 6,000 per cell. The progeny particles from HeLa cells were, like the infecting particles, restricted in the HeLa cell host. Despite the great difference in final yields of virus from BHK-21 and HeLa cells, the times when maximal yields were reached were similar. GDVII virus stock grown in BHK-21 cells was designated HeLa(-). A variant of GDVII virus which is capable of extensive growth in HeLa cells was obtained. This variant, designated HeLa(+) GDVII virus, was passaged serially in HeLa cells. Virus yields of 50 to 150 infective virus particles per cell were obtained from infection of HeLa cells with HeLa(+) GDVII virus. The major species of HeLa(+) virus-specific ribonucleic acid (RNA) produced was single stranded and sedimented with an S value of 35S. The rate of accumulation of HeLa(+) virus-specific RNA in HeLa cell cultures was about four times that of HeLa(-) RNA. The amount of virus-specific HeLa(+) RNA formed in HeLa cells was several-fold greater than that of HeLa(-) RNA. With HeLa(-) parent GDVII virus undergoing productive replication in BHK-21 cells or abortive replication in HeLa cells, the major species of virus-specific RNA produced was single stranded and sedimented with an approximate S value of 35S. The amount of HeLa(-) virus-specific RNA extracted from BHK-21 cells was several-fold greater than the amount obtained from HeLa cells.