Unique morphological alterations of the HTLV-I transformed C8166 cells by infection with HIV-1.

C8166 cells express T lymphocyte markers, a monocyte-specific esterase, taxpolypeptide of HTLV-I. In spite of this transactivator, their HIV-1 yield is low. Their culture conditions were modified, and infected cells were immobilized on a poly-L-lysine sheet under semisolid overlays to study their phenotypic alterations and HIV-1 production by microscopy and electron microscopy. Another lymphoid cultures (MT-4, CEM, CEM-ss, AdCEM) similarly treated were infected with either HIV-1/RF or IIIB. Specificity of HIV-1 was compared to the effects of vesicular stomatitis virus (VSV). Unlike other cultures, HIV-1/RF infected C8166 cells in Eagle s MEM exhibited surface projections resembling hairy leukemia cells, which was followed by balloon degeneration and apoptosis. Immobilized HIV-1 infected cultures formed flat syncytia with several interdigitating dendritic projections. Syncytia shrunk with condensed nuclear material and axon-like filaments characteristic for infected macrophages. VSV induced enlargement and necrotic lysis of all cell types. Early postinfection with HIV-1, electron microscopy revealed irreversible membrane fusion above cell nuclei, and transient fusion between filaments. Transient presence of coated vesicles containing intact HIV-1 particles, Birbeck granule-like structures of Langerhans cells, fibrillar-lamellar structures resembling hairy leukemia or Sézary cells were detected. Late postinfection, high proportion of HIV-1 bud from polarized cytoplasm was empty particle, while that bud and entrapped in cytoplasmic vacuoles contained two or multiple cores in a fused envelope. The effect of early gene products of HIV-1 on HTLV-I and C8166 cells might elicit their latent potentials for monocyte or interdigitating dendritic cells, while in the later phase HTLV-I products might alter HIV-1 virion assembly.

Herpes simplex virus L particles contain spherical membrane-enclosed inclusion vesicles.

The fine structure of light (L) particles of herpes simplex virus type 1 was examined by cryo-electron microscopy and compared to that of virions. The L particles appeared to be spherical entities with significant variation in size, on average smaller in diameter than virions (140 nm compared to 180 nm). The technique confirmed that L particles are composed of an outer envelope, i.e. a bilaminar membrane with protruding glycoprotein spikes, and a uniformly granular tegument, but lack any nucleocapsid. In addition it revealed the presence of one or occasionally more spherical objects, termed inclusion vesicles (IVs), embedded in the tegument of a large proportion of L particles but not observed in virions, suggesting that presence of IVs is unique to the L particles. The IVs vary in size and appear to be composed of a bilaminar membrane without surface projections and filled with material of relatively low electron density, suggesting that the composition of IVs is distinct from that of the envelope and tegument of the L particles.

The influence of cell culture and storage conditions on HIV-1 infectivity and fusogenic activity.

We have previously demonstrated that acidic medium inhibits the replication of HIV-1. The present study was designed to examine the effects of other growth conditions and infection of fibroblasts by coculture with HIV infected lymphoid cells. Several lymphoblastoid cell lines normally grown in RPMI-1640 were grown in Eagle's MEM. These cells supported virus replication to higher titres than did RPMI-1640. Peak viral titres were achieved within 24-48 h after newly infected or chronically infected cells were placed in fresh medium. When virus was stored in liquid medium either frozen or at higher temperatures, virus titres were retained for several months while frozen but decreased upon storage at 4 degrees C or higher. If cells were passaged after trypsinization in Ca(++)-depleted medium, then a decreased susceptibility of cells for HIV-1 by 2 log10 at 24 h post infection was observed. Infectivity of cell-free and cell-associated HIV-1 was measured using syncytium formation, reverse transcriptase activity and p24 antigen. No fusion between HIV-1 infected CD4+ lymphoblasts and CD4- fibroblasts was observed but HIV-1 infected lymphoid cells, even in the absence of syncytium formation, exerted a strong toxic effect on fibroblasts. This study extends previous findings that medium acidity was inhibitory to virus replication and survival. Thus, conditions for study of HIV must be well controlled in buffered medium so that misleading results are not obtained regarding virus multiplication and possibly regarding transmission to and pathogenesis in CD4- cells.

Identification and characterization of a novel non-infectious herpes simplex virus-related particle.

During gradient purification of herpes simplex virus type 1 (HSV-1) two bands of particles were observed: a sharp lower band and a more diffuse upper band. The lower band contained almost exclusively HSV-1 virions (H particles) whereas the upper band consisted of membrane-enclosed particles (L particles). These L particles resembled the virions in appearance, but lacked the viral nucleocapsid and were not infectious. Many polypeptides of the viral envelope and the tegument were common to both types of particles. The H particles had polypeptide profiles typical of HSV virions. The L particles contained at least three phosphoproteins (175K, 92K and 55K) and a further two phosphorylated polypeptides not normally observed in virion profiles which comigrated with the 134K and 60K glycoproteins. This clearly indicates that the novel L particles were not merely virions which had formed without the inclusion of a nucleocapsid or virions which had subsequently lost their nucleocapsid during preparative handling. Thus these novel L particles are genuine products of the infectious processes occurring when HSV-1 replicates.

Viral RNAs synthesized in cells infected with Germiston Bunyavirus.

A rapidly growing strain of Germiston virus was used to study intracellular viral RNA synthesis in BHK cells. The RNAs were separated by electrophoresis into seven bands which fell into three size classes: large (bands L1 and L2), medium (bands M1 and M2), and small (bands S1, S2, and S3). Blot hybridisation established that bands L1, M1, and S1 contained the negative-sense genomic RNAs, while bands L2, M2, S2, and S3 contained positive-sense RNAs complementary to the genomic RNAs within the same size class. After glyoxal treatment the RNAs separated into a large, a medium, and two small bands, indicating that the positive-sense RNAs originally present in bands L2, M2, and S2 are similar in size to their genomic RNAs, while the RNA in S3 is shorter than the small genomic segment. These results suggest that band S2 contains the replicative intermediate RNA and band S3 the messenger RNA of the small genomic segment and also that bands L2 and M2 contain both replicative intermediate and messenger RNAs. Long after virus development had ceased in the infected cells the amounts of RNAs in bands L1, M1, S1, and S2 remained the same, those in bands L2 and M2 were reduced, while only trace amounts of RNAs were observed in band S3, suggesting that the genomic RNAs and the replicative intermediate RNAs form ribonuclease-resistant ribonucleoprotein complexes while the messenger RNAs do not form such complexes. Synthesis of RNA in the infected cells was first evident in bands S3 and M2, after which synthesis was soon observed in all seven bands reaching a maximum rate at the logarithmic phase of growth, suggesting that the pattern of Germiston virus development resembles that of other negative-strand RNA viruses. The presence of defective-interfering particles was indicated by the observation that purified virus preparations contained a minor RNA component originating from the large RNA segment.

Temperature sensitivity of the transcriptase of mutants tsB1 and tsF1 of vesicular stomatitis virus New Jersey is a consequence of mutation affecting polypeptide L.

Two conditional transcriptase-negative mutants of vesicular stomatitis virus (VSV) serotype New Jersey, tsB1 and tsF1, their revertants tsB1/R1 and tsF1/R1 and the wildtype virus were dissociated into pellet, NS and L fractions and, after reconstitution of these in various combinations, the transcriptase activities were assayed in vitro at the permissive (31 degrees C) and restrictive (39 degrees C) temperatures. The pellet fractions contained the virion RNA-polypeptide N complexes, while the NS and L fractions were essentially pure preparations of these polypeptides. The synthesis of RNA by the reconstituted pellet and L fractions was inhibited at 39 degrees C only when the L fractions of tsB1 or tsF1 were used. Addition of the NS fractions to the reconstituted pellet and L fractions did not alter the rates of RNA synthesis. These results demonstrate that polypeptide L is the temperature-sensitive polypeptide of both mutants tsB1 and tsF1 and support previous observations that polypeptide L is the transcriptase itself. The fact that a second mutant of complementation group F, tsF2, is transcriptase-positive but replicase-negative suggests that polypeptide L is involved both in transcription and replication. Intracistronic complementations may account for the observation that the temperature-sensitive mutations affect polypeptide L in complementation groups B and F.

The role of polypeptides L and NS in the transcription process of vesicular stomatitis virus New Jersey using the temperature-sensitive mutant tsE1.

The roles of the L and NS polypeptides in transcription by vesicular stomatitis virus New Jersey were studied using a mutant, tsE1, which contains a temperature-sensitive transcriptase and an altered NS polypeptide, both phenotypic changes being the consequence of the ts mutation. Mutant tsE1, its revertant (tsE1/R1) and the wild-type virus were dissociated into sub-viral fractions and, after reconstitution of these fractions in all combinations, the transcriptase was assayed in vitro at the permissive (31 degrees C) and restrictive (39 degrees C) temperatures. Reconstitution of the pellet fractions (containing polypeptide N complexed with the virion RNA) and the supernatant fractions (containing polypeptides L and NS) restored transcriptase activity at 31 degrees C in all combinations, but at 39 degrees C transcription was observed only in the presence of the supernatant fractions of wild-type and revertant viruses but not in the presence of the supernatant fractions of tsE1. When the pellet fractions and the L fractions were reconstituted, the transcriptase activity was restored in all combinations both at 31 degrees C and 39 degrees C. However, in vitro transcription at 39 degrees C by reconstituted pellet and L fractions was strongly inhibited when the NS fraction of tsE1 was also added, while addition of the NS fractions of wild-type and revertant viruses had no effect. Since only traces of polypeptide NS were present in the L fractions and none in the pellet fractions, the results strongly suggest that polypeptide L is the transcriptase itself while polypeptide NS exerts some control over transcription.

Temperature-sensitive mutants of complementation group E of vesicular stomatitis virus New Jersey serotype possess altered NS polypeptides.

In vesicular stomatitis virus New Jersey serotype polyacrylamide gel electrophoresis was unable to distinguish the polypeptides of the temperature-sensitive (ts) mutants of complementation groups A, B, C, and F from those of the wild-type virus. However, the NS polypeptide of the representative mutant of group E, ts E1, had a significantly greater electrophoretic mobility than that of the wild-type virus NS polypeptide. The electrophoretic mobilities of the NS polypeptides of the three mutants of complementation group E varied, being greatest in the case of ts E1, slightly less for ts E2, and only a little greater than that of wild-type virus NS polypeptide in the case of ts E3. Since the NS polypeptides of the revertant clones ts E1/R1 and ts E3/R1 have mobilities identical to that of wild-type NS polypeptide, the observed altered mobilities of the group E mutants are almost certainly the direct result of the ts mutations in the E locus. The electrophoretic mobilities of the intracellular NS polypeptides of the group E mutants were indistinguishable from those of their virion NS polypeptides. The electrophoretic mobilities of the NS polypeptides of the group E mutants synthesized in vitro using mRNA synthesized in vitro by TNP were identical to those of the NS polypeptides of their purified virions. The NS polypeptides of all three mutants were labeled with (32)P(i) to approximately the same extent as wild-type virus NS polypeptide, indicating that gross differences in phosphorylation of this polypeptide are unlikely to account for the altered mobilities. We propose a model in which the NS polypeptide consists of at least three loops held in this configuration by hydrophobic or ionic forces or both and stabilized by phosphodiester bridges. If a mutation affects one of the amino acids to which the phosphate is covalently linked, the phosphodiester bridge cannot be formed, and, as a result, in the presence of sodium dodecyl sulfate the affected loop opens and thus the NS polypeptide migrates further into the gel. Such a configuration may also explain the multifunctional nature of the NS polypeptide.

Effect of temperature-sensitive mutation on activity of the RNA transcriptase of vesicular stomatitis virus New Jersey.

The virion-associated RNA transcriptase activity of vesicular stomatitis virus New Jersey temperature-sensitive (ts) mutants was assayed in vitro at the permissive (31 degrees C) and restrictive (39 degrees C) temperatures. RNA synthesis at 39 degrees C by the RNA-negative ts A1 and the RNA-positive ts C1 and ts D1 mutants was similar to that of wild-type virus. The RNA-negative ts B1 synthesized only small amounts of RNA in vitro at 39 degrees C. The three mutants of complementation group E were dissimilar in the amounts of RNA they synthesized at 39 degrees C: ts E1 synthesized very little RNA, ts E2 synthesized moderate amounts, and RNA synthesis by ts E3 was not inhibited. The two mutants of group F were also dissimilar, since ts F1 synthesized very little RNA at 39 degrees C, whereas ts F2 synthesized as much RNA as wild-type virus. The revertant clones ts B1/R1, ts E1/R1, and ts F1/R1 synthesized RNA at 39 degrees C in amounts comparable to wild-type virus, indicating that the heat sensitivity of the transcriptase activity of the mutants ts B1, ts E1, and ts F1 was associated with temperature sensitivity. Similar heat sensitivities were observed when transcribing nucleoprotein complexes were used in the assays, showing that the mutated polypeptides were part of the viral core. The heat stability of the mutant ts B1 was similar to that of wild-type virus, and in vitro RNA synthesis was fully restored when the temperature was lowered to 31 degrees C after 30 min of preincubation at 39 degrees C, showing that the inhibition was due to reversible configurational change of the mutated polypeptide. When virions of the mutant ts E1 were heated for 5 h at 39 degrees C, their infectivity and transcriptase activity were as stable as those of the wild-type virus, whereas transcriptase activity became very heat labile after disruption of the viral coat with a neutral detergent. This suggests an interaction between the mutated polypeptide and a coat polypeptide which stabilizes the activity of the transcriptase. The RNA transcriptase activity of the mutant ts F1 was also heat labile, although to a lesser extent than that of ts E1. Thus, the defects in transcriptase activity of groups B, E, and F suggest that all three polypeptides of the virus core, polypeptides L, N, and NS, are involved in the transcription. In addition, we postulate that the mutated gene products of groups E and F are multifunctional, being required both in transcription and replication, and that the gene product of group E may also be involved in some late stage of virus development.

Temperature-dependent host range mutation in vesicular stomatitis virus affecting polypeptide L.

We established previously that the temperature-dependent host range mutant, td CE 3, of vesicular stomatitis virus (VSV) New Jersey possesses temperature-sensitive RNA transcriptase activity. In this paper, we describe dissociation and reconstitution experiments designed to determine which VSV polypeptide is affected by the td CE 3 mutation. Wild-type VSV New Jersey (ts+), the temperature-dependent host range mutant (td CE 3), and the revertant of this mutant (td CE/R1) were used. Transcribing nucleoprotein preparations, isolated from purified virus particles, were treated in the presence of digitonin with either 0.9 M LiCl to produce supernatants containing virtually only the L polypeptide or 2.0 M LiCl to produce ribonucleoprotein pellets containing only the polypeptides N and NS. Supernatant and pellet fractions synthesized either no or only trace amounts of RNA in vitro. Reconstitution of the supernatants with the pellets in all combinations at 31 degrees C restored much of the transcriptase activity of the transcribing nucleoprotein preparations. RNA synthesis occurred at 39 degrees C when the three pellets were reconstituted with wild-type and revertant supernatants. However, supernatant of the mutant td CE 3 reconstituted with any of the three pellets resulted in little or no detectable transcriptase activity at 39 degrees C. This implies that the polypeptide affected by the td CE 3 mutation is the L polypeptide.

Cell-free translation of RNA synthesized in vitro by a transcribing nucleoprotein complex prepared from purified vesicular stomatitis virus.

The RNA species synthesized in vitro by a transcribing nucleoprotein (TNP) complex of vesicular stomatitis virus (VSV) were translated with high efficiency in a fractionated cell-free system derived from reticulocytes. The use of TNP complexes isolated from VSV Indiana, VSV New Jersey, and Chandipura viruses showed that in each case the predominant polypeptides synthesized had electrophoretic mobilities identical to their virion N, NS, and M polypeptides in proportions reflecting those found in infected cells rather than purified virions. A minor polypeptide corresponding to unglycosylated polypeptide G was also observed, but the in vitro synthesis of polypeptide L was not detected. The addition of RNase inhibitor to transcription mixtures markedly increased the rate of RNA synthesis. Furthermore, the messenger activity of the RNA was significantly enhanced. The inclusion of S-adenosyl L-methionine during transcription substantially increased the messenger activity of the product RNA, suggesting a requirement for methylation. Fractionation by oligodeoxythymidylic acid-cellulose chromatography revealed that the RNA required a polyadnylic acid tract for messenger activity.

Virion trascriptase activity differences in host range mutants of vesicular stomatitis virus.

Three types of conditional lethal mutant were isolated from wild-type vesicular stomatitis virus, New Jersey serotype, after mutagenization by 5-fluorouracil: (i) conventional temperature-sensitive (ts) mutants, which form plaques at 31 C but not at 39 C; (ii) conventional host range mutants (hr CE), which grow in BHK but not in secondary chicken embryo cells; and (iii) temperature-dependent host range mutants (td CE), which form plaques both at 31 and 39 C on BHK cells but only at 31 C on chicken embryo cells. To determine whether the mutation in hr CE and td CE mutants affected the virion-associated RNA transcriptase, this enzyme was assayed in vitro at 31 and 39 C, and the results were compared with those obtained for the wild-type virus. The RNA trascriptase activity of hr CE mutants did not appear to be affected by the mutation. The td CE mutants fall into two classes: those that synthesized RNA at 39 C similar to the wild-type virus and those that did not. One mutant of the latter category, td CE 3, had heat-sensitive transcriptase regardless of whether it was grown in BHK or chicken embryo cells. A revertant to the wild-type phenotype isolated from this mutant had regained the ability to synthesize RNA at 39 C. These results strongly suggest that a polypeptide that is either the transcriptase itself or part of the transcriptase complex was made temperature sensitive by the mutation in the second class of td CE mutants. The inhibition of the transcriptase activity of the mutant td CE 3 was fully reversible by lowering the temperature of incubation from 39 to 31 C, and both inhibition and reactivation appeared to be instantaneous.

Isolation of an infectious ribonucleoprotein from vesicular stomatitis virus containing an active RNA transcriptase.

A ribonucleoprotein complex (TNP) containing an active RNA polymerase was isolated from purified vesicular stomatitis virus particles. The TNP sedimented through a sucrose gradient as a single band and appeared under the electron microscope as discrete long filaments in a spiral configuration. TNP contained one major and two minor polypeptides, but not the polypeptides associated with the outer coat of vesicular stomatitis virus. BHK-21 clone 13 cells could be infected with TNP, yielding infectious virus particles.

Effect of rifamycins and related antibiotics on the deoxyribonucleic acid-dependent ribonucleic acid polymerase of vaccinia virus particles.

A number of compounds related to rifampin which act as expected in the Escherichia coli system have been tested for their ability to inhibit the vaccinia particle deoxyribonucleic acid-dependent ribonucleic acid (RNA) polymerase in vitro. Some compounds are inactive even at concentrations of 500 mug/ml, others are able to produce partial inhibition, and others strongly inhibit the enzyme activity at 150 mug/ml or less. The inhibition, where present, operates immediately but appears to be at least partially reversible. At least one compound which is without effect against bacterial RNA polymerase is a potent inhibitor of the viral RNA polymerase. As the enzyme activity of rifampin-resistant mutants of vaccinia virus is inhibited to the same extent as that of the wild type, the observed in vitro effect on vaccinia virus RNA polymerase is not identical with the in vivo effect specifically directed against a vaccinia-specified protein.

16s and 23s components in the ribosomal ribonucleic acid of Rhodopseudomonas spheroides.

1. Ribosomal RNA was extracted from lysates of Rhodopseudomonas spheroides without prior isolation of ribosomes. 2. The composition of this RNA was investigated by using gradient centrifugation, showing that the proportion present as 23s component depended on the method of extraction. 3. The highest proportion of 23s component was found when cells were disrupted by ultrasonic treatment in the presence of ribonuclease inhibitors. 4. The results indicated that a ribonuclease is active in the cell lysate; this could account for the previous report (Lessie, 1965) that ribosomes of Rhodopseudomonas spheroides do not contain a 23s component.