Characterization of a temperature-sensitive, hexon transport mutant of type 5 adenovirus

Infection of KB cells at 39.5 degrees C with H5ts147, a temperature-sensitive (ts) mutant of type 5 adenovirus, resulted in the cytoplasmic accumulation of hexon antigen; all other virion proteins measured, however, were normally transported into the nucleus. Immunofluorescence techniques were used to study the intracellular location of viral proteins. Genetic studies revealed that H5ts147 was the single member of a nonoverlapping complementation group and occupied a unique locus on the adenovirus genetic map, distinct from mutants that failed to produce immunologically reactive hexons at 39.5 degrees C ("hexon-minus" mutants). Sedimentation studies of extracts of H5ts147-infected cells cultured and labeled at 39.5 degrees C revealed the production of 12S hexon capsomers (the native, trimeric structures), which were immunoprecipitable to the same extent as hexons synthesized in wild type (WT)-infected cells. In contrast, only 3.4S polypeptide chains were found in extracts of cells infected with the class of mutants unable to produce immunologically reactive hexon protein at 39.5 degrees C. Hexons synthesized in H5ts147-infected cells at 39.5 degrees C were capable of being assembled into virions, to the same extent as hexons synthesized in WT-infected cells, when the temperature was shifted down to the permissive temperature, 32 degrees C. Infectious virus production was initiated within 2 to 6 h after shift-down to 32 degrees C; de novo protein synthesis was required to allow this increase in viral titer. If ts147-infected cells were shifted up to 39.5 degrees C late in the viral multiplication cycle, viral production was arrested within 1 to 2 h. The kinetics of shutoff was similar to that of a WT-infected culture treated with cycloheximide at the time of shift-up. The P-VI nonvirion polypeptide, the precursor to virion protein VI, was unstable at 39.5 degrees C, whereas the hexon polypeptide was not degraded during the chase. It appears that there is a structural requirement for the transport of hexons into the nucleus more stringent than the acquisition of immunological reactivity and folding into the 12S form.     

Modification of venous stasis thrombosis in the rat by platelet-active drugs and by heparin

Incubation of adenovirus type 2 infected cells at 42 degrees C resulted in an inhibition of assembly of virus particles although all the major viral structural polypeptides and virus-induced cellular polypeptides so far identified were detected by electrophoretic analysis. Selective high salt-acid-urea extraction of low mol. wt. polypeptides revealed the absence of protein VII at 42 degrees C whereas precursor polypeptide P-VII and core protein V were found. Pulse-chase and temperature shift experiments indicated that cleavage of P-VII into VII was a reversible thermosensitive process, requiring de novo protein synthesis after shift-down to 37 degrees C. Virus particles assembled at 37 degrees C after transfer from 42 to 37 degrees C contained both viral DNA and polypeptides pre-labelled during the eclipse phase at 42 degrees C, including core protein VII.     

Comparison of the temperature sensitivity of protein synthesis by cell-free systems from liver of rat and skate (Raja ocellata)

Studies were undertaken to determine the component(s) responsible for the temperature optimum characteristic of the protein-synthesizing system from skate and rat. 1. The macromolecular constituents of rat and skate liver ribosomes are compared. The number of ribosomal proteins is similar in the two species, although most proteins display different electrophoretic mobilities on polyacrylamide gels. The RNAs from the small subunit of skate and rat have similar sedimentation coefficients; however, the RNA from the large subunit of skate ribosomes appeared to be slight smaller than the comparable RNA from the rat. 2. Ribosomes from either rat or skate were capable of supporting poly(U)dependent polyphenylalanine synthesis with soluble factors from either species. 3. Maximal leucine incorporation directed by endogenous mRNA occurred at 35--40 degrees C with post-mitochondrial supernatant from the rat liver and at 20--30 degrees C with that from skate liver. 4. The characteristic temperature sensitivity of protein synthesis was dependent upon the source of cell sap and independent of the source of ribosomes. 5. Elongation factor 1 from both the rat and skate exhibited maximum activity at approx. 30 degrees C. 6. Phenylalanyl-tRNA synthetase from skate liver showed maximum activity at 30 degrees C while that from rat was maximally active at 37 degrees C. The rat enzyme, however, was active at 0--10 degrees C, at which temperature protein synthesis in the reconstructed rat system is virtually absent. 7. The protein-synthesizing capacity of the reconstituted system at various temperatures was closely correlated with the activity of Elongation factor 2 (translocase). Elongation factor 2 from rat liver displayed an optimum at 30 degrees C and lost all activity below 10 degrees C, while this same factor from skate liver showed an optimum at 20 degrees C and significant activity below 10 degrees C. At this low temperature the reconstituted skate liver system continued to exhibit the ability to synthesize protein. These studies suggest that Elongation factor 2 is the component responsible for determining the temperature at which the protein-synthesizing system displays its characteristic maximum activity.     

Biochemical nature and cellular distribution of the paired immunoglobulin-like receptors, PIR-A and PIR-B

Feline immunodeficiency virus (FIV) multiplication is totally blocked by incubation of infected cells at 41 degrees. This inhibition does not take place with a thermoresistant strain of FIV, designated m41, indicating the role played by the viral genome in temperature sensitivity. We have investigated the steps in the life cycle of wild-type FIV that are thermosensitive and found that they depend on the host cells infected. In CrFK cells, FIV replication was inhibited after the penetration step at 41 degrees. Synthesis of viral RNA and DNA was barely detectable and no viral antigen appeared in the extracellular medium. Nevertheless, viral multiplication resumed on incubation at 37 degrees, suggesting a state of latency at the elevated temperature. In peripheral blood mononuclear cells (PBMCs), the FIV cycle was inhibited at 41 degrees after the synthesis of viral DNA. Several viral mRNAs failed to appear as fully spliced products and no viral antigen was found in the extracellular medium. As in CrFK cells, viral multiplication occurred in PBMCs after a shift to the permissive temperature. These results suggest that at least two steps in the viral life cycle are sensitive to 41 degrees and that two different viral functions of the thermoresistant mutant m41 are modified to overcome temperature sensitivity.     

Effects of Ca(2+)-channel drugs on K(+)-induced respiration in skeletal muscles

The immunogenicity and pathogenicity of a strain of respiratory syncytial (RS) virus modified by sequential induction of three temperature-sensitive (ts) mutations have been evaluated by intranasal administration to 22 adult volunteers. This modified virus, a triple ts mutant designated ts1C, was derived from a double mutant ts1B evaluated in a previous trial. The original isolate (strain RSS-2) and all its derivatives were propagated throughout in human diploid cells in a specially assigned laboratory. The triple mutant ts1C is unable to multiply in MRC-5 cells at 37 degrees C and above. Following nasal administration of ts1C, immune responses were observed in volunteers with low pre-existing neutralizing antibody titres. The ability of mutant ts1C to induce upper respiratory tract disease in adults was greatly diminished in comparison with the non-ts wild-type virus, but not markedly more so than a previously tested double ts mutant (ts1B) which replicates at 37 degrees C. Mutant ts1C, however, may have greater potential as a live vaccine in view of its inherently greater genetic stability.     

Increased Survival and Inhibition of Mammary Tumors in Hypothyroid Mice

The polytene cells of Drosophila melanogaster larvae are ideally suited for the study of gene information flow during differentiation, in that gene derepression is visualized in the form of chromosomal modifications called puffs. One special group of temperature-sensitive puffs can be experimentally induced within 5 min after transfer of salivary glands from 24-37 degrees C. Glands at 37 degrees C also begin synthesizing several new polypeptides which are of higher molecular weight than the prominent polypeptides being synthesized in glands at 24 degrees C [1]. The present study demonstrates that there is also a shift toward the utilization of heavier or more rapidly sedimenting polyribosomes for protein synthesis in glands transferred from 24-37 degrees C. As a result of such studies it is reasonable to assume that the polyribosome redistribution after a heat shock results from the translation of larger mRNA molecules synthesized by the temperature-sensitive loci. Further studies are suggested to describe the role of heat-induced gene activity in maintaining homeostasis, as well as the manner by which such activity is induced, in cells subjected to dramatic temperature fluctuations.     

Herpes simplex virus type 1 alkaline nuclease is required for efficient processing of viral DNA replication intermediates

Mutations in the alkaline nuclease gene of herpes simplex type 1 (HSV-1) (nuc mutations) induce almost wild-type levels of viral DNA; however, mutant viral yields are 0.1 to 1% of wild-type yields (L. Shao, L. Rapp, and S. Weller, Virology 195:146-162, 1993; R. Martinez, L. Shao, J.C. Bronstein, P.C. Weber, and S. Weller, Virology 215:152-164, 1996). nuc mutants are defective in one or more stages of genome maturation and appear to package DNA into aberrant or defective capsids which fail to egress from the nucleus of infected cells. In this study, we used pulsed-field gel electrophoresis to test the hypothesis that the defects in nuc mutants are due to the failure of the newly replicated viral DNA to be processed properly during DNA replication and/or recombination. Replicative intermediates of HSV-1 DNA from both wild-type- and mutant-infected cells remain in the wells of pulsed-field gels, while free linear monomers are readily resolved. Digestion of this well DNA with restriction enzymes that cleave once in the viral genome releases discrete monomer DNA from wild-type virus-infected cells but not from nuc mutant-infected cells. We conclude that both wild-type and mutant DNAs exist in a complex, nonlinear form (possibly branched) during replication. The fact that discrete monomer-length DNA cannot be released from nuc DNA by a single-cutting enzyme suggests that this DNA is more branched than DNA which accumulates in cells infected with wild-type virus. The well DNA from cells infected with wild-type and nuc mutants contains XbaI fragments which result from genomic inversions, indicating that alkaline nuclease is not required for mediating recombination events within HSV DNA. Furthermore, nuc mutants are able to carry out DNA replication-mediated homologous recombination events between inverted repeats on plasmids as evaluated by using a quantitative transient recombination assay. Well DNA from both wild-type- and mutant-infected cells contains free U(L) termini but not free U(S) termini. Various models to explain the structure of replicating DNA are considered.     

Adenovirus transcription. IV. Synthesis of viral-specific RNA in human cells infected with temperature-sensitive mutants of adenovirus 5

Cytoplasmic RNA sequences produced in HeLa cells infected with the adeno-virus 5 temperature-sensitive mutants ts1, ts2, ts9, ts17, ts18, ts19, ts20, ts22, ts49, ts36, and ts125 were characterized by hybridization to DNA probes generated by strand separation of restriction endonuclease fragments of adenovirus 5 DNA. Two "early' mutants defective in DNA synthesis, ts125 and ts36, fail to make wild-type levels of all previously reported classes of late RNA at the nonpermissive temperature. At 40.5 degrees C, both ts125 and ts36 synthesize a wild-type complement of early cytoplasmic RNA 16 h after infection. Under these conditions, no "late' cytoplasmic RNA sequences were observed. Similarly, nuclear RNA present in these cells resembled early cytoplasmic RNA rather than late nuclear RNA. All the late adenovirus 5 temperature-sensitive mutants synthesized normal wild-type levels of late cytoplasmic RNA at the nonpermissive temperature, except ts2, which appears to overproduce certain cytoplasmic species.     

Characterization of a temperature-sensitive yeast vacuolar ATPase mutant with defects in actin distribution and bud morphology

The 27-kDa E subunit, encoded by the VMA4 gene, is a peripheral membrane subunit of the yeast vacuolar H+-ATPase. We have randomly mutagenized the VMA4 gene in order to examine the structure and function of the 27-kDa subunit. Cells lacking a functional VMA4 gene are unable to grow at pH > 7 or in elevated concentrations of CaCl2. Plasmid-borne, mutagenized vma4 genes were screened for failure to complement these phenotypes. Mutants producing Vma4 proteins detectable by immunoblot were selected; one (vma4-1(ts)) is temperature conditional, exhibiting the Vma- phenotype only at elevated temperature (37 degreesC). Sequencing revealed that a single point mutation, D145G, was responsible for the phenotypes of the vma4-1(ts) allele. The unassembled 27-kDa subunit made in the vma4-1(ts) cells is rapidly degraded, particularly at 37 degreesC, but can be protected from degradation by prior assembly into the V-ATPase complex. In purified vacuolar vesicles from the mutant cells, the peripheral subunits are localized to the vacuolar membrane at decreased levels and a comparably decreased level of ATPase activity (14% of the activity in wild-type vesicles) is observed. When vma4-1(ts) mutant cells are shifted to pH 7.5 medium at 37 degrees C, the cells become enlarged and exhibit multiple large buds, elongated buds, and other abnormal morphologies, together with delocalization of actin and chitin, within 4 h. These phenotypes suggest connections between the vacuolar ATPase, bud morphology, and cytokinesis that had not been recognized previously.     

Role of early and late replication events in induction of apoptosis by baculoviruses

Autographa californica nuclear polyhedrosis virus (AcMNPV) mutants that lack the apoptotic suppressor gene p35 cause apoptosis in Spodoptera frugiperda SF21 cells. To identify a viral signal(s) that induces programmed cell death, we first defined the timing of apoptotic events during infection. Activation of a P35-inhibitable caspase, intracellular fragmentation of host and AcMNPV DNA, and cell membrane blebbing coincided with the initiation of viral DNA synthesis between 9 and 12 h after infection and thus suggested that apoptotic signaling begins at or before this time. Virus entry was required since binding of budded virus to host cell receptors alone was insufficient to induce apoptosis. To therefore determine the contribution of early and late replication events to apoptotic signaling, we used the AcMNPV mutant ts8 with a temperature-sensitive lesion in the putative helicase gene p143. At the nonpermissive temperature at which viral DNA synthesis was conditionally blocked, ts8 caused extensive apoptosis of the SF21 cell line p3576D, which dominantly interferes with anti-apoptotic function of viral P35. Confirming that apoptosis can be induced in the absence of normal viral DNA synthesis, parental SF21 cells also underwent apoptosis when infected with a ts8 p35 deletion mutant at the nonpermissive temperature. However, maximum levels of ts8 p35 deletion mutant-induced apoptosis required a temperature-sensitive event(s) that included the initiation of viral DNA synthesis. Collectively, these data suggested that baculovirus-induced apoptosis can be triggered by distinct early (pre-DNA synthesis) and late replicative events, including viral DNA synthesis or late gene expression.     

Activation of homologous complement via the alternative complement pathway by quail cells transformed by Rous sarcoma virus

After incubation of the cells with fresh quail serum, deposition of the third component of complement (C3) was demonstrated on the cell surface of various quail cell lines transformed by Rous sarcoma virus (RSV) as well as on that of primary quail embryo (QE) cells transformed by RSV. The C3 deposition occurred irrespective of virus production. On the other hand, the C-3 deposition was not observed on two quail cell lines transformed by a chemical carcinogen, QE cells infected with avian leukosis virus or normal QE cells. Moreover, QE cells infected with a temperature-sensitive mutant of RSV activated the complement at 37 degrees C but not at 41 degrees C. Since the progeny virus was generated even at 41 degrees C, viral molecules on the cell surface may not play an essential role for the activation. The activation of complement was blocked by EDTA but not by EGTA-Mg++. Therefore, the complement activation on the transformed cells appears to be mediated via the alternative complement pathway (ACP). Similar results were obtained with the complement consumption test; the residual cytolytic activity of fresh quail serum via ACP was markedly reduced by pre-incubation of the serum with transformed cells.     

The S/M checkpoint at 37 degrees C and the recovery of viability of the mutant poldeltats3 require the crb2+/rhp9+ gene in fission yeast

We have isolated a mutant in fission yeast, in which mitosis is uncoupled from completion of DNA replication when DNA synthesis is impaired by a thermosensitive mutation in the gene encoding the catalytic subunit of DNA polymerase delta. By functional complementation, we cloned the wild-type gene and identified it as the recently cloned checkpoint gene crb2+/rhp9+. This gene has been implicated in the DNA damage checkpoint and acts in the Chk1 pathway. Unlike the deleted strain dcrb2, cells bearing the crb2-1 allele were not affected in the DNA repair checkpoint after UV or MMS treatment at 30 degrees C, but were defective in this checkpoint function when treated with MMS at 37 degrees C. We analysed the involvement of Crb2 in the S/M checkpoint by blocking DNA replication with hydroxyurea, by using S phase cdc mutants, or by overexpression of the mutant PCNA L68S. Both crb2 mutants were unable to maintain the S/M checkpoint at 37 degrees C. Furthermore, the crb2+ gene was required, together with the cds1+ gene, for the S/M checkpoint at 30 degrees C. Finally, both the crb2 deletion and the crb2-1 allele induced a rapid death phenotype in the poldeltats3 background at both 30 degrees C and 37 degrees C. The rapid death phenotype was independent of the checkpoint functions.     

The early region 1B 55-kilodalton oncoprotein of adenovirus relieves growth restrictions imposed on viral replication by the cell cycle

The E1B 55-kDa oncoprotein of adenovirus enables the virus to overcome restrictions imposed on viral replication by the cell cycle. Approximately 20% of HeLa cells infected with an E1B 55-kDa mutant adenovirus produced virus when evaluated by electron microscopy or by assays for infectious centers. By contrast, all HeLa cells infected with a wild-type adenovirus produced virus. The yield of E1B mutant virus from randomly cycling HeLa cells correlated with the fraction of cells in S phase at the time of infection. In synchronously growing HeLa cells, approximately 75% of the cells infected during S phase with the E1B mutant virus produced virus, whereas only 10% of the cells infected during G1 produced virus. The yield of E1B mutant virus from HeLa cells infected during S phase was sevenfold greater than that of cells infected during G1 and threefold greater than that of cells infected during asynchronous growth. Cells infected during S phase with the E1B mutant virus exhibited severe cytopathic effects, whereas cells infected with the E1B mutant virus during G1 exhibited a mild cytopathic effect. Viral DNA synthesis appeared independent of the cell cycle because equivalent amounts of viral DNA were synthesized in cells infected with either wild-type or E1B mutant virus. The inability of the E1B mutant virus to replicate was not mediated by the status of p53. These results define a novel property of the large tumor antigen of adenovirus in relieving growth restrictions imposed on viral replication by the cell cycle.     

Effect of mutagens on RNA-containing phages and its infectious RNA. VII. Genetic nature of morphologic mutants of RNA-containing phage MS2

Temperature-sensitive "leaky" mutants of phage MS2 having white dense ring around negative colonies are described. As these mutants are used for quantitative genetic studies, the white ring presents interest. Typical mutant 40 is used as a model for investigation. Light microscopy has shown, that cells from white ring zone have spore-like inclusions, which determine the characteristic structure of surrounding mutant negative colonies. Cytochemical reactions for the presence of glicogen, lipids, volutin, nuclear material and spores were negative. Electrone microscopy of negatively stained samples and ultrathin sections has revealed that cells from white ring zone, unlike phage-infected wild type cells, have two types of electron dense inclusions: 1) crystalline structures formed with great number of closely packed mature phage particles, and 2) large amorphic bodies. Electrone microscope-cytochemical data showed that inclusions remain intact under treatment of ultrathin sections of white zone ring with DNase and perchloric acid, while nuclear material was completely destroyed. Amorphic bodied were completely destructed after the treatment with RNase, while nuclear material and crystalline phage aggregated remained unchanged. Therefore, amorphic bodies consist of RNA, which has not been used to form virions. Single cycle of the development of mutant 40 at 37 degrees and 43 degrees C and under the temperature of incubation 37 degrees leads to 43 degrees C and 43 degrees leads to 37 degrees C in the course of intracellular reproduction is investigated. Influence of the phage on growth on infected culture is studied. The data obtained draw to a conclusion that the impaired function belongs to cystron protein of the phage membrane. As certain mutations in this cystrone of RNA-containing phage result in the depression of cystrone RNA polymerase, it is supposed that the formation of RNA containing bodies in infected cells, determining the formation of white rings in NA, together with cristalline aggregates of cells, is a result of mutation damage of cystrone protein of the phage MS2 membrane.     

Further characterization of the complementation group B temperature-sensitive mutant of respiratory syncytial virus

ts-2, a temperature-sensitive and plaque morphology mutant of respiratory syncytial virus and sole representative of complementation group B, was compared with members of the other complementation groups of respiratory syncytial virus (group A [ts-1] and group C [ts-7]). ts-2 was found to be 10- to 1,000-fold more restricted in growth and ability to spread at restrictive temperatures (37, 38, and 39 degrees C) than at the permissive temperature (32 degrees C). In temperature shift-up experiments, the ts defect of ts-1 and other members of complementation group A was found to effect a late function that was required for at least 13 h in the replicative cycle. The ts lesion of ts-7 affected a function early in the replication cycle. In contrast, ts-2 was not temperature sensitive when studied by the shift-up technique. The discrepancy between the ts plaque property and failure to detect temperature sensitivity during the shift-up experiment was resolved when it was shown that ts-2 had a defect in adsorption or penetration or both at the restrictive temperature. Clonal analysis of revertant ts-2 showed a coordinate restoration of ts+ phenotype ans syncytium-forming capacity. It appears that ts-2 has a defect in a protein that is involved in adsorption and/or penetration of virus and is also responsible for cell fusion activity.     

Low molecular weight viral RNAs transcribed by RNA polymerase III during adenovirus 2 infection

Nuclei isolated from human cells productively infected with adenovirus 2 have been shown to synthesize four low molecular weight RNA species which hybridize efficiently to viral DNA. One species corresponds to the 5.5S or VA RNA (Ohe, Weissman, and Cooke, 1969), and is designated V156. The other three species are novel and have been designated V200, V140, V130, since they are approximately 200, 140, and 130 nucleotides in length, respectively. These viral RNAs retain their distinct electrophoretic properties after denaturation with formamide. RNA species with electrophoretic mobilities similar to those of the V200, V156, and V140 RNAs have been found in the cytoplasmic fraction of cells at late times after adenovirus infection. In isolated nuclei, the V200, V156, V140, and V130 RNAs are all synthesized by DNA-dependent RNA polymerase III, since synthesis is sensitive to high but not to low concentrations of alpha-amanitin. The synthesis of these low molecular weight RNAs continues for a prolonged period of time in isolated nuclei, suggesting that reinitiation occurs. Adenovirus 2 DNA fragments obtained by digestion with restriction endonucleases Eco RI and Sma I were used to map the location of the DNA sequences which encode the RNAs. All the low molecular weight RNAs hybridized to a region of the genome between o.18 and 0.38 fractional lengths from the left end of the adenovirus genome, suggesting that the respective DNA sequences are clustered. Other nonviral low molecular weight RNAs are synthesized in nuclei isolated from infected cells. These include the cellular 5S rRNA species which was minitored by its hybridization to purified 5S DNA from Xenopus laevis.