Homologous interference mediated by defective interfering influenza virus derived from a temperature-sensitive mutant of influenza virus.

A temperature-sensitive group II mutant of influenza virus, ts-52, with a presumed defect in viral RNA synthesis, readily produced von Magnus-type defective interfering virus (DI virus) when passed serially (four times) at high multiplicity in MDBK cells. The defective virus (ts-52 DI virus) had a high hemagglutinin and a low infectivity titer, and strongly interfered with the replication of standard infectious viruses (both ts-52 and wild-type ts+) in co-infected cells. Progeny virus particles produced by co-infection of DI virus and infectious virus were also defective and also had low infectivity, high hemagglutinating activity, and a strong interfering property. Infectious viruses ts+ and ts-52 were indistinguishable from ts-52 DI viruses by sucrose velocity or density gradient analysis. Additionally, these viruses all possessed similar morphology. However, when the RNA of DI viruses was analyzed by use of polyacrylamide gels containing 6 M urea, there was a reduction in the amount of large RNA species (V1 to V4), and a number of new smaller RNA species (D1 to D6) with molecular weights ranging from 2.9 X 10(5) to 1.05 X 10(5) appeared. Since these smaller RNA species (D1 to D6) were absent in some clones of infectious viruses, but were consistently associated with DI viruses and increased during undiluted passages and during co-infection of ts-52 with DI virus, they appeared to be a characteristic of DI viruses. Additionally, the UV target size of interfering activity and infectivity of DI virus indicated that interfering activity was 40 times more resistant to UV irradiation than was infectivity, further implicating small RNA molecules in interference. Our data suggest that the loss of infectivity observed among DI viruses may be due to nonspecific loss of a viral RNA segment(s), and the interfering property of DI viruses may be due to interfering RNA segments (DIRNA, D1 to D6). ts-52 DI virus interfered with the replication of standard virus (ts+) at both permissive (34 degrees C) and nonpermissive temperatures. The infectivity of the progeny virus was reduced to 0.2% for ts+ and 0.05% for ts-52 virus without a reduction in hemagglutinin titer. Interference was dependent on the concentration of DI virus. A particle ratio of 1 between DI virus (0.001 PFU/cell) and infectious virus (1.0 PFU/cell) produced a maximal amount of interference. Infectious virus yield was reduced 99.9% without any reduction of the yield of DI viruses Interference was also dependent on the time of addition of DI virus. Interference was most effective within the first 3 h of infection by infectious virus, indicating interference with an early function during viral replication.     

Genetic analysis of influenza virus NS1 gene: a temperature-sensitive mutant shows defective formation of virus particles

To perform a genetic analysis of the influenza A virus NS1 gene, a library of NS1 mutants was generated by PCR-mediated mutagenesis. A collection of mutant ribonucleic proteins containing the nonstructural genes was generated from the library that were rescued for an infectious virus mutant library by a novel RNP competition virus rescue procedure. Several temperature-sensitive (ts) mutant viruses were obtained by screening of the mutant library, and the sequences of their NS1 genes were determined. Most of the mutations identified led to amino acid exchanges and concentrated in the N-terminal region of the protein, but some of them occurred in the C-terminal region. Mutant 11C contained three mutations that led to amino acid exchanges, V18A, R44K, and S195P, all of which were required for the ts phenotype, and was characterized further. Several steps in the infection were slightly altered: (i) M1, M2, NS1, and neuraminidase (NA) accumulations were reduced and (ii) NS1 protein was retained in the nucleus in a temperature-independent manner, but these modifications could not justify the strong virus titer reduction at restrictive temperature. The most dramatic phenotype was the almost complete absence of virus particles in the culture medium, in spite of normal accumulation and nucleocytoplasmic export of virus RNPs. The function affected in the 11C mutant was required late in the infection, as documented by shift-up and shift-down experiments. The defect in virion production was not due to reduced NA expression, as virus yield could not be rescued by exogenous neuraminidase treatment. All together, the analysis of 11C mutant phenotype may indicate a role for NS1 protein in a late event in virus morphogenesis.     

Characterization of ts 16, a temperature-sensitive mutant of vaccinia virus.

We have characterized a temperature-sensitive mutant of vaccinia virus, ts16, originally isolated by Condit et al. (Virology 128:429-443, 1983), at the permissive and nonpermissive temperatures. In a previous study by Kane and Shuman (J. Virol 67:2689-2698, 1993), the mutation of ts16 was mapped to the I7 gene, encoding a 47-kDa protein that shows partial homology to the type II topoisomerase of Saccharomyces cerevisiae. The present study extends previous electron microscopy analysis, showing that in BSC40 cells infected with ts16 at the restrictive temperature (40 degrees C), the assembly was arrested at a stage between the spherical immature virus and the intracellular mature virus (IMV). In thawed cryosections, a number of the major proteins normally found in the IMV were subsequently localized to these mutant particles. By using sucrose density gradients, the ts16 particles were purified from cells infected at the permissive and nonpermissive temperatures. These were analyzed by immunogold labelling and negative-staining electron microscopy, and their protein composition was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While the ts16 virus particles made at the permissive temperature appeared to have a protein pattern identical to that of wild-type IMV, in the mutant particles the three core proteins, p4a, p4b, and 28K, were not proteolytically processed. Consistent with previous data the sucrose-purified particles could be labelled with [3H]thymidine. In addition, anti-DNA labelling on thawed cryosections suggested that most of the mutant particles had taken up DNA. On thawed cryosections of cells infected at the permissive temperature, antibodies to I7 labelled the virus factories, the immature viruses, and the IMVs, while under restrictive conditions these structures were labelled much less, if at all. Surprisingly, however, by Western blotting (immunoblotting) the I7 protein was present in similar amounts in the defective particles and in the IMVs isolated at the permissive temperature. Finally, our data suggest that at the nonpermissive temperature the assembly of ts16 is irreversibly arrested in a stage at which the DNA is in the process of entering but before the particle has completely sealed, as monitored by protease experiments.     

Role of neuraminidase in the morphogenesis of influenza B virus.

When ts7, a temperature-sensitive (ts) mutant of influenza B/Kanagawa/73 virus, infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating activity and enzymatic activity of neuraminidase (NA) were negligible. However, viral protein synthesis and transport of hemadsorption-active hemagglutinin to the cell surface were not affected. When the cell lysate was treated with bacterial NA, hemagglutinating activity was recovered but infectivity was not, even after further treatment with trypsin. It was found that ts7 was defective in transport of NA to the cell surface and formation of virus particles. Analysis of the genomes of non-ts recombinants obtained by crossing ts7 and UV-inactivated B/Lee showed that ts7 had the ts mutation only in RNA segment 6 coding for NA and the glycoprotein NB. Nucleotide sequence analysis of the RNA segment revealed that ts7 had four amino acid changes in the NA molecule but not in NB. We suggest that assembly or budding of influenza B virus requires the presence of NA at the plasma membrane, unlike influenza A virus.     

A temperature-sensitive mutant of Newcastle disease virus defective in intracellular processing of fusion protein.

A temperature-sensitive mutant (ts3) of Newcastle disease virus was physiologically characterized. All major viral structural proteins were synthesized at the permissive (37 degrees C) and nonpermissive (42 degrees C) temperatures, but the fusion (F) glycoprotein was not cleaved at 42 degrees C. In immunocytochemical electron microscopy, the F protein was abundant in the rough endoplasmic reticulum but not in cytoplasmic membrane at 42 degrees C. Noninfectious hemagglutinating virus particles containing all major structural proteins except the F protein were released at 42 degrees C from infected cells. We concluded that the defect in ts3 resides in the intracellular processing of the F protein.     

Identification of hemagglutinin and neuraminidase genes of influenza B virus.

The genome of influenza B viruses was shown by electrophoresis to consist of eight RNA segments. The fifth largest segment coded for hemagglutinin and the sixth coded for neuraminidase.     

Physiology of a temperature-sensitive mutant of Saccharomyces cerevisiae defective in phosphofructokinase activity.

In this paper, we describe a temperature-sensitive mutant of the yeast Saccharomyces cerevisiae (P5-9) which at a restrictive temperature (36 degrees C) shows a pleiotropic defect for transport of many different metabolites. The temperature sensitivity of the mutant is closely related to a reduction in phosphofructokinase activity. This conclusion is based on the following criteria. (i) Both the primary isolate, designated P5-9 (ts [rho-] Ino-), which is an inositol auxotroph and respiration deficient, and a purified derivative, SB4 (ts [rho+] Ino+ ), which is respiration competent and capable of growing in the absence of inositol, are temperature sensitive for growth and ethanol production in media containing glucose or fructose as the sole carbon source. (ii) The respiration-competent derivative SB4 is not temperature sensitive in media containing glycerol or glycerol-pyruvate; glucose inhibits its growth at 36 degrees C in these media. (iii) Assays of glycolytic enzymes in P5-9 and SB4 extracts, prepared from cells incubated for 1 to 2 h at 36 degrees C before harvesting, show selective reduction in phosphofructokinase activity. Analysis of tetrads derived from the cross of mutant and nonmutant haploids indicates that temperature sensitivity for growth is due to a single gene or to two closely linked genes. The biochemical analysis of spores from seven such tetrads revealed a uniform cosegregation of temperature sensitivity for growth and phosphofructokinase activity. Transport and ATP levels were drastically reduced in SB4 cells incubated at 36 degrees C for 1 to 2 h with glucose as the carbon source, but not when glycerol-pyruvate or lactate was the energy source. Therefore, depletion of energy as a result of phosphofructokinase inactivation appears to be the cause of the pleiotropic transport defect observed in the mutant.     

A temperature-sensitive mutant defective in mitosis and cytokinesis

A temperature-sensitive mutant, ts2, of murine leukemic cells (L5178Y) loses its viability gradually at the non-permissive temperature (39 °C) but resumes normal growth when shifted to the permissive temperature (33 °C). At 39 °C the incorporation rate of thymidine is reduced on a per-cell-basis, whereas that of uridine and leucine is unchanged.Autoradiographic study indicates that the fraction of cells which can synthesize DNA decreases steadily with time of incubation at 39 °C. Accumulation of mitotic and multinucleate cells suggests that ts2 cells are defective in both mitosis and cytokinesis. Experiments using synchronized culture demonstrate that the cells shifted up atthe G2, but not at the G1 phase pass through the first mitotic phase normally.     

Characterization of T antigen in cells infected with a temperature-sensitive mutant of simian virus 40.

T antigen induced in African green monkey kidney cells by a temperature-sensitive mutant of simian virus 40, defective in a function required for cell transformation, was characterized. The number of T antigen-positive cells estimated by an immunofluorescent techniques was almost equal at permissive (32.5 C) and restrictive (38.5 C) temperatures, but was slightly reduced when the infected cells were incubated at a higher temperature (40.5 C). However, a complement fixation test indicated that the amount of T antigen induced by the mutant is not significantly different from that induced by wild-type virus at 40.5 C. These results suggest that the T antigen-inducing ability of the mutant is not defective. Two distinct molecular species of T antigen were induced by the mutant at the permissive temperature, whereas only one form was observed at the restrictive temperature. The larger molecular form (14 to 15S) induced by the mutant at the permissive temperature was more heat labile than that induced by wild-type virus, suggesting that the mutated gene product is a component of the larger molecular form.     

Characterization of a temperature-sensitive membrane alteration in chick embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus

The intramembrane particles of freeze-fractured chick embryo fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (TS68) are distributed differently at the permissive and non-permissive temperatures if, and only if, the cells are treated with glycerol before fixation. Few aggregates of intramembrane particles are present in glycerol-treated cells grown at the permissive temperature for transformation (36 degrees C), while numerous large aggregates of particles are present at the non-permissive temperature (41 degrees C). Changes in the distribution of particles after cells are shifted from 36 to 41 degrees C are observed after 20 min, while a temperature shift from 41 to 26 degrees C causes changes in glycerol-induced redistributions after 1 h. The changes observed in temperature shifts from 36 to 41 degrees C and from 41 to 36 degrees D do not require protein synthesis or RNA synthesis.     

The neuraminidase of influenza virus

It is the enzyme neuraminidase, projecting from the surface of influenza virus particles, which allows the virus to leave infected cells and spread in the body. Antibodies which inhibit the enzyme limit the infection, but antigenic variation of the neuraminidase renders it ineffective in a vaccine. This article describes the crystal structure of influenza virus neuraminidase, information about the active site which may lead to development of specific and effective inhibitors of the enzyme, and the structure of epitopes (antigenic determinants) on the neuraminidase. The 3-dimensional structure of the epitopes was obtained by X-ray diffraction methods using crystals of neuraminidase complexed with monoclonal antibody Fab fragments. Escape mutants, selected by growing virus in the presence of monoclonal antibodies to the neuraminidase, possess single amino acid sequence changes. The crystal structure of two mutants showed that the change in structure was restricted to that particular sidechain, but the change in the epitope was sufficient to abolish antibody binding even though it is known in one case that 21 other amino acids on the neuraminidase are in contact with the antibody.     

Characterization of a cleavage mutant of the measles virus fusion protein defective in syncytium formation.

Membrane fusion caused by measles virus (MV) is a function of the fusion (F) protein. This process is essential for penetration into the host cell and subsequent initiation of the virus replicative cycle. The biological activity of the MV F protein is generated by endoproteolytic cleavage of a precursor protein (F0) into a large F1 subunit and a smaller F2 subunit held together by disulfide bonds. The cleavage site consists of a cluster of five basic amino acids (amino acids 108 to 112) within the predicted primary structure of the F protein. To investigate the role of the arginine residue at the carboxy terminus of the F2 subunit (arginine 112), site-directed mutagenesis was used to construct a cleavage mutant of the MV F protein in which this arginine residue was changed to a leucine residue. The mutated F gene, encoding four out of the five basic amino acids at the cleavage site, was inserted into the genome of vaccinia virus. The resulting recombinant virus was used to study expression of the mutant F protein in infected cells. Analysis of the Leu-112 mutant protein made in infected cells demonstrated that this single-amino-acid substitution resulted in a reduced rate of transport of the mutant protein to the cell surface, despite its efficient cleavage to yield F1 and F2 subunits. However, the electrophoretic mobilities of the Leu-112 polypeptides suggested that the protein was cleaved incorrectly. This aberrant cleavage appears to have abolished the ability of the F protein to cause syncytium formation. The data indicate that the arginine 112 residue is critical for the correct proteolytic cleavage that is required for the membrane fusion activity of the MV F protein.     

Steps in maturation of influenza A virus neuraminidase.

We have studied the maturation of the influenza A virus neuraminidase (NA), using monoclonal antibodies (MAbs) with different conformational specificities against the head domains of the N8 NA. The results obtained with radioimmunoprecipitation, together with previously published information, suggest the following steps in maturation of this molecule. First, the folding of the nascent NA leads to formation of the epitope recognized by MAb N8-10, a step that depends on the formation of intramolecular disulfide bonds. Second, monomers form dimers by an intermolecular disulfide linkage in the stalk, with a t1/2 of 2.5 min. Third, the epitope recognized by MAb N8-82 appears after dimerization, suggesting that oligomeric NAs may undergo conformational change with a t1/2 of 8 min. Finally, a tetramer-specific epitope recognized by MAb N8-4 appears on the NA with a t1/2 of 13 min. Epitope detection by MAb N8-4 was inhibited by tunicamycin treatment, suggesting that glycosylation of this molecule is required for proper tetramerization. Each of these proposed steps occurs in the endoplasmic reticulum of host cells, as demonstrated by treatment of virus-infected cells with brefeldin A or carbonyl cyanide m-chlorophenylhydrazine; subsequently, tetrameric NA is transported to the Golgi apparatus, where oligosaccharide processing is completed. Our findings also provide a possible explanation--lack of a functionally active conformation--for the absence of enzymatic function by NA monomers.     

Recombination between a temperature-sensitive mutant and a deletion mutant of Rous sarcoma virus.

Cells doubly infected with two mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), ts68, which is temperature sensitive for cell transformation (srcts), and a deletion mutant, N8, which is deficient in the envelope glycoprotein (env-), produced a recombinant which carried the defects of both parents. The frequency of formation of such a recombinant was exceptionally high and made up 45 to 55% of the progeny carrying the srcts marker. By contrast, the reciprocal recombinant, which is wild type in transformation (srcts) and contains the subgroup A envelope glycoprotein (envA), was almost undetectable. This remarkable difference in the frequency of the formation of the two possible recombinants suggests that a unique mechanism may be involved in the genetic interaction of the two virus genomes, one of which has a large deletion. When an RNA-dependent DNA polymerase-negative variant of the N8 (N8alpha) was crinants also became deficient in the polymerase. Cells infected by the srctsenv- recombinant were morphologically normal at the nonpermissive temperature (41 degrees C) and susceptible to all subgroups of RSV. The rate by which the wild-type RSV transformed the recombinant-preinfected cells was indistinguishable from that of transformation of uninfected chicken cells by the same wild-type virus. This indicates that no detectable interference exists at postpenetration stages between the preinfected and superinfecting virus genomes and confirms that the expression of the transformed state is dominant over the suppressed state.     

Characterization of influenza virus neuraminidase with hemagglutinin activity and its comparison with that of viral neuraminidase

The neuraminidase associated with the bifunctional protein, hemagglutinin-neuraminidase, of influenza virus has been characterized. The enzyme has a pH optimum of 4.5, does not require Ca2+ and is inactivated (98%) by incubation at 50 degrees C. The enzyme has a Km of 2.00 X 10(-3) M and 0.06 X 10(-3) M with the substrates 2-(3-methoxyphenyl)-N-acetylneuraminic acid and fetuin, respectively. The Ki is 400 X 10(-6) with the inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. The incorporation of labeled cysteine, valine and leucine in the hemagglutinin-neuraminidase protein is different from that of viral neuraminidase. A comparison of the properties of the neuraminidase associated with protein hemagglutinin-neuraminidase with that of viral neuraminidase or sialidase showed that the former is biochemically different and an antigenically distinct enzyme. The unique feature of the new enzyme is that it has the hemagglutinin activity as well. The two biological activities could not be separated from each other in all systems used. Apparently, protein hemagglutinin-neuraminidase is genetically transferable and it is detectable in a laboratory recombinant virus E-2971 (H3 Aichi X N7). These results suggest that protein hemagglutinin-neuraminidase is a unique surface protein of the influenza virus A/Aichi/2/68 (H3N2).     

Molecular genetics of herpes simplex virus. VII. Characterization of a temperature-sensitive mutant produced by in vitro mutagenesis and defective in DNA synthesis and accumulation of gamma polypeptides.

We report on the properties of a temperature-sensitive mutant produced by transfection of cells with intact DNA and a specific DNA fragment mutagenized with low levels of hydroxylamine. The plating efficiency of the mutant at 39 degrees C relative to that at 33.5 degrees C was 5 X 10(-6). The pattern of polypeptides produced at the nonpermissive temperature was similar to that seen with wild-type virus in infected cells treated with inhibitory concentrations of phosphonoacetic acid in that alpha and beta polypeptides were produced, whereas most gamma polypeptides were either reduced or absent. Consistently, the mutant did not make viral DNA, although temperature sensitivity of the viral DNA polymerase could not be demonstrated. Marker rescue studies with herpes simplex virus type 2 (HSV-2) DNA mapped the mutant in the L component within map positions 0.385 and 0.402 in the prototype (P) arrangement of the HSV-1 genome. Analysis of the recombinants permitted the mapping of the genes specifying infected cell polypeptides 36, 35, 37, 19.5, 11, 8, 2, 43, and 44, but only the infected cell polypeptide 8 of HSV-2 was consistently made by all recombinants containing demonstrable HSV-2 sequences. Marker rescue studies with cloned HSV-1 DNA fragments mapped the temperature-sensitive lesion within less than 10(3) base pairs between 0.383 and 0.388 map units. Translation of the RNA hybridizing to cloned HSV-1 DNA, encompassing the smallest region containing the mutation, revealed polypeptide 8 (128,000 molecular weight), which was previously identified as a beta polypeptide with high affinity for viral DNA, and a polypeptide (25,000 molecular weight) not previously identified in lysates of labeled cells.     

Isolation and analysis of a mammalian temperature-sensitive mutant defective in G2 functions

A temperature-sensitive (ts) mutant, designated tsFT210, was isolated from a mouse mammary carcinoma cell line, FM3A. The tsFT210 cells grew normally at 33 degrees C (permissive temperature), but more than 80% of the cells were arrested at the G2 phase at 39 degrees C (non-permissive temperature) as revealed by flow-microfluorimetric analysis. DNA replication and synthesis of other macromolecules by this mutant seemed to be normal at 39 degrees C for at least 10 h. However, in this mutant, hyperphosphorylation of H1 histone from the G2 to M phase, which occurs in the normal cell cycle, could not be detected at the non-permissive temperature. This suggests that a gene product which is temperature-sensitive in tsFT210 cells is necessary for hyperphosphorylation of H1 histone and that this gene product may be related to chromosome condensation.