Effect of Host Cell on Distribution of a Lysosomal Enzyme During Virus Infection

The time of appearance of a lysosomal enzyme, beta-glucuronidase, in the medium of cells infected with either measles virus or echovirus 6 varied with the host cell system. Replication and release of virus preceded leakage of beta-glucuronidase from green monkey kidney cells. In contrast, extracellular enzyme appeared before replication and release of virus in human amnion cells. Hydrocortisone depressed enzyme leakage but did not retard replication of measles virus or viral-induced cytopathology. The intracellular distribution of beta-glucuronidase in uninfected and measles virus-infected cells was also studied. Measles virus infection altered the position of particulate-bound beta-glucuronidase in linear sucrose gradients prior to substantial release of this enzyme intra- and extracellularly. At early stages in infection, most of the cell-associated virus banded with particulate-bound enzyme in the middle of the gradient. As infection progressed, separation of measles virus infectivity from enzyme activity occurred, and intracellular virus was recovered near the meniscus of sucrose gradients.     

Functional Significance of Sialidase During Influenza Virus Multiplication: an Electron Microscope Study

Morphological evidence has been obtained by electron microscopy in support of previous findings that one of the most important functions of sialidase is associated with the release of virus from infected host cells. Highly specific antiserum against fowl plague virus enzyme and specific antiserum against X7 recombinant influenza virus enzyme were shown to influence the morphology of cells infected with their homologous virus. In the presence of enzyme antiserum, an accumulation and aggregation of virus particles were evident on the cell surface and in the extracellular space of infected host cells. The aggregation of virus particles was interpreted to result from the inhibition of the release of virus.     

Isolation of a protein kinase induced by herpes simplex virus type 1

We have isolated a new cyclic AMP-independent protein kinase activity induced in HeLa cells by infection with herpes simplex virus type 1. Induction of the enzyme does not occur in cells treated with cycloheximide at the time of infection, or in cells infected with UV-inactivated herpes simplex virus type 1. The amount of enzyme induced in infected cells is dependent upon the multiplicity of infection. An enzyme with identical properties to the appearing in infected HeLa cells is also induced by herpes simplex virus type 1 in BHK cells.     

Photoreactivation of a Cytoplasmic Virus

Ultraviolet light-inactivated frog virus 3 is efficiently photoreactivated by chick embryo cells. A cellular enzyme is presumably responsible for this repair of viral deoxyribonucleic acid, for the phenomenon is insensitive to an inhibitor of protein synthesis and is not seen in mammalian cells that are known to lack photoreactivating enzyme. Since frog virus 3 is a cytoplasmic virus, functionally significant amounts of photoreactivating enzyme are probably present in the cytoplasm of chick embryo cells.     

Induction of deoxypyrimidine kinase activity in human embryonic lung cells infected with varicella-zoster virus.

Deoxypyrimidine kinase (deoxythymidine [TdR] kinase and deoxycytidine kinase) activity was induced in human embryonic lung cells after infection with varicella-zoster virus (VZ virus). Increased enzyme activity was also produced by using cell-associated virus as inoculum instead of cell-free virus. Anti-VZ virus serum inhibited both the appearance of cytopathic effect and the induction of enzyme activity. The induced TdR kinase activity was more thermostable than that induced by herpes simplex virus type 1. Also, the TdR kinase activity of VZ virus-infected cells was inhibited by dTTP less than in mock-infected cells and more than in herpes simplex virus type 1-infected cells.     

Introduction of the herpes simplex virus thymidine kinase gene into mouse cells using virus DNA or transformed cell DNA.

Cells lacking the enzyme thymidine kinase (LMTK- cells) have been transformed to a kinase-positive phenotype using sheared herpes simplex virus (HSV) DNA, and the enzyme found in these transformed cells is HSV-specific. One of the cell lines is able to complement the functional defect found in two temperature-sensitive mutants of HSV 1, and reversion of the cells to a thymidine kinase-negative phenotype results in the loss of this capability. The HSV thymidine kinase gene can also be introduced into LMTK- cells using DNA extracted from transformed cells, and the high efficiency of this procedure suggests that the state of the virus DNA in transformed cells is different from that of DNA in virus particles.     

Ribonucleic Acid Polymerase Induced in Cells Infected with Sendai Virus

A ribonucleic acid (RNA)-dependent RNA polymerase was induced in chick embryo fibroblast cells after infection with Sendai virus (parainfluenza 1 virus). The enzyme was associated with the microsomal fraction of infected cells and reached maximum detectable activity at 18 hr after virus infection. The activity of the enzyme in vitro was dependent on the presence of added magnesium ions and all four nucleoside triphosphates and was not inhibited by actinomycin D. The RNA synthesized by the enzyme in vitro was sensitive to ribonuclease and consisted of a complex mixture of RNA species including 34S, 24S, and 18S components. Similar RNA components were detected in the microsomal fraction of Sendai virus-infected cells by labeling with (3)H-uridine from 17 to 18 hr postinfection in the presence of actinomycin D. Of the RNA synthesized by Sendai virus-induced RNA polymerase in vitro, 98% became insensitive to ribonuclease after annealing with RNA extracted from purified Sendai virus particles.     

Vpr-mediated incorporation of UNG2 into HIV-1 particles is required to modulate the virus mutation rate and for replication in macrophages

Human immunodeficiency virus type 1 is able to infect nondividing cells, such as macrophages, and the viral Vpr protein has been shown to participate in this process. Here, we investigated the impact of the recruitment into virus particles of the nuclear form of uracil DNA glycosylase (UNG2), a cellular DNA repair enzyme, on the virus mutation rate and on replication in macrophages. We demonstrate that the interaction of Vpr with UNG2 led to virion incorporation of a catalytically active enzyme that is directly involved with Vpr in modulating the virus mutation rate. The lack of UNG in virions during virus replication in primary monocyte-derived macrophages further exacerbated virus mutant frequencies to an 18-fold increase compared with the 4-fold increase measured in actively dividing cells. Because the presence of UNG is also critical for efficient infection of macrophages, these observations extend the role of Vpr to another early step of the virus life cycle, e.g. viral DNA synthesis, that is essential for replication of human immunodeficiency virus type 1 in nondividing cells.     

Alkaline DNase activity in cells infected with a temperature-sensitive mutant of herpes simplex virus type 2.

BHK cells infected with the temperature-sensitive mutant ts13 of herpes simplex virus type 2 at a nonpermissive temperature lack the alkaline nuclease activity, which is induced by the mutant at a permissive temperature and by wild-type virus at either temperature. For ts13, enzyme activity could be induced by a temperature shift to permissive conditions, but not in the presence of cycloheximide. After a shift from permissive to nonpermissive conditions in the presence of cycloheximide, the activity was stable in wild-type, but not in mutant-infected, cells. After extensive purification, the wild-type nuclease was fourfold more heat stable in the presence of substrate than was the mutant enzyme. Mixtures of both purified enzymes showed the predicted intermediate stabilities. The results strongly suggest that the enzyme is virus coded and that the mutant possesses a lesion in the structural gene of the enzyme.     

Characterization of a virion protein kinase as a virus-specified enzyme.

Antibodies which completely inhibited the enzymatic activity of the protein kinase associated with virions of frog virus were obtained by immunization of rabbits with the purified enzyme. This inhibition provided a specific probe for the frog virus protein kinase, since this antiserum had no inhibitory effect on a variety of other protein kinases, including the activity of uninfected cells, or the protein kinase associated with vesicular stomatitis virus or vaccinia virus cultivated in the same cell line as frog virus. The frog virus protein kinase was characterized as a virus-specified protein on the basis of the following observations: (a) the virion protein kinase was antigenically distinct from essentially all of the protein kinase expressed in uninfected cells; (b) following infection by frog virus more than a 15-fold increase was detected in the specific activity of intracellular protein kinase and most of this activity was antigenically related to the virion enzyme; (c) when frog virus was grown in cells derived from widely different species, the antigenic and biochemical specificities of the virion protein kinase remained identical; and (d) screening of cells infected with different temperature-sensitive mutants of frog virus indicated that certain viral mutants failed to synthesize this protein kinase when cultivated at the nonpermissive temperature.     

African Swine Fever Virus dUTPase Is a Highly Specific Enzyme Required for Efficient Replication in Swine Macrophages

The African swine fever virus (ASFV) gene E165R, which is homologous to dUTPases, has been characterized. A multiple alignment of dUTPases showed the conservation in ASFV dUTPase of the motifs that define this protein family. A biochemical analysis of the purified recombinant enzyme showed that the virus dUTPase is a trimeric, highly specific enzyme that requires a divalent cation for activity. The enzyme is most probably complexed with Mg(2+), the preferred cation, and has an apparent K(m) for dUTP of 1 microM. Northern and Western blotting, as well as immunofluorescence analyses, indicated that the enzyme is expressed at early and late times of infection and is localized in the cytoplasm of the infected cells. On the other hand, an ASFV dUTPase-deletion mutant (vDeltaE165R) has been obtained. Growth kinetics showed that vDeltaE165R replicates as efficiently as parental virus in Vero cells but only to 10% or less of parental virus in swine macrophages. Our results suggest that the dUTPase activity is dispensable for virus replication in dividing cells but is required for productive infection in nondividing swine macrophages, the natural host cell for the virus. The viral dUTPase may play a role in lowering the dUTP concentration in natural infections to minimize misincorporation of deoxyuridine into the viral DNA and ensure the fidelity of genome replication.     

Induction of a new ribonucleotide reductase after infection of mouse L cells with pseudorabies virus.

The mammalian ribonucleotide reductase consists of two nonidentical subunits, protein M1 and M2. M1 binds nucleoside triphosphate allosteric effectors, whereas M2 contains a tyrosine free radical essential for activity. The activity of ribonucleotide reductase increased 10-fold in extracts of mouse L cells 6 h after infection with pseudorabies virus. The new activity was not influenced by antibodies against subunit M1 of calf thymus ribonucleotide reductase, whereas the reductase activity in uninfected cells was completely neutralized. Furthermore, packed infected cells (but not mock-infected cells) showed an electron paramagnetic resonance spectrum of the tyrosine free radical of subunit M2 of the cellular ribonucleotide reductase. These data given conclusive evidence that on infection, herpesvirus induces a new or modified ribonucleotide reductase. The virus-induced enzyme showed the same sensitivity to inhibition by hydroxyurea as the cellular reductase. The allosteric regulation of the virus enzyme was completely different from the regulation of the cellular reductase. Thus, CDP reduction catalyzed by the virus enzyme showed no requirement for ATP as a positive effector, and no feedback inhibition was observed by dTTP or dATP. The virus reductase did not even bind to a dATP-Sepharose column which bound the cellular enzyme with high affinity.     

Formation of heterosymplasts in human reticular cell cultures]

The cell fusion has been studied in human reticular cell cultures J-96 and J-41 treated with the Sendai virus or with polyethylene glycol 1000 and 6000. The J-96 cells have a high alkaline phosphatase activity, in J-41 cells the enzyme is not detectable. No heterogenous alkaline phosphatase activity was seen in the protoplasm of symplasts 18 hours after virus cell fusion. It has been shown with polyethylene glycol treatment that during the fusion of cells J-96 and J-41 the enzyme activity was spreading over the symplast protoplasm.     

Differential cell membrane labilizing effect of adenovirus type 5 and 12 observed during productive and abortive infection.

Culture fluid of human epitheloid (HEp-2) cells was examined for extracellular lactate dehydrogenase activity as an indicator of cell damage during a 48 h period in which virus replication and changes in cell morphology occurred. Uninfected and adenovirus type 5-infected cells had the same levels of extracellular enzyme activity both before and after the appearance of morphological changes in cells due to virus infection, whereas adenovirus type 12-infected cells showed increased extracellular enzyme activity. Cells infected with either adenovirus type 5 or type 12 had the same total cellular and extracellular lactate dehydrogenase activity. Hydrocortisone, a membrane stabilizing agent, prevented abnormal leakage of lactate dehydrogenase from adenovirus type 12-infected cells, but had no effect on virus replication or total enzyme activity of infected cells. After inoculation of monkey kidney (Vero) cells the yield of progeny adenovirus type 5 virions was greatly reduced and there was no production of adenovirus type 12 virions. The pattern of extracellular lactate dehydrogenase activity of uninfected and adenovirus type 5- and type 12-infected Vero cells was like that with HEp-2 cells. Therefore, production of adenovirus type 12 virions is not necessary for the virus-cell interaction causing cell membrane labilization.     

Virus Replication, Cytopathology, and Lysosomal Enzyme Response of Mitotic and Interphase Hep-2 Cells Infected with Poliovirus

Mitotic Hep-2 cells, selected by the PEL (colloidal silica) density gradient method and held in mitosis with Colcemid, are readily infected by poliovirus type I (Mahoney). They produce and release the same amount of virus as interphase, random-growing cells. In contrast to interphase cells, mitotic cells show no detectable virus-induced cytopathic effect at the light microscopy level and only slight alterations, consisting of small clusters of vacuoles, at the electron microscopy level. Mitotic cells contain the same total amount of lysosomal enzymes per cell as interphase cells, but they display no redistribution of lysosomal enzymes during the virus infection as interphase cells do. This supports the view that lysosomal enzyme redistribution is associated with the cytopathic effect in poliovirus infection but shows that virus synthesis and release is not dependent on either the cytopathic effect or lysosomal enzyme release. The possible reasons for the lack of cytopathic effect in mitotic cells are discussed.     

Vaccinia DNA ligase complements Saccharomyces cerevisiae cdc9, localizes in cytoplasmic factories and affects virulence and virus sensitivity to DNA damaging agents.

The functional compatibility of vaccinia virus DNA ligase with eukaryotic counterparts was demonstrated by its ability to complement Saccharomyces cerevisiae cdc9. The vaccinia DNA ligase is a 63 kDa protein expressed early during infection that is non-essential for virus DNA replication and recombination in cultured cells. This implies complementation by a mammalian DNA ligase, yet no obvious recruitment of host DNA ligase I from the nucleus to the cytoplasm was observed during infection. An antiserum raised against a peptide conserved in eukaryotic DNA ligases identified the virus enzyme in discrete cytoplasmic 'factories', the sites of virus DNA synthesis, demonstrating immunological cross-reactivity between host DNA ligase I and the vaccinia enzyme. DNA ligase was not detected in the factories of a mutant virus lacking the ligase gene. Despite this, no difference in growth between wild-type (WT) and mutant virus was detectable even in Bloom's syndrome cells which have reduced DNA ligase I activity. However, DNA ligase negative virus showed an increased sensitivity to UV or bleomycin in cultured cells, and the importance of DNA ligase for virus virulence in vivo was demonstrated by the attenuated phenotype of the deletion mutant in intranasally infected mice.     

Phage formation in Staphylococcus muscae cultures; the effect of the yeast fraction on virus synthesis

1. A non-dialyzable fraction from fresh bakers' yeast stimulates the formation of S. muscae virus in cells in synthetic medium in the log phase of multiplication. 2. A similar fraction was not found in calf thymus, pancreas, or liver. 3. The active substance in this fraction has been partially purified. 4. This substance is taken up by the cells. In the absence of virus the added substance is metabolized to a form no longer available for virus formation. 5. A purified yeast fraction, which stimulates adaptive enzyme formation in yeast, has been found to stimulate virus formation in the S. muscae system. 6. The similarities between the yeast fraction that stimulates adaptive enzyme formation and the yeast fraction that stimulates virus formation are discussed.     

Genetic evidence for vaccinia virus-encoded DNA polymerase: isolation of phosphonoacetate-resistant enzyme from the cytoplasm of cells infected with mutant virus.

Phosphonoacetate (PAA), at concentrations of 200 micrograms/ml or more, prevented growth of vaccinia virus in HeLa and BSC-1 cells. Spontaneous vaccinia virus mutants, selected at high PAA levels, were resistant to the antiviral effects of the drug. The action of PAA was directed toward an early viral function, since the drug was inhibitory only during the first 4 h of the approximately 15-h growth cycle. Conversely, significant reversal of the antiviral effects was obtained only when the drug was removed at or before the fourth hour of infection. Incorporation of [3H]thymidine into cytoplasmic viral DNA was severely inhibited in cells infected with wild-type virus but not in cells infected with mutant virus. Virus-induced DNA polymerase isolated from the cytoplasm of cells infected with wild-type or mutant virus had indistinguishable chromatographic properties on DEAE-cellulose and phosphocellulose columns. However, the wild-type enzyme was inhibited by relatively low concentrations of PAA, whereas 10-fold higher concentrations were needed for equivalent inhibition of the mutant enzyme. Kinetic analysis indicated that PAA inhibition was noncompetitive with deoxyribonucleoside triphosphates; Ki values for wild-type and mutant DNA polymerases were approximately 25 and 300 microM, respectively. Inhibition of wild-type DNA polymerase was immediate and complete even when PAA was added after initiation of DNA synthesis in vitro, suggesting that chain elongation was affected. These results established that the DNA polymerase is a target of the antiviral action of PAA and provided genetic evidence that this enzyme is virus encoded.