Victor S. Mikhailov

New insights into the induction of the heat shock proteins in baculovirus infected insect cells.

Eight members of the HSP/HSC70 family were identified in Spodoptera frugiperda Sf9 cells infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by 2D electrophoresis followed by mass spectrometry (MALDI/TOF) and a Mascot search. The family includes five HSP70s induced by AcMNPV-infection and three constitutive cognate HSC70s that remained abundant in infected cells. Confocal microscopy revealed dynamic changes in subcellular localization of HSP/HSC70s in the course of infection. At the early stages (4 to 10 hpi), a fraction of HSPs is localized in distinct speckles in cytoplasm. The speckles contained ubiquitinylated proteins suggesting that they may be aggresomes where proteins targeted by ubiquitin are sequestered or processed for proteolysis. S. frugiperda HSP90 was identified in the 2D gels by Western blotting. Its amount was unchanged during infection. A selective inhibitor of HSP90, 17-AAG, decreased the rate of viral DNA synthesis in infected cells suggesting a supportive role of HSP90 in virus replication.

An important role of the heat shock response in infected cells for replication of baculoviruses.

Baculoviruses serve as a stress factor that can activate both death-inducing and cytoprotective pathways in infected cells. In this report, induction of heat shock proteins (HSPs) of the 70-kDa family (HSP/HSC70) in Sf-9 cells after infection with AcMNPV was monitored by Western blot analysis. Two-dimensional electrophoresis in polyacrylamide gel revealed changes in the cellular pattern of HSP/HSC70s and synthesis of a new member of the HSP/HSC70 family in the infected cells. Although infection with AcMNPV moderately increased the HSP/HSC70 content in cells under standard conditions, the infection potentiated the response to heat shock boosting the HSP/HSC70s content in infected cells several-fold in comparison with uninfected cells. Addition of KNK437, a known inhibitor of inducible HSPs, decreased the rate of viral DNA synthesis in infected cells more than one order of magnitude and markedly suppressed the release of budded viruses indicating the importance of the heat shock response for baculovirus replication.

Heterogeneous rapidly labeled DNA with the properties of replicating form in isolated rat liver mitochondria

Abstract Deproteinized DNA preparations from rat liver mitochondria incubated in vitro with [3H]dATP or [3H]thymidine contain three labeled components sedimenting at 38, 27 and 9–10 S. The label in 38-S and 27-S components is associated with covalently closed and open circular molecules of mitochondrial DNA (mtDNA), respectively. 9–10-S material represents DNA fragments split from circular molecules during purification, it is not present in lysates prepared under mild conditions. Analysis of mitochondrial lysates prepared with minimal shear in CsCl-ethidium bromide density gradients, revealed the existence of a heterogeneous “intermediate fraction” with a high specific activity located as a broad zone between the peaks of covalently closed and open circular molecules. The bulk of this intermediate fraction is represented by closed circular template molecules associated with varying proportions of linear DNA strands. The sedimentation coefficient of this fraction in neutral sucrose is 35–38 S, its density in neutral CsCl is 3–4 mg/cm3 higher than that of native DNA suggesting the presence of single-stranded DNA regions. Heating of this fraction at 90 °C for 30 s with subsequent quenching in ice liberates part of its radioactivity as a heterogeneous population of fragments sedimenting in the region 10–20 S. The remaining radioactivity sediments at 38 and 27 S. It is concluded that the described intermediate fraction contains replicating molecules of mtDNA.

The redox state of the baculovirus single-stranded DNA-binding protein LEF-3 regulates its DNA binding, unwinding, and annealing activities.

The single-stranded (ss) DNA-binding protein LEF-3 of Autographa californica multinucleocapsid nucleopolyhedrovirus promoted Mg2+-independent unwinding of DNA duplexes and annealing of complementary DNA strands. The unwinding and annealing activities of LEF-3 appeared to act in a competitive manner and were determined by the ratio of protein to DNA. At subsaturating and saturating concentrations, LEF-3 promoted annealing, whereas it promoted unwinding at oversaturation of DNA substrates. The LEF-3 binding to ssDNA and unwinding activity were sensitive to redox agents and were inhibited by oxidation of thiol groups in LEF-3 with 1,1′-azobis(N,N-dimethylformamide) (diamide) or by modification with the thiol-conjugating agent N-ethylmaleimide. Both oxidation and alkylation increased the dissociation constant of the interaction with model oligonucleotides indicating a decrease in an intrinsic affinity of LEF-3 for ssDNA. These results proved that free thiol groups are essential both for LEF-3 interaction with ssDNA and for DNA unwinding. In contrast, oxidation or modification of thiol groups stimulated the annealing activity of LEF-3 partially due to suppression of its unwinding activity. Treatment of LEF-3 with the reducing agent dithiothreitol inhibited annealing, indicating association of this activity with the oxidized protein. Thus, the balance between annealing and unwinding activities of LEF-3 was determined by the redox state of protein with the oxidized state favoring annealing and the reduced state favoring unwinding. An LEF-3 mutant in which the conservative cysteine Cys214 was replaced with serine showed both a decreased binding to DNA and a reduced unwinding activity, thus indicating that this residue might participate in the regulation of LEF-3 activities.

Proteotoxic stress induced by Autographa californica nucleopolyhedrovirus infection of Spodoptera frugiperda Sf9 cells

Baculovirus AcMNPV causes proteotoxicity in Sf9 cells as revealed by accumulation of ubiquitinated proteins and aggresomes in the course of infection. Inhibition of proteasomes by lactacystin increased markedly the stock of ubiquitinated proteins indicating a primary role of proteasomes in detoxication. The proteasomes were present in Sf9 cells as 26S and 20S complexes whose protease activity did not change during infection. Proteasome inhibition caused a delay in the initiation of viral DNA replication suggesting an important role of proteasomes at early stages in infection. However, lactacystin did not affect ongoing replication indicating that active proteasomes are not required for genome amplification. At late stages in infection (24-48 hpi), aggresomes containing the ubiquitinated proteins and HSP/HSC70s showed gradual fusion with the vacuole-like structures identified as lysosomes by antibody to cathepsin D. This result suggests that lysosomes may assist in protection against proteotoxicity caused by baculoviruses absorbing the ubiquitinated proteins.

Protein synthesis in pupae of the silkworm Bombyx mori after infection with nuclear polyhedrosis virus: resistance to viral infection acquired during pupal period.

Protein synthesis has been studied in pupae of the silkworm Bombyx mori (Bm) infected with nuclear polyhedrosis virus (BmNPV) at various stages of the pupal period. Nascent proteins were labelled by injection of [35S]methionine into pupae and then analysed by SDS-PAGE. Temporal regulation of synthesis of infected cell-specific proteins (ICSPs) in pupae was demonstrated by electrophoretic analysis of the proteins labelled at different times post-infection (p.i.). The rate of ICSP synthesis reached a maximum at 4 to 5 days p.i., exceeding the rate of synthesis of cellular proteins in uninfected pupae by about twofold. The viral proteins p10 and polyhedrin were the most abundant products synthesized late in the infection. Both proteins were found to be associated with the nuclear matrix after fractionation of nuclei from infected pupae. Two virus-induced phosphoproteins, pp35 and ppB, were found to be the major acceptors of labelled phosphate from [gamma-32P]ATP during in vitro phosphorylation of proteins in pupal homogenates, nuclei and nuclear extracts. These proteins had electrophoretic mobilities comparable to those of structural phosphoproteins of BmNPV virions with M(r)s of 35K and 11K to 16K, respectively. The latter polypeptide was identified as the major DNA-binding protein of the virus. The susceptibility of silkworms to BmNPV decreased markedly during the pupal period. Following injection of BmNPV all young pupae acquired polyhedrosis and finally died whereas most of the older pupae did not exhibit disease and completed metamorphosis normally. Moreover, the later in the pupal period the silkworms were infected, the lower the production of polyhedrin in diseased pupae.

Induction of a novel protein kinase in pupae of the silkworm Bombyx mori after infection with nuclear polyhedrosis virus

Protein kinases induced by Bombyx mori nuclear polyhedrosis virus in pupae of the silkworm B. mori were examined by activity gel analysis using phosvitin as a protein substrate. The method involved PAGE of the soluble fraction from pupae under native conditions and in the presence of SDS, followed by in situ renaturation of proteins and recovery of protein kinase activity in the intact gel. A novel protein kinase able to phosphorylate phosvitin was detected in the infected pupae from 2 days post-infection. This enzyme was not present in uninfected silkworms at any stage of the pupal period. The novel kinase activity was found by SDS-PAGE to be associated with a single polypeptide with an apparent M(r) of 50K. However, on electrophoresis under native conditions its activity was associated with a set of polypeptides with similar but not identical electrophoretic mobilities. Microheterogeneity of the catalytically active polypeptides suggests that the virus-induced protein kinase undergoes post-translational modification during the course of infection.

State of the DNA-synthesizing system in isolated mitochondria from the mature eggs of the loach (Misgurnus fossilis)

Abstract 1. Isolated mitochondria from unfertilized loach eggs incubated in the reaction mixture for DNA synthesis with deoxynucleoside triphosphates incorporate 0.03 pmole of [3H]dTTP into the acid-insoluble fraction per mg of mitochondrial protein per h. This incorporation is at least 10 times lower than for mitochondria from later developmental stages of the loach when the biosynthesis of mitochondrial DNA is switched on. 2. Sedimentation analysis of the labeled incorporation product in CsCl-ethidiumbromide density gradients shows that essentially all radioactivity is present in the upper band of such gradients containing open DNA molecules. In contrast up to 80 % of radioactivity incorporated by mitochondria from later developmental stages of the loach is associated with lower and intermediate zones of the gradient containing molecules replicating on the covalently closed templates. 3. On the basis of 1 and 2 we conclude that loach egg mitochondria are inactive in replicative DNA synthesis and the small observed incorporation represents repair associated with spontaneous induction of single-strand breaks in mitochondrial DNA in the course of isolation and/or incubation of mitochondria. 4. Induction of single-strand breaks in mitochondrial DNA by treatment of isolated mitochondria with bruneomycin, known to produce among others 3′-OH termini in DNA, stimulates the incorporation of [3H]dTTP into acid-insoluble products 30–50 times. DNA labeled under these conditions is native, 90 % of the labeled molecules is present in the upper band of CsCl-ethidiumbromide gradients. The sedimentation coefficient of the labeled material in neutral sucrose is 27 S. 5. About 10 % of DNA labeled in the presence of a low concentration of bruneomycin (0.1 μg/ml) is associated with covalently closed molecules of mitochondrial DNA. Formation of this material is probably a consequence of sealing of labeled open circular DNA by a joining enzyme. This result may be evidence for the presence of DNA ligase in mitochondria from unfertilized eggs. 6. At least one explanation for the inactivity of egg mitochondria in replicative DNA synthesis is the absence of primer containing the 3′-OH terminal group necessary for the manifestation of DNA polymerase activity.

Preferential interaction of loach DNA polymerase δ with DNA duplexes containing single-stranded gaps.

We studied the interaction of DNA polymerase δ (pol δ) purified from the eggs of the teleost fish Misgurnus fossilis (loach) with DNA duplexes containing single‐stranded gaps of 1–13 nucleotides (nt). In the absence of processivity factors (PCNA, RF‐C, and ATP), pol δ elongated primers on single‐stranded DNA templates in a distributive manner. However, the enzyme was capable of processive synthesis by filling gaps of 5–9 nt in DNA duplexes. These data suggest that, upon filling a small gap, pol δ interacts with the 5′‐terminus downstream of the gap as well as with the 3′‐terminus of the primer. Interaction of pol δ with the proximal 5′‐terminus restricting the gap was confirmed by electrophoretic mobility shift assay. Analysis of the enzyme binding to DNA duplexes containing gaps of various sizes showed a much higher affinity of pol δ for duplexes with gaps of about 10 nt than for DNA substrates with primers annealed to single‐stranded templates. The most efficient pol δ binding was observed in experiments with DNA substrates containing unpaired 3′‐tails in primers. The data obtained suggest that DNA molecules with small gaps and single‐stranded tails may serve as substrates for direct action of pol δ in the course of DNA repair.

CHARACTERIZATION OF SHORT-LIVED INTERMEDIATES PRODUCED DURING REPLICATION OF BACULOVIRUS DNA

In this report the short-lived DNA replication intermediates produced in both uninfected and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infected Spodoptera frugiperda cells were characterized. The methods used included pulse-labeling of DNA in permiabilized cells, treatment of nascent DNA with Mung bean nuclease, and electrophoresis in neutral and alkaline agarose gels. In contrast to uninfected cells that produced a population of small DNA fragments of about 200bp, a population of heterogeneous fragments of up to 5kb with an average size of 1-2kb derived randomly from the virus genome was identified as the short-lived intermediates produced during AcMNPV replication. The intermediates likely include Okazaki fragments derived from the lagging strands in viral replication forks as well as fragments produced during the recombination-dependent replication.