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M Marschall

Publications and source records attributed to M Marschall.

At least 19 recordsLinked to original sources
PubMed2004

Expression and localization of the Epstein-Barr virus-encoded protein kinase.

The protein kinase (PK) encoded by the Epstein-Barr Virus (EBV) BGLF4 gene is the only EBV protein kinase. The expression pattern of EBV PK during the reactivation of the viral lytic cycle and the subcellular localization of the protein were analyzed with a polyclonal antiserum raised against a peptide corresponding to the N terminus of EBV PK. Based on previously published data (E. Gershburg and J. S. Pagano, J. Virol. 76:998-1003, 2002) and the expression pattern described here, we conclude that EBV PK is an early protein that requires viral-DNA replication for maximum expression. By biochemical fractionation, the protein could be detected mainly in the nuclear fraction 4 h after viral reactivation in Akata cells. Nuclear localization could be visualized by indirect immunofluorescence in HeLa cells transiently expressing EBV BGLF4 in the absence of other viral products. Transient expression of 3'-terminal deletion mutants of EBV BGLF4 resulted in cytoplasmic localization, confirming the presence of a nuclear localization site in the C-terminal region of the protein. In contrast to the wild-type EBV PK, all of the mutants were unable to hyperphosphorylate EA-D during coexpression or to phosphorylate ganciclovir, as measured by an in-cell activity assay. Thus, the results demonstrate that the nuclear localization, as well as the kinase activity, of BGFL4 is dependent on an intact C-terminal region.

Active Transport, Cell Nucleus↗
PubMed2000

Inhibition of influenza C viruses by human MxA protein.

Human MxA protein was analyzed for its ability to inhibit the replication of different influenza C viruses. Three laboratory derivatives of viral strain C/Ann Arbor/1/50 were investigated, namely the parental wild-type virus C/AA-wt, the persistent variant C/AA-pi and the highly cytopathogenic variant C/AA-cyt. In addition, strain C/Paris/214/91 isolated from an influenza patient was used. Multiplication of all four viruses was suppressed in MxA-expressing Vero cells, as indicated by a decrease in viral RNA synthesis, viral protein synthesis, virion production and induction of a cytopathic effect. Inhibition correlated with the level of MxA expression. Furthermore, inhibition was independent of cell clone-specific differences in expression of virus receptors, as demonstrated by receptor reconstitution experiments. Thus, human MxA protein has antiviral activity against influenza C viruses.

Animals↗
PubMed2000

Recombinant green fluorescent protein-expressing human cytomegalovirus as a tool for screening antiviral agents.

A recombinant human cytomegalovirus (AD169-GFP) expressing green fluorescent protein was generated by homologous recombination. Infection of human fibroblast cultures with AD169-GFP virus produced stable and readily detectable amounts of GFP signals which were quantitated by automated fluorometry. Hereby, high levels of sensitivity and reproducibility could be achieved, compared to those with the conventional plaque reduction assay. Antiviral activities were determined for four reference compounds as well as a set of putative novel cytomegalovirus inhibitors. The results obtained were exactly in line with the known characteristics of reference compounds and furthermore revealed distinct antiviral activities of novel in vitro inhibitors. The fluorometric data could be confirmed by GFP-based flow cytometry and fluorescence microscopy. In addition, laboratory virus variants derived from the recombinant AD169-GFP virus provided further possibilities for study of the characteristics of drug resistance. The GFP-based antiviral assay appeared to be very reliable for measuring virus-inhibitory effects in concentration- and time-dependent fashions and might also be adaptable for high-throughput screenings of cytomegalovirus-specific antiviral agents.

Antiviral Agents↗
PubMed1999

The ORF, regulated synthesis, and persistence-specific variation of influenza C viral NS1 protein.

The open reading frame (ORF) and the regulated synthesis of the influenza C viral NS1 protein were analyzed in view of viruses possessing different biological activities. We provide evidence for a 246-amino-acid NS1-ORF, encoded by five viral strains and variants. Prokaryotic expression of the prototype NS1-ORF resulted in a product of 27 kDa, confirming the predicted molecular weight. Using an antiserum raised against recombinant NS1 protein, nonstructural proteins of wild-type virus were detected in infected cells for a limited course of time, whereas a persistent virus variant was characterized by a long-term nonstructural gene expression. As examined by infection experiments, the intracellular distribution of nonstructural protein was nuclear and cytoplasmic, whereas in NS1 gene-transfected cells, the cytoplasmic localization occurred in a fine-grained structure, suggesting an analogy to influenza A viral NS1 protein. Concerning persistent infection, NS1 protein species differing in sizes and posttranslational modifications were observed for a persistent virus variant, as particularly illustrated by a high degree of NS1 phosphorylation. Virus reassortant analyses proved the importance of the NS-coding genomic segment: the minimal viral properties required for the establishment of persistence were transferred with this segment to a monoreassortant virus. Thus the influenza C viral NS1 protein is a 246-amino-acid nuclear-cytoplasmic phosphoprotein that can be subject to specific variations being functionally linked to a persistent virus phenotype.

Amino Acid Sequence↗
PubMed1999

Influenza C virus persistence depends on exceptional steps in viral RNA synthesis and transport.

The cell line MDCK-pi, which is persistently infected with a variant of influenza C/AnnArbor/1/50 virus (C/AA-pi), was studied as a long-term persistence model by means of a strand-specific in situ hybridization assay. As a typical feature of the persistence, we identified a continuous synthesis of antigenomic positive-strand RNA encoded by segment 7 (NS) during virus production. In contrast, infection with the parental wild-type virus led to a rapid reduction of antigenomic RNA as observed in the late period of replication particularly for RNA segment 7. Furthermore, the replication cycle of the persistent variant did not show the switch from early to late replication events followed by clearance of intracellular virus, but was regulated in terms of productive and nonproductive phases. Nonproductive phases were reversible and characterized by a low level of virus-specific RNA signals. In the productive phase, a difference in cytoplasmic RNA transport was detected for the two viruses: a marked cytoplasmic accumulation of negative- and positive-strand wild-type virus RNAs stood in contrast to a RNA localization in different cellular compartments for the persistent virus. Also, Vero cells infected with the C/AA-pi variant were restricted to a transient, non-persistent replication cycle and produced a wild-type-like course of virus-specific RNA transport. These data indicate that influenza C virus persistence depends on a distinctly modified and cell type-specific regulation of virus-specific RNA synthesis and transport.

Animals↗
PubMed1999

A persistent variant of influenza C virus fails to interact with actin filaments during viral assembly.

C/AA-pi virus, a variant of influenza C/Ann Arbor/1/50 virus, establishes persistent infections in MDCK cells, characterized by low levels of progeny production. During viral assembly, nucleoprotein (NP) was found homogeneously distributed over cytoplasmic and nuclear compartments and matrix (M) protein was likewise localized in a barely structured fashion. In contrast, infections with nonpersistent influenza A, B and C viruses produced cytoplasmic granular structures, which typically consisted of colocalized NP and M proteins. Studies on the in vitro interaction between NP and M proteins revealed identical binding capacities comparing influenza C wild-type virus with the persistent variant. Cytochalasin D treatment of infected cells demonstrated that NP protein of the wild-type virus, but not of the persistent variant, was distinctly associated with cellular actin filaments. Moreover, the assembly characteristics of wild-type virus were modulated in the presence of recombinant persistent-type NP protein towards a behaviour similar to persistent infection. Cell type specificity was particularly illustrated in C/AA-pi virus-infected Vero cells, which did not support viral persistence, but produced granular wild-type-like complexes. Thus, interaction between NP, M and actin proteins (i) is a basic part of the viral assembly process, (ii) is dominantly modulated by NP protein and (iii) is specifically altered in the case of persistent infection.

Actins↗
PubMed1998

Persistent infection with an influenza C virus variant is dominantly established in the presence of the parental wild-type virus.

Two influenza C viruses were used for double-infection experiments to investigate the dominance of their phenotypes. The wild-type virus (C/AA-wt) had been characterized by its short-lived productive cycle, whereas a distinct variant derived from it (C/AA-pi) was demonstrated to persist in long-term passages of infected MDCK cultures. Here we show that the persistent virus C/AA-pi is capable of replicating in the presence of abundant amounts of wild-type virus: the persistent virus could be diluted to 10(-9) within wild-type inoculum, still developing a stable form of persistence. This behaviour was reflected by permanent virus release and by continuous enzymatic activity of the viral HEF glycoprotein in infected cells. All long-term cultures tested remained positive for viral NS protein and vRNA. On the vRNA level, it was shown that viral segments originated from the persistent-type genome, while wild-type vRNAs were not maintained after double-infection. Thus, the genotype of the persistent variant was dominantly selected in serial passages. These results indicate a specific intracellular advantage of persistent influenza C virus over the parental wild-type.

Animals↗
PubMed1997

The cell receptor level is reduced during persistent infection with influenza C virus.

Persistent influenza C virus infection of MDCK cells perpetuates the viral genome in a cell-associated form. Typically, virus production remains at a low level over extended periods, in the absence of lytic effects of replication. In this study, we demonstrate that persistently infected cells are very restricted in permissiveness for superinfection. By reconstitution experiments, using bovine brain gangliosides as artificial receptors, the degree of super-infection was markedly increased. Analysis of cellular receptor expression revealed reduced concentrations of sialoglycoproteins in general and a limited presentation of the major receptor gp40. Cocultures of persistently infected and uninfected cells (the latter carrying normal receptor levels) initiated a transient rise in virus titers. This kind of induction of virus synthesis appeared to be mainly receptor-linked, since a receptor-deprived subline, MDCK II, did not give rise to a similar effect. Susceptibility of MDCK II cocultures could be partly restored by ganglioside treatment. In accordance to related virus systems, these findings on influenza C virus suggest a role of cell receptor concentrations in the regulation of long-term persistence.

Animals↗
PubMed1997

A highly cytopathogenic influenza C virus variant induces apoptosis in cell culture.

An influenza C virus variant, C/AA-cyt, was identified as the agent responsible for highly effective induction of cytopathogenicity in MDCK cells. The cytopathogenic effect was manifested by cell rounding, cell shrinkage and foci of cell destruction leading finally to disruption of the monolayer in a virus dose-dependent manner. Virus-induced cytopathogenicity was suppressed by temperatures nonpermissive for virus replication. Maintenance of plasma membrane integrity post-infection, in connection with induction of a DNA fragmentation ladder, revealed the characteristic picture of apoptosis. In support of this, quantitative analysis demonstrated high levels of apoptosis-like oligonucleosomal DNA. The results indicate that influenza C viruses can induce programmed cell death, as formerly reported for influenza type A and B viruses.

Animals↗
PubMed1997

[Inline filtration of erythrocyte concentrates with the Leucoflex LCR4 T/B system].

The in-line-filtration of red cell concentrates (RCC) is an effective method to reduce white-cell-induced reactions. SAG-M RCCs were prepared in quadruple top/bottom bag systems and filtered after storage at 4 degrees C (study: n = 12). The mean white blood cell (WBC) reduction was 99.9% with the number of residual WBCs at about 0.67 x 10(6) per unit and with a loss of Hb mass of 10.64%. No cell fragments of WBCs and monocytes were detected (MAIPA). After storage of 42 days, mean values of hemolysis level (0.64%) and ATP concentration (2.27 mumol/g Hb) were improved compared with unfiltered RCCs. Comparably good results of leukocyte depletion were achieved by preparation of about 3,000 in-line filtrated RCCs for clinical use, measured by routine quality control (n = 25). Our experience shows that the systems are easy to handle and to use. The Leucoflex LCR4 in-line T/B system (Maco Pharma) can be recommended in the clinical practice by virtue of the effective leukocyte elimination, particularly in those blood banks with platelet concentrate production from buffy coats beside the RCC in-line filtration.

Equipment Design↗
PubMed1996

Sodium ion translocation by N5-methyltetrahydromethanopterin: coenzyme M methyltransferase from Methanosarcina mazei Gö1 reconstituted in ether lipid liposomes.

The N5-methyltetrahydromethanopterin (H,MPT):coenzyme M methyltransferase is a membrane associated, corrinoid-containing protein that uses the methylation of coenzyme M (HS-CoM) by methyl-tetrahydromethanopterin to drive an energy-conserving sodium ion pump. The enzyme was purified from acetate-grown Methanosarcina mazei Gö1 by a two-step solubilization with n-octyl-beta-glucoside, chromatography on hydroxyapatite, and by gel filtration on Superdex 200 or Sepharose CL-6B. The highly purified protein was apparently composed of six different subunits of 34, 28, 20, 13, 12, and 9 kDa. The N-terminal amino acid sequences of these polypeptides were determined. The native enzyme exhibited an apparent molecular mass of about 380 kDa. During purification, the enzyme was stabilized with 10 microM hydroxocobalamin. The highest specific activity reached during purification was 10.4 U/mg. The purified enzyme was reconstituted in monolayer liposomes prepared from ether lipids of M. mazei Gö1. In experiments with radioactive sodium ions, it was shown that the methyltransferase catalyzes the vectorial translocation of sodium ions across the membrane. Methyltransferase activity was stimulated by sodium ions. 1.7 mol Na-/mol methyl groups transferred were translocated. Methyltetrahydrofolate and methyl-cobalamin could substitute for methyl-H,MPT.

Amino Acid Sequence↗
PubMed1996

Comparative analysis of six European influenza vaccines.

Three split-virion vaccines (Vaxigrip, Begrivac, and Influsplit/Fluarix) and three subunit vaccines containing only viral surface glycoproteins (Influvac, Agrippal, and Fluvirin) available for the 1994-95 season were analysed by biological, molecular, and biochemical methods. Although all vaccines are required by health authorities to contain 15 micrograms haemagglutinin per dose of each virus strain, there were significant differences in haemagglutination titres among the examined vaccines of both types. The enzymatic activity of neuraminidase was present in all vaccines except Fluvirin. Total protein content was lower for subunit vaccines. Viral nucleoprotein was detected in all split vaccines but to varying levels according to SDS-PAGE and Western blot analyses. The ovalbumin content was low in general but was about tenfold higher for Influvac than for the other vaccines analysed. This protein may induce hypersensitive reactions among persons with severe egg allergy. All three split-virion vaccines were found to contain the matrix protein; however, it was not detected in the subunit vaccines. Differences in influenza antigen variety in currently available vaccines may affect efficacy, whereas differences in concentrations of nonviral compounds such as ovalbumin and endotoxin may lead to different postvaccination reactogenicity profiles.

Antigens, Viral↗
PubMed1996

Influenza C virus RNA is uniquely stabilized in a steady state during primary and secondary persistent infections.

The ability to establish persistent infections in vitro and in vivo has been illustrated for different human RNA viruses. However, little insight has been gained regarding the intracellular state of viral RNA species and the regulatory processes governing their long-term continuance. In this report, primary persistence of a variant of influenza C/Ann Arbor/1/50 virus in infected MDCK cells and secondary infections in human cell lines were investigated. Different PCR and staining techniques were applied for the description of low viral loads. The RNA pattern in primary persistence indicates that viral RNA synthesis is quantitatively linked to productive and non-productive phases, with negative-strand RNA being present continuously. In single cells cultures, derived from the primary line, all clones tested were positive by nested PCR and Southern blot screening. This suggests that a true steady-state persistence of influenza C virus is established in each individual cell of the infected population. Secondary infection experiments, in terms of transfer of the persistent virus variant to different cell types, showed that a re-establishment of persistence can be accomplished in vitro. The stable persistent status remained reserved for distinct host cell lines. Hereby, vRNA is stably maintained in cell-type specific manner, whereas gene expression (e.g. HEF glycoprotein production) occurs in a variable fashion. These data point out novel characteristics in the understanding of influenza virus persistence.

Animals↗
PubMed1996

[Distribution of influenza C virus infection in dogs and pigs in Bavaria].

150 dog and 240 pig sera were tested for antibodies against influenza C virus in the hemagglutination inhibition test and confirmed by immunofluorescence and Western blotting tests. Virus strain C/JHB/1/66 was utilized as a standard antigen. It was found that 50.6% of the total number of dogs in the age between one month and 14 years possessed antibody titers of 1:20 or more, which was set as the general cut-off. In comparison, 24.0% of pigs in the age between one day and one month (n = 90) and 25.9% of pigs in the age between one month and one year (n = 150) were found to be positive. The highest hemagglutination inhibition titer was determined as 1:320 for of the dog sera and 1:160 for of the pig sera, respectively.

Age Distribution↗
PubMed1996

Experimental infection with a persistent influenza C virus variant leads to prolonged genome detection in the chicken lung.

A persistent variant of influenza C virus was used to infect chickens by intraamniotic (i.a.) inoculation. The infected hatchings were analyzed for virus production in different tissues and for the continuous presence of viral RNA genomes. The permissiveness for infection was demonstrated primarily for the chicken lung, besides other organs. Viral antigens could be detected by indirect immunofluorescence staining for a period of 8 days and reisolates were obtained mainly at early time points post infection (p.i.). Nested reverse transcription-polymerase chain reaction (RT-PCR) directed to 3 genomic sequences was positive at least until day 53, whereby no distinct end point was determined. These experiments provide first evidence for the long-term stability of influenza C virus RNA segments in vivo.

Animals↗
PubMed1995

Nucleotide-specific PCR for molecular virus typing.

Nucleotide sequence studies detected a double-point mutation in the genomic RNA segment 4 (nt 871 and 872) of the persistent variant C/AA-pi of influenza C/Ann Arbor/1/50 virus. The 3'-end-points of two distinct PCR primers were positioned exactly at this genome location and thereby adjusted the priming determinant complementary to the varied strain or to its wild-type counterpart. Consequently, positive RT-PCR products strictly referred to one of the two viruses examined, in both cases, using either virion or infected-cell RNA templates. Artificial virus mixtures could easily be distinguished by this method in a subsequent qualitative gel analysis. PCR annealing conditions and control reactions were optimized, for the monitoring of influenza virus isolates throughout multifold passages. Thus, sequence diversity in just two neighbouring nucleotides is sufficient to determine whether or not successful PCR amplification takes place, and this method can be used as a reliable means of virus strain differentiation.

Animals↗