Manfred Marschall

Hepatitis B virus surface antigen as a reporter of promoter activity

The coding sequence for the hepatitis B virus surface antigen (HBsAg) was used as a new reporter gene for studies on eukaryotic promoter activity and upstream regulatory sequences. The data observed in transfection assays were comparable to results obtained with conventional chloramphenicol acetyltransferase (CAT) assays, as was demonstrated using various transcriptional regulation sequences. The expression of HBsAg as a reporter protein offered some advantages: (i) In transient expression assays, a time course of promoter activity depending on variable culture conditions could be monitored over a period of time, since the HBsAg was secreted into the culture supernatant. (ii) Evaluation of HBsAg from supernatant aliquots and quantification of the corresponding promoter activities could be performed easily, using the very sensitive and readily available diagnostic HBsAg kits. (iii) In contrast to the conventional CAT assay, the cells remained available for further tests, e.g., Western blot, immunofluorescence or transcript analysis. Characteristics of several Epstein-Barr virus (EBV) promoters, depending on the virus state of EBV-positive B-cells (latency, chemical induction, lytic superinfection, trans-activation), were assayed using the HBsAg reporter system.

The lytic transition of Epstein-Barr virus is imitated by recombinant B-cells

SummaryLytic transition of Epstein-Barr virus (EBV) is initiated by distinct immediate early regulators of the viral cycle, in synchronization to temporary, permissive conditions during host cell differentiation. We developed eukaryotic vectors suitable to imitate the processes involved in lytic transition in cell culture systems. Two stable B cell lines were established: R59Z activator cells were used to induce lytic EBV expression in a constitutive manner by the production of the BZLF 1trans-activator (Zta). R7-57 reporter cells, on the other hand, signaled induced activity of the lytic origin of EBV replication (oriLyt). Different modes, like chemical induction, lytic superinfection with EBV and single genetrans-activation converted the recombinant oriLyt element in R7-57 reporter cells. BZLF 1, transiently expressed in R7-57 reporter cells, was the only EBVtrans-activator found, sufficient in inducing the viral lytic cycle. Basing on these experiments,trans-cellular activation of EBV was tested by cocultivation of BZLF 1-expressing R59Z activator cells with the R7-57 reporter line. No lytic effect on the reporter cells could be measured, neither by cocultivation of activator cells nor by coincubation of BZLF 1-containing cell lysates. Latency breaking activity, however, was transferred from activator to reporter cells when active, exogenous virus was added. The cell system described in these experiments provides a tool for the detection of EBV reactivation and demonstrates the potential of the lytic regulatory gene BZLF 1.

The persistent variant of influenza C virus carries one characteristic point mutation in RNA segment 1

Influenza C/ Ann Arbor/1/50-pi(C/AA-pi) virus causes persistent infections in MDCK and Wi38 cells, but sets limited, wild-type like infections in other cells. Concluding that persistence itself it dependent on the host environment, we determined the nucleotide sequence of the C/AA-pi analogous gene to the basic polymerase 2 (PB2) of influenza A virus, which is known to be a determinant for the host range. C/AA-pi and the parental wild-type virus (C/AA-wt) have 16 nucleotides in common that are different to a previously published PB2 sequence (C/JJ/50). These variations, which are probably due to divergent passages histories, are scattered along the sequence and are partially found in another published isolate (C/Berlin/1/85). One single mutation, however, is unique to the persistent variant. Nucleotide 28 mutated from T to C which leads to a change of amino acid 3 from Leu to Phe. This substitution is stably associated with the persistent phenotype throughout multiple passages in different cells lines and eggs and cannot be found in any other influenza C viruses.

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

SummaryThe 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 atypical feature of the persistence, we identified a continuous synthesis of anti-genomic 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, nonpersistent replication cycle and produced a wild-type-like course of virus-specific RNA transport. These data indicate that influenza C virus persistence depends ona distinctly modified and cell type-specific regulation of virus-specific RNAsynthesis and transport.

Transcription and Protein Expression Pattern of EBV in Freshly Infected Lymphoid Cells

Infection of EBV negative B-lymphoid cells is a more relevant model to study the early events after fresh infection with EBV than infection of Raji cells already carrying EBV genomes and expressing latent gene products. We used BJA-B and BL02 cells for infection experiments with P3HR1 virus, which has no immortalizing capacity for umbilical cord blood lymphocytes, and B95–8, which is able to immortalize. We investigated the transcription and protein expression pattern and could demonstrate a lytic infection of BJA-B cells. The same viral genes known to be immediate early genes with transactivating properties from superinfection of Raji cells (1, 2) and which are also active in transient expression systems (3, 4, 5) seem to have a similar role in infected BJA-B cells.

Trans-regulation of the Early MS Gene of EBV and Its Dependence on the Host Cell

The transition from latency to the lytic cycle of viral replication appears to be highly dependent on the type and differentiation of the host cell. Transiently active cellular factors control the expression of lytic viral proteins. Besides activation of the repressed BZLF1 immediate early gene (F. Schwarzmann, manuscript in preparation) additional regulatory events are involved, e.g. the expression of two further immediate early products, BRLF1 (1,4) and BI’LF4 (11). All three trans-activators share at least one common target, the BMLF1BSLF2 (MS) promoter region, which controls the expression of the posttranscriptional MS regulator (2,6) consequently leading to the cascade-like lytic pattern. We analysed the MS promoter and upstream element for its responsiveness to EBV trans-activators or TPA in different cell types and investigated its dependence to the stage of cell differentiation.