Biomedical subjects

Meike Hass

Publications and source records attributed to Meike Hass.

4 recordsLinked to original sources

PubMed2006

Mutational analysis of the lassa virus promoter.

The promoter sequences directing viral gene expression and genome replication of arenaviruses reside within the 3' and 5' termini of each RNA segment. The terminal 19 nucleotides at both ends are highly conserved among all arenavirus species and are almost completely complementary to each other. This study aimed at characterizing the Lassa virus promoter in detail. The relevance of each position in the promoter was studied by site-directed mutagenesis using the Lassa virus minireplicon system. The data indicate that the Lassa virus promoter functions as a duplex, regulates transcription and replication in a coordinated manner, and is composed of two functional elements, a sequence-specific region from residue 1 to 12 and a variable complementary region from residue 13 to 19. The first region appears to interact with the replication complex mainly via base-specific interactions, while in the second region solely base pairing between 3' and 5' promoter ends is important for promoter function.

Base Pairing↗

PubMed2005

Functional analysis of hepatitis B virus reactivating in hepatitis B surface antigen-negative individuals.

The biological properties of latent or occult hepatitis B virus (HBV) have been poorly characterized as a result of the extremely low virus concentration. This report describes the phenotype of HBV reactivating in two patients after an HBsAg-negative latency period. One patient had latent HBV infection for at least 12 years without detectable viremia and symptoms of liver disease. Several full-length HBV genomes were cloned at reactivation, sequenced, and functionally tested by transfection into HuH7 cells. Genomes from both patients showed a low replication phenotype. It was caused at the level of RNA encapsidation or HBV DNA synthesis, but was not attributable to uncommon mutations in the terminal protein domain of P protein. A substantial subpopulation ( approximately 50%) of genomes from one patient did not express pre-S2/S mRNA and HBsAg. Site-directed mutagenesis identified a single G-A mutation within the S gene (position 458) to be responsible for this effect. The G458A mutation was also effective if the S gene was placed under control of a heterologous promoter. Furthermore, nuclear run-on transcription showed that the G458A mutation acts at the posttranscriptional level. The mutation affected a 5' splice site and prevented splicing of the pre-S2/S mRNA from position 458 to 1305. In conclusion, HBV latency may be characterized by viruses with reduced replication competence and antigen expression. In one patient, HBsAg expression was terminated by an as yet undescribed posttranscriptional mechanism. A single mutation inactivated a 5' splice site that is obviously essential for pre-S2/S mRNA accumulation. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Carrier State↗

PubMed2004

Replicon system for Lassa virus.

Lassa virus is endemic to West Africa and causes hemorrhagic fever in humans. To facilitate the functional analysis of this virus, a replicon system was developed based on Lassa virus strain AV. Genomic and antigenomic minigenomes (MG) were constructed consisting of the intergenic region of S RNA and a reporter gene (Renilla luciferase) in antisense orientation, flanked by the 5' and 3' untranslated regions of S RNA. MGs were expressed under the control of the T7 promoter. Nucleoprotein (NP), L protein, and Z protein were expressed from plasmids containing the T7 promoter and internal ribosomal entry site. Transfection of cells stably expressing T7 RNA polymerase (BSR T7/5) with MG in the form of DNA or RNA and plasmids for the expression of NP and L protein resulted in high levels of Renilla luciferase expression. The replicon system was optimized with respect to the ratio of the transfected constructs and by modifying the 5' end of the MG. Maximum activity was observed 24 to 36 h after transfection with a signal-to-noise ratio of 2 to 3 log units. Northern blot analysis provided evidence for replication and transcription of the MG. Z protein downregulated replicon activity close to background levels. Treatment with ribavirin and alpha interferon inhibited replicon activity, suggesting that both act on the level of RNA replication, transcription, or ribonucleoprotein assembly. In conclusion, this study describes the first replicon system for a highly pathogenic arenavirus. It is a tool for investigating the mechanisms of replication and transcription of Lassa virus and may facilitate the testing of antivirals outside a biosafety level 4 laboratory.

Animals↗

PubMed2004

Inhibition of different Lassa virus strains by alpha and gamma interferons and comparison with a less pathogenic arenavirus.

The high pathogenicity of Lassa virus is assumed to involve resistance to the effects of interferon (IFN). We have analyzed the effects of alpha IFN (IFN-alpha), IFN-gamma, and tumor necrosis factor alpha (TNF-alpha) on replication of Lassa virus compared to the related, but less pathogenic, lymphocytic choriomeningitis virus (LCMV). Three low-passage Lassa virus strains (AV, NL, and CSF), isolated from humans with mild to fulminant Lassa fever, were tested. Lassa virus replication was inhibited by IFN-alpha and IFN-gamma, but not TNF-alpha, in Huh7 and Vero cells. The degree of IFN sensitivity of a Lassa virus isolate did not correlate with disease severity in human patients. Furthermore, cytokine effects observed for Lassa virus and LCMV (strains CH-5692, Armstrong, and WE) were similar. To address the mechanisms involved in the IFN effect, we used cell lines in which overexpression of IFN-stimulated proteins promyelocytic leukemia protein (PML) and Sp100 could be induced. Both proteins reside in PML bodies, a cellular target of the LCMV and Lassa virus Z proteins. Overexpression of PML or Sp100 did not affect replication of either virus. This, together with the previous finding that PML knockout facilitates LCMV replication in vitro and in vivo (M. Djavani, J. Rodas, I. S. Lukashevich, D. Horejsh, P. P. Pandolfi, K. L. Borden, and M. S. Salvato, J. Virol. 75:6204-6208, 2001; W. V. Bonilla, D. D. Pinschewer, P. Klenerman, V. Rousson, M. Gaboli, P. P. Pandolfi, R. M. Zinkernagel, M. S. Salvato, and H. Hengartner, J. Virol. 76:3810-3818, 2002), describes PML as a mediator within the antiviral pathway rather than as a direct effector protein. In conclusion, the high pathogenicity of Lassa virus compared to LCMV is probably not due to increased resistance to the effects of IFN-alpha or IFN-gamma. Both cytokines inhibit replication which is relevant for the design of antiviral strategies against Lassa fever with the aim of enhancing the IFN response.

Animals↗