Biomedical subjects

Verena Gauss-Müller

Publications and source records attributed to Verena Gauss-Müller.

10 recordsLinked to original sources

PubMed2006

Immunogenicity of a chimeric hepatitis A virus (HAV) carrying the HIV gp41 epitope 2F5.

Its stable particle structure combined with its high immunogenicity makes the hepatitis A virus (HAV) a perfect carrier to expose foreign epitopes to the host immune system. In an earlier report [Beneduce, F., Kusov, Y., Klinger, M., Gauss-Müller, V., Morace, G., 2002. Chimeric hepatitis A virus particles presenting a foreign epitope (HIV gp41) at their surface. Antiviral Res. 55, 369-377] chimeric virus-like particles (HAV-gp41) were described that carried at their surface the dominant gp41 epitope 2F5 (2F5e) of the human immunodeficiency virus HIV-1. Extending this work, we now report that chimeric virus HAV-gp41 replicates in HAV-susceptible cells as well as in non-human primates. Infected marmosets developed both an anti-HAV and anti-2F5 epitope immune response. Furthermore, an HIV-neutralizing antibody response was elicited in guinea pigs immunized with HAV-gp41 chimeric particles. The results demonstrate that the replication-competent chimeric HAV-gp41 can serve as either a live or a subunit vaccine for eliciting of antibodies directed against a foreign antigenic epitope.

AIDS Vaccines↗

PubMed2006

Silencing of hepatitis A virus infection by small interfering RNAs.

Infection by hepatitis A virus (HAV) can cause acute hepatitis and, rarely, fulminant liver failure, in particular in patients chronically infected with hepatitis C virus. Based on our previous observation that small interfering RNAs (siRNAs) can silence translation and replication of the firefly luciferase-encoding HAV replicon, we now exploited this technology to demonstrate the effect of siRNAs on viral infection in Huh-7 cells. Freshly and persistently infected cells were transfected with siRNAs targeting various sites in the HAV nonstructural genes. Compared to a single application, consecutive siRNA transfections targeting multiple sequences in the viral genome resulted in a more efficient and sustained silencing effect than a single transfection. In most instances, multiple applications of a single siRNA led to the emergence of viral escape mutants with mutated target sites that rendered these genomes resistant to RNA interference (RNAi). Efficient and sustained suppression of the viral infectivity was achieved after consecutive applications of an siRNA targeting a computer-predicted hairpin structure. This siRNA holds promise as a therapeutic tool for severe courses of HAV infection. In addition, the results provide new insight into the structural bases for sequence-specific RNAi.

Cell Line, Tumor↗

PubMed2005

Suppression of hepatitis A virus genome translation and replication by siRNAs targeting the internal ribosomal entry site.

Small interfering RNAs (siRNAs) targeting the coding region of hepatitis A virus (HAV) were shown to specifically inhibit viral genome replication. Compared to the coding region, the HAV internal ribosomal entry site (IRES) in the 5' non-coding region is highly sequence-conserved and folds into stable secondary structures. Here, we report efficient and sustained RNA interference mediated by both RNase III-prepared siRNA (esiRNA) and vector-derived short hairpin RNAs (shRNAs) that are targeted to various domains of the HAV IRES. Using reporter constructs, and the DNA-based HAV replicon system, we found that shRNAs targeting the HAV IRES domains IIIc and V sustainably suppressed genome translation and replication whereas the IRES domains IIIa and IV were resistant to RNA interference. Our study suggests that some HAV IRES domains might be used as a universal and effective target for specific inhibition of HAV infection.

Base Sequence↗

PubMed2005

Hepatitis A virus proteinase 3C binding to viral RNA: correlation with substrate binding and enzyme dimerization.

Proteinase 3C of hepatitis A virus (HAV) plays a key role in the viral life cycle by generating mature viral proteins from the precursor polyprotein. In addition to its proteolytic activity, 3C binds to viral RNA, and thus influences viral genome replication. In order to investigate the interplay between proteolytic activity and RNA binding at the molecular level, we subjected HAV 3C and three variants carrying mutations of the cysteine residues [C24S (Cys-24-->Ser), C172A and C24S/C172A] to proteolysis assays with peptide substrates, and to surface plasmon resonance binding studies with peptides and viral RNA. We report that the enzyme readily forms dimers via disulphide bridges involving Cys-24. Dissociation constants (K(D)) for peptides were in the millimolar range. The binding kinetics for the peptides were characterized by k(on) and k(off) values of the order of 10(2) M(-1) x s(-1) and 10(-2) to 10(-1) s(-1) respectively. In contrast, 3C binding to immobilized viral RNA, representing the structure of the 5'-terminal domain, followed fast binding kinetics with k(on) and k(off) values beyond the limits of the kinetic resolution of the technique. The affinity of viral RNA depended strongly on the dimerization status of 3C. Whereas monomeric 3C bound to the viral RNA with a K(D) in the millimolar range, dimeric 3C had a significantly increased binding affinity with K(D) values in the micromolar range. A model of the 3C dimer suggests that spatial proximity of the presumed RNA-binding motifs KFRDI is possible. 3C binding to RNA was also promoted in the presence of substrate peptides, indicating co-operativity between RNA binding and protease activity. The data imply that the dual functions of 3C are mutually dependent, and regulate protein and RNA synthesis during the viral life cycle.

Carcinoma, Hepatocellular↗

PubMed2005

Replication and in vivo repair of the hepatitis A virus genome lacking the poly(A) tail.

The precise role of the poly(A) tail at the 3' end of the picornavirus RNA genome and the cellular factors that control its homeostasis are unknown. To assess the importance of the poly(A) tail for virus replication, the genome of the slowly replicating hepatitis A virus (HAV) with and without a poly(A) tail was studied after transfection into cells maintained under various conditions. A tailless HAV genome had a shorter half-life than a poly(A)-containing genome and was unable to replicate in quiescent cells. In dividing cells, the tailless RNA gave rise to infectious virus with a restored poly(A) tail of up to 60 residues. Cells arrested at the G(0) and the G(2)/M phase produced lower amounts of infectious HAV than cells in the G(1) phase. These data suggest that the 3' poly(A) tail of HAV can be restored with the help of a cellular and/or viral function that is regulated during the cell cycle.

Cell Cycle↗

PubMed2004

Interference of hepatitis A virus replication by small interfering RNAs.

The rate of acute liver failure due to hepatitis A virus (HAV) has not decreased, and therapy of severe infections is still of major interest. Using a DNA-based HAV replicon cell culture system, we demonstrate that small interfering RNAs (siRNAs) targeted against viral sequences or a reporter gene contained in the viral genome specifically inhibit HAV RNA replication in HuhT7 cells. Combinations of siRNAs were more effective suppressors of HAV RNA replication. Also, siRNAs targeted against HAV 2C and 3D inhibited the expression of the respective protein. Expressions of endogenous beta-actin and double-stranded-specific RNA-activated serin/threonine kinase (PKR) were unaltered, demonstrating that the siRNA inhibitory effect was not connected to interferon inhibition, but rather was specifically targeted against HAV RNA. These results suggest that RNA interference might ultimately be useful in treatment of severe HAV infection with or without chronic liver diseases.

Actins↗

PubMed2003

Homogeneous hepatitis A virus particles. Proteolytic release of the assembly signal 2A from procapsids by factor Xa.

Among the picornaviridae, hepatitis A virus (HAV) is unique in that its assembly is driven by domain 2A of P1-2A, the precursor of the structural proteins (Probst, C., Jecht, M., and Gauss-Müller, V. (1999) J. Biol. Chem. 274, 4527-4531). Whereas infected individuals excrete in stool mature HAV capsids with VP1 as the major structural protein, its C-terminal extended form VP1-2A is the main component of immature procapsids produced in HAV-infected cells in culture. Obviously, a postassembly proteolytic step is required to remove the primary assembly signal 2A from VP1-2A of procapsids. Mutants of VP1-2A were expressed in COS7 cells to determine the cleavage site in VP1-2A and to test for the cleavage potential of viral and host proteinases (factor Xa and thrombin). Site-specific in vitro cleavage by factor Xa and thrombin occurred in procapsids that contained VP1-2A with engineered cognate cleavage sites for these proteinases. Interestingly, factor Xa but not thrombin liberated mature VP1 also from native procapsids in an assembly-dependent manner. The data show that domain 2A, which is required for pentamerization of its precursor polypeptides and thus for the primary step of HAV assembly, is removed from the surface of immature procapsid by a host proteinase. Moreover, our data open a novel avenue to produce homogeneous HAV particles from recombinant intermediates by in vitro treatment with exogenously added proteases such as factor Xa or thrombin.

Amino Acid Sequence↗

PubMed2002

Chimeric hepatitis A virus particles presenting a foreign epitope (HIV gp41) at their surface.

Hepatitis A virus (HAV) protein 2A has been demonstrated to be involved in virus morphogenesis and suggested to be located on the surface of the particle. To determine whether this protein can function as a target structure to harbor and expose foreign epitopes on HAV particles, a full-length HAV cDNA, containing a seven amino acid stretch of human immunodeficiency virus type 1 (HIV-1) envelope protein gp41, was constructed. Following vaccinia virus MVA-T7-mediated expression of the cDNA in COS7 and Huh-T7 cells, chimeric HAV particles, exposing the foreign epitope gp41 on their surface, were produced. These particles were found to be empty capsids (70S), as judged by immunospecific enzyme linked immunosorbent assay (ELISA) on sucrose gradient fractions and immunoelectron microscopy. The immunological detection of VP1-2A harboring the gp41 epitope of HIV suggests that the 2A domain of HAV is suitable to present foreign antigenic epitopes.

Antigens, Viral↗

PubMed2002

A vaccinia virus MVA-T7-mediated recovery of infectious hepatitis A virus from full-size cDNA or from two cDNAs, both by themselves unable to complete the virus life cycle.

The replication-deficient vaccinia virus (VV) MVA-T7 produces large amounts of T7 RNA polymerase and permits efficient protein expression from cDNA of T7-promoted genes. Yet, unlike recombinant VV vTF7-3, (VV) MVA-T7 produces no cytopathic effect in primate cells, thus allowing the study of processes with slow kinetics. We have applied MVA-T7 to aid genome expression of HAV, a representative of the Picornaviridae family that is well known for its inefficient replication in mammalian cell cultures. After cDNA transfection and MVA-T7 infection, empty capsids and mature HAV particles were formed with different kinetics and were characterized by their morphology, protein content, and infectivity. The data suggests that HAV genome replication is initiated from RNA, which was transcribed in vivo by the MVA-T7-encoded T7 RNA polymerase. HAV genome replication was also demonstrated in a recombination assay. After co-expression of two subgenomic HAV cDNAs, both by themselves unable to complete the viral life cycle, infectious HAV was rescued, indicating that replication-dependent genetic recombination has occurred. We propose that the high-level genome expression mediated in vivo by the VV-encoded T7 RNA polymerase augments the amount of viral RNA, such that replication of viruses poorly replicating in cell cytoplasm is detectable.

Bacteriophage T7↗

PubMed2002

Replication of a hepatitis A virus replicon detected by genetic recombination in vivo.

Unlike other picornaviruses, hepatitis A virus (HAV) replicates so inefficiently in cell culture that the study of its RNA biosynthesis presents a major experimental challenge. To assess viral RNA replication independent of particle formation, a subgenomic replicon representing a self-replicating RNA was constructed by replacing the P1 domain encoding the capsid proteins with the firefly luciferase sequence. Although translation of the HAV replicon was as efficient as a similar poliovirus replicon, the luciferase activity derived from replication of the HAV construct was more than 100-fold lower than that of poliovirus. The replication capacity of the HAV replicon was clearly demonstrated by its ability to recombine genetically with a non-viable, full-length HAV genome that served as capsid donor and thus to rescue a fully infectious virus. In contrast to a replication-deficient replicon, co-expression of the genetically marked and replication-competent HAV replicon with several lethally mutated HAV genomes resulted in the successful rescue of infectious HAV with a unique genetic marker. Our data suggest: (i) that autonomous HAV RNA replication does not require sequences for the HAV structural proteins; and (ii) that low-level genome replication can unequivocally be demonstrated by the rescue of infectious virus after co-expression with non-viable genomes.

Animals↗