Development of a Sensitive Detection Method for Alphaviruses and Its Use as a Virus Neutralization Assay
Alphaviruses have a single-stranded, positive-sense RNA genome that contains two open reading frames encoding either the non-structural or the structural genes. Upon infection, the genomic RNA is translated into the non-structural proteins (nsPs). NsPs are required for viral RNA replication and transcription driven from the subgenomic promoter (sgP). Transfection of an RNA encoding the luciferase gene under the control of the sgP into cells enabled the detection of replication-competent chikungunya virus (CHIKV) or Mayaro virus (MAYV) with high sensitivity as a function of the induced luciferase activity. This assay principle was additionally used to analyze virus-neutralizing antibodies in…
A Trans-amplifying RNA Vaccine Strategy for Induction of Potent Protective Immunity
Here, we present a potent RNA vaccine approach based on a novel bipartite vector system using trans-amplifying RNA (taRNA). The vector cassette encoding the vaccine antigen originates from an alphaviral self-amplifying RNA (saRNA), from which the replicase was deleted to form a transreplicon. Replicase activity is provided in trans by a second molecule, either by a standard saRNA or an optimized non-replicating mRNA (nrRNA). The latter delivered 10- to 100-fold higher transreplicon expression than the former. Moreover, expression driven by the nrRNA-encoded replicase in the taRNA system was as efficient as in a conventional monopartite saRNA system. We show that the superiority of nrRNA- ov…
Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins.
Among nucleic acid–based delivery platforms, self-amplifying RNA (saRNA) vectors are of increasing interest for applications such as transient expression of recombinant proteins and vaccination. saRNA is safe and, due to its capability to amplify intracellularly, high protein levels can be produced from even minute amounts of transfected templates. However, it is an obstacle to full exploitation of this platform that saRNA induces a strong innate host immune response. In transfected cells, pattern recognition receptors sense double-stranded RNA intermediates and via activation of protein kinase R (PKR) and interferon signaling initiate host defense measures including a translational shutdow…
APOBEC4 Enhances the Replication of HIV-1
APOBEC4 (A4) is a member of the AID/APOBEC family of cytidine deaminases. In this study we found a high mRNA expression of A4 in human testis. In contrast, there were only low levels of A4 mRNA detectable in 293T, HeLa, Jurkat or A3.01 cells. Ectopic expression of A4 in HeLa cells resulted in mostly cytoplasmic localization of the protein. To test whether A4 has antiviral activity similar to that of proteins of the APOBEC3 (A3) subfamily, A4 was co-expressed in 293T cells with wild type HIV-1 and HIV-1 luciferase reporter viruses. We found that A4 did not inhibit the replication of HIV-1 but instead enhanced the production of HIV-1 in a dose-dependent manner and seemed to act on the viral L…