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Due to the zoonotic nature of many coronaviruses, we are interested in understanding functional differences in which viruses deal with host cell defenses across different host species. By the example of SARS-coronavirus papain-like protease and the MERS-coronavirus accessory proteins 4a and 4b we investigate protein variants from epidemic and reservoir-associated coronavirus strains. We use in-vitro assays and reverse genetic systems to explore the functional diversity of these immune modulating factors.
Modulation of infection and defense by MERS-coronavirus protein 4b
The Middle East respiratory syndrome-coronavirus (MERS-CoV) is a prototypic prepandemic agent for which no approved treatment or vaccine is available. It causes lower respiratory tract infection with hospital based mortality as high as 30%. A study conducted by Hocke et al. in 2013 shows a unique cellular tropism in that the virus infects virtually all cells of the human alveolar wall and induces tissue damage including junctional deterioration. As human infection may cause viral adaptation with increase of transmissibility, MERS-CoV is considered a serious threat to global health security.
Within the MERS-CoV genome, protein 4b (p4b) has a dual role. On the one hand, p4b acts as a 2´,5´-phosphodiesterase that degrades 2´,5´- oligoadenylate and thus prevents activation of RNAse L, an important interferon-stimulated antiviral effector and enhancer of interferon induction. On the other hand, p4b encodes a functional nuclear localization signal and competes with the transcription factor NF-κB for nuclear translocation.
By this mechanism, p4b prevents the activation of NF-κB, which controls the production of pro-inflammatory cytokines and cell survival.
Although the main site of activity of p4b´s RNAse L antagonism should be the cytosol, the 4b gene encodes a functional nuclear localization signal that is lost in some virus variants. The loss of nuclear localization may provide additional p4b activity in the cytosol, thereby conferring additional interferon antagonism. On the other hand, the NFkB-dependent immune response may also increase, as p4b no longer competes with NFkB for nuclear localization.
However, so far p4b has only been studied using heterologous expression systems. This approach identifies the protein´s mechanistic function but does not provide any insight into the protein´s phenotypic relevance in the context of the full MERS-CoV replication cycle. Moreover, these variants with alterations of the gene encoding p4b in MERS-CoV have not been examined in relevant models of human lung infection that allow for assessments of replication level, cell tropism and organ-specific cytokine expression.
Because these changes may have relevance in the context of viral infection of humans, the aim of this project is to understand functional consequences of p4b variability particularly in the human lung. Therefore, artificial and naturally occurring p4b variants will be engineered into the MERS-CoV genome by reverse genetics and phenotypic consequences will be studied in human lung infection models. These mutations will include genetic changes that may occur, or have already occurred, during human-to-human transmission chains. Particular focus will be on lung specific replication levels in the different human alveolar cell lines and host gene expression of cytokines depending on p4b´s nuclear localization.
Our work on MERS-CoV protein 4b is funded by the Transregio Collaborative Research Centre “Innate Immunity of the Lung: Mechanisms of Pathogen Attack and Host Defence in Pneumonia” (Principle investigator: Christian Drosten).
Risk assessment in pre-pandemic respiratory infectious diseases (RAPID)
The project will determine whether MERS-CoV can increase its replication level upon adaptation to human cells. We will create a MERS-CoV that is deficient in its error correction function and thereby shows an increased error frequency that attenuates the virus, but enables it to adapt faster to cells than wild type. We will conduct serial passaging in cell cultures, air liquid interface cultures, as well as lung explant cultures. If MERS-CoV has adaptive capability to optimize human infection, the attenuated virus will show a limited recovery of replication level after passage. In spite of experimental adaptation, the virus will continue to be severely attenuated due to the lack of essential enzyme function.
Our work on MERS-CoV error correction enzyme is funded by a grant from the Federal Ministry of Education and Research (Principle investigator: Christian Drosten).