CONSEJO DE VIGILANCIA

To get an overview how the host cell reacts on an RNA level to an infection by MV, microarray experiments were conducted using rMV vac2 infected B-cells. The viruses used for these experiments were purified by pelletation to remove any cytokines from the supernatant of the stock-producing cells, which could alter the behaviour of infected cells. A B-cell line (DG75) was chosen to represent a cell type which is naturally infected in its host by wildtype measles virus as well as the vaccine strains. The changes in the total mRNA content compared to mock infected cells were subsequently analysed (Fig. 23). They are represented as fold changes of the viral infected cells compared to the mock infected cell population harvested at the same timepoint. Two distinct timepoints were analysed: 24h and 48h post infection with rMV vac2 using an MOI of 1. The total cellular RNA content then was hybridized on Affymetrix Human GeneChip 1.0 arrays and these arrays then analysed for binding of cellular RNAs in the microarray facility of the Cramer group (Genecenter, Munich).

The RNAs detected were 81 fold upregulated or 6 fold downregulated at most compared to mock infected cells after 48h of infection. The differentially regulated cellular RNAs (regulation > 2, p < 0.5) could be clustered into three basic clusters according to their regulation in infected cells over time: Genes which were downregulated (Fig. 23 A), genes which were upregulated (Fig. 23 B) and genes which were strongly upregulated temporarily after 24h, but then declined after 48h (Fig. 23 C).

Figure 23: RNAs regulated by infection with MV vac2 at different timepoints in human B- cells.

DG75 cells were infected with MV vac2 at an MOI of 1 and the total RNA harvested after 24h and 48h subjected to microarray analysis. (A, B, C) Clusters of regulated RNAs according to the scheme above the lists. The official gene symbol and the affymetrix ID of the probes on the microarray are display in the first two columns of the table. The last two columns indicate the fold changes of the RNA detected in comparison to mock infected cells after 24h or 48h. Negative values mean a downregulation, positive values an upregulation. The colour coding was done according to the blue-white-red scheme, blue meaning downregulation, red meaning upregulation. The RNAs displayed showed at least 2 fold regulation and have a p value smaller than 0.5. Only parts of the regulated genes are displayed here, representing the highest changes compared to mock. The complete list of significantly regulated RNAs clustered and unclustered is in the appendix. This experiment represents duplicates. (D) Detection of the L mRNA of measles virus in the samples and relative quantification by qRT-PCR. At 24h the amount of L mRNA was to 1, the value for 48h p.i calculated relatively. The values represent the mean of the duplicates. The mock infected control was negative for L mRNAs and is therefore not displayed here.

The infection was monitored by quantitative realtime PCR using primers specific for the MV L mRNA (Fig. 23 D) and the 24h value was set to 1, as L mRNA could not be detected in mock infected cells. After 48h, the intracellular content of MV L mRNA doubled, indicating that viral multiplication indeed took place.

To characterize these clusters in further detail, pathway analysis was performed using the web based DAVID bioinformatics server. The first cluster could not be annotated automatically (Fig. 24 A). However, it is obvious that most of these are small nucleolar RNAs (SNORDs). Some of these SNORDs play an important role in the 2’-O-methylation pathway, especially the mainly downregulated SNORDs: 78, 26 and 50B. 4-Hydroxyphenylpyruvate Dioxygenase-Like (HPDL), the second most downregulated RNA is largely uncharacterized in literature. RAB3A mRNA encodes a Ras-related protein involved in exocytosis probably by regulating a late step in synaptic vesicle fusion (Zahraoui et al., 1989).

The upregulated genes (Fig. 23 B) can easily be characterized as interferon and infection stimulated genes (Fig. 24 B), their products representing chemotactic signals for immune cells. Most of these genes encode proteins that are classical interferon stimulated genes (ISGs): e.g. IFIT1, IFIT2 or chemokines (CCL5, CXCL11 & 10). These are part of the typical inflammatory and anti-viral response of a cell and are used to attract the adaptive immune system or as an alert signal to neighbouring cells. IFIT1 binds triphosphorylated RNA and inhibits expression of 2’ O-methylation defective RNAs along with IFIT2. They both exhibit direct antiviral activity (Zhou et al., 2013). Secreted CCL5 attracts blood monocytes, memory T helper cells and eosinophils, whereas CXCL11 and 10 are chemotactics for T cells mainly. Notably, the upregulation of the interferon beta mRNA is significant only after 48h to a value of 6-fold, which is fairly low, compared to a 81-fold induction of IFIT2.

Interestingly, the third cluster of genes (Fig. 23 C) contains genes responsible for amino acid metabolism and biosynthesis (Fig. 24 C). Unfortunately there is no annotation for FAM106A (Family With Sequence Similarity 106, Member A), and its function remains unknown. Phosphoserine Phosphatase (PSPH) plays an important role in the biosynthesis of serine, catalysing the last step of L-serine formation. Solute Carrier Family 7 (Anionic Amino Acid Transporter Light Chain, Xc-System), Member 11 (SLC7A11) is an amino acid carrier specific for glutamate and cystein. Cation Transport Regulator Homolog 1 (CHAC1) is a negative regulator of the notch signalling pathway. The Cystathionase (CTH) protein catalyses the conversion of cystathione into cysteine and therefore is also involved in amino

acid metabolism. For ROCK1P1 (Rho-Associated, Coiled-Coil Containing Protein Kinase 1 Pseudogene 1) no detailed annotation or description could be found.

Figure 24: Functional annotation and clustering of the RNAs found to be regulated more than 2-fold in microarray experiments.

Three different clusters were made according to the regulation of the RNAs over time (24h and 48h post infection) and the list of genes submitted for functional annotation and clustering using the DAVID websuite. The full profiles are in the appendix. The pathways are indicated next to the different clusters (A, B and C as in Fig. 23), Count displays the number of genes matching to the pathway which represents the percentage fraction in the next column. Both the p-value and benjamini are indicators of the significance of the overrepresentation of the pathway in the subset of genes submitted for analysis.

In summary, MV infection of B cells induced the expected innate immunity responses apparently, lacking however a robust induction of interferon beta. Notably, the transcription of genes coding for small nucleolar RNAs was severely downregulated.