Fig. 4: Successful HCMV maturation requires induction of dormant neuronal proteins.
From: Human cytomegalovirus induces neuronal gene expression through IE1 for viral maturation
A Schematic of targeted siRNA screen. (B) RT-qPCR quantification of RNA levels of target genes during HCMV infection. These genes include APOE, a lipoprotein associated with Alzheimer’s disease and synaptic vesicle release67,68; CNTFR, a ciliary neurotrophic factor receptor that supports motor neuron axons69; DOC2B a calcium sensor that promotes synaptic vesicle release70; ERC1, a cellular scaffolding protein71; IFI27, an interferon-induced gene expressed in the cerebellum in response to viral CNS infection72; KIF1A, a neuronal kinesin33; LAMA1, a laminin essential for neurite growth73,74; LRRC4B, a transmembrane protein that regulates synapse formation75; MYO5B, a myosin associated with polarity and axon development76; NPTX2 (formerly NARP), a small molecule released in excitatory synapses77; SOX11, a transcription factor associated with neuron development78; and WWC1, a synaptic scaffolding protein79. Knockdown of each gene is normalized to its expression in cells treated with the non-targeting control (NC) at 4 dpi. Bar graphs show mean with error bars indicating SEM. n = 3 biological replicates. P-values by unpaired t-test as follows: APOE**** p = 7.4×10−5, CNTFR* p = 1.14×10−3, ERC1* p = 3.83×10−2, IFI27* p = 1.62×10−2, KIF1A** p = 4.09×10−3, LRRC4B*** p = 9.3×10−4, MYO5B* p = 1.62×10−2, NPTX2**** p = 1.5×10−5, SOX11* p = 3.80×10−2, WWC1* p = 1.62×10−2. C Representative western blot of siRNA knockdown of KIF1A in WT cells during HCMV infection at 4 dpi compared to mock (M). Asterisk indicates a non-specific band. Tubulin is shown as loading control and pp28 is shown as infection control. D Quantification of plaque area produced from supernatant harvested from cells treated with siRNA indicated. Those that reach statistical significance are bolded. Bar graphs show mean with error bars indicating SEM. * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001 by one way ANOVA with follow up Dunnett’s test as follows: NC/IFI27*** p = 9.5×10−4, NC/KIF1A* p = 2.52×10−2, NC/LAMA1* p = 4.3×10−2, NC/NPTX2** p = 8.12×10−3, NC/WWC1*** p = 3.2×10−4, and n values: NC n = 652, APOE n = 593, CNTFR n = 863, DOC2B n = 599, ERC1 n = 591, IFI27 n = 332, KIF1A n = 455, LAMA1 n = 478, LRRC4B n = 546, MYO5B n = 408, NPTX2 n = 514, SOX11 n = 651, WWC1 n = 459. E Representative images of plaque sizes for those with significant differences as indicated. Yellow dashed line frames plaque example. Scale bar indicates 150 µm.
