Virus Attachment

Counting a viral transcript in a cell does not mean the cell is infected, as this count can come from a virus attached to the surface of the cell, ambient virus in the suspension, or from read misalignment. Given the reported shared 3′ poly(A) tail in coronavirus transcripts [78 (link)], we were unsure whether we could correctly capture the different ORFs using the 10x Genomics 3′ gene expression library. Therefore, we aligned the viral reads to a genome-wide single “exon,” i.e., a count is given for a read mapped to SARS-CoV-2 ORFs and intergenic regions. These counts were used to infer individual cells’ infectious state. To filter out cells with viral genome transcript counts that may result from viral cell surface attachment, ambient virus in the droplet suspension, or read misalignment, we considered infected cells to have 10 viral transcripts counts. This threshold of 10 viral transcripts to define an infected cell was determined empirically as it represents an inflection point (S1A Fig). While the mock condition is not expected to have viral counts, we did observe a small number of reads that could be attributed to misalignment or transcript leakage. We observed only 5 mock cells with full SARS-CoV-2 viral genome transcript counts 10 transcripts. These criteria allowed us to find 144 infected cells at 1 dpi, 1,428 cells at 2 dpi, and 3,173 cells at 3 dpi. To quantify the extent to which an individual cell is transcriptionally similar to an infected cell, we used a previously developed graph signal processing approach called Manifold Enhancement of Latent Dimensions (MELD) [79 (link)]. We encoded a raw experimental score for each cell in the dataset such that −1 represents a bystander or uninfected cell, and +1 represents an infected cell. Using the kernel from the BB-kNN graph (described above), these raw scores were smoothed in the graph domain, yielding a metric for transcriptomic similarity to infected cells per cell that represents the extent to which an individual cell is transcriptionally similar to infected cells. For example, if an infected cell is more transcriptionally similar to bystander cells, it will have a low value of the metric, closer to −1. Cells in a cluster of transcriptionally similar cells that are infected will have values closer to +1, indicating similar transcriptomic signatures to infected cells. For summary statistics, this score was stratified by cell type and condition.

A 6-well plate with 6 × 10⁵ RD cells/well was prepared and incubated overnight at 37 °C in 5% CO₂. Prior to viral infection, the complete growth medium (DMEM supplemented with 10% FBS) was removed and approximately 1 mL serial 10-fold dilutions of virus inoculum was added to the cells for 1 h at room temperature with gentle shaking to allow for virus attachment. After 1 h incubation, the inoculum was removed and replaced with 2 mL of 1.2% w/v carboxylmethylcellulose. After 72 h incubation, the plaque medium was removed and the cells were fixed with 4% formaldehyde and stained with 0.5% crystal violet. The plaques were visible against a white background.

For the gene delivery we used AAV2 serotype that has a high tropism to RGC, long-term expression and low immunogenic profile. The high tropism of AAV2 could be due to the high expression of heparin sulfate proteoglycan by RGC, which mediates attachment of the AAV2 virus. After general and topical anesthesia was administered, the right eye of each animal was rinsed with a 10% povidone-iodine solution (Betadine, EGIS, Hungary). Animals from the experimental group received a 3 μL intravitreal injection of 10⁸ AAV-shRNA-HuR. The control group received 3 μL injections containing 10⁸ AAV-shRNA-scramble control. The injection was performed using a 5 μL Hamilton syringe with a 6 mm-long 34 G needle. The microneedle was gently introduced into the vitreous cavity, avoiding contact with the lens. After injection, the eye was topically treated with 2% chloramphenicol ointment (Detreomycin 2%, Chema-Elektromet, Poland) and secured with a clean dressing. Animals were housed singly in new cages for the next 24 h to avoid injury or infection after the procedure. The intravitreal injection of MT was performed in a similar manner as described above.

Across all sets (feature selection, training, extra-familial validation), six families of respiratory viruses were included in this study: Coronaviridae, Paramyxoviridae, Pneumoviridae, Adenoviridae, Orthomyxoviridae, and Herpesviridae. Each of the viruses within these families has a protein responsible for viral attachment and host cell entry, which will be referred to herein as the “spike” protein (see Fig 1A). For Coronaviruses, it is the Spike S Glycoprotein which is aptly named because it projects from the surface of the virion (Fig 1B) as do the other “spike” proteins. Note that for Influenza Virus A within the Orthomyxoviridae family, we selected Hemagglutinin as the equivalent of the “spike” over Neuraminidase as the latter primarily prevents virion aggregation and as such serves more as a helper protein to the role of the former in determining cell entry [25 (link)].
A total of 50 viral sequences (ranging from 4 to 12 for each virus family) encoding 360 proteins were utilized (see Table 1 for a list of sequences). Specifically, in the feature selection set we included 7 Coronaviridae sequences representing 7 viruses; in the training set, we included 7 different Coronaviridae sequences representing 7 viruses, 4 Paramyxoviridae sequences representing 4 viruses, 12 Pneumoviridae sequences representing 2 viruses, 8 Adenoviridae sequences representing 1 virus, and 8 Orthomyxoviridae sequences representing 1 virus. Finally, for the extra-familial validation set, we included 4 Herpesviridae sequences representing 4 viruses. See Table 2 for the number of “spike” vs. non-spike proteins for each virus family.

MDCK cells were pre-seeded on a 96-well plate at the density of 2 × 10⁴ cells per well 24 h in advance. 0.005 MOI of H1N1 was used for all inhibition experiments. For the virus pretreatment groups, 10 μg/mL CD-6′SLN were incubated with H1N1 at 37 °C for 1 h and the mixture was added to the cell cultures. For the cell pretreatment groups, the cells were first treated with 10 μg/mL CD-6′SLN for 1 h and then infected with the virus. For the virus co-treatment groups, 10 μg/mL CD-6′SLN were added simultaneously with the virus to the cells. For the post-infection treatment groups, the virus was added to the cells, after 1 h incubation for viral attachment, the inoculum was removed and a medium containing 10 μg/mL CD-6′SLN was added to the cells. After 24 h incubation, the medium was removed and cells were fixed with methanol for 1 h. Then the LIGHT DIAGNOSTICS Flu A monoclonal antibody (1:100 dilution, Merck Millipore, Burlington, MA, USA) was added and incubated for 1 h at 37 °C. The cells were washed with wash buffer (PBS + Tween 0.05%) three times, followed by adding anti-mouse lgG, HRP-linked antibody (1:500 dilution, Cell Signaling Technology, Danvers, MA, USA). After 1 h, the cells were washed and the DAB solution was added. Infected cells were counted and percentages of infection were calculated by comparing the number of infected cells in treated and untreated conditions.
The EC₅₀ measurements of CD-6′SLN, CD-(S-C11-COOH)₇, and CD-(Mal-PEG8)7-6′SLN were performed using the virus pretreatment method. Prism 9 (GraphPad, San Diego, CA, USA) was used to estimate the EC₅₀ from the dose-response curve (Figure S1).