Results

4. ANTIVIRAL RESPONSES DIFFERENTIALLY MEDIATE SUPERINFECTION

4.3. Results

4.3.1. Type I interferon drives reovirus superinfection exclusion in vitro.

Type 3 reoviruses are known to potently induce type I interferon signaling in response to infection in vitro. In Chapter 3, I show that primary infection with a type 3 reovirus can limit replication of superinfecting viruses in L cells (Fig. 3-7B-C). To determine whether reovirus superinfection exclusion is mediated by type I interferon signaling, I primarily infected wild-type, IFNAR knockout (KO), and MAVS KO SVECs with a genetically- barcoded T3D^I reovirus (BC T3D^I)or T1L reovirus for 24 h before superinfecting with WT T3D^I. After a 24 h superinfection, I harvested cells and quantified superinfecting virus transcript abundance by RT-qPCR. As shown with L cells, in wild-type SVECs, the abundance of superinfecting virus transcripts was significantly decreased following primary infection with the BC T3D^I virus, compared to mock-infected controls (Fig. 4-1).

Transcripts were also reduced following T1L primary infection, although not to the extent observed following T3D^I primary infection (Fig. 4-1). In IFNAR KO cells,

superinfecting virus transcripts were significantly reduced by primary infection, but the magnitude of this decrease was smaller than that observed in wild-type cells. There was no significant difference between superinfecting virus transcript levels following T1L and T3D^I primary infection in IFNAR KO SVECs (Fig. 4-1). Together, these data suggest that amplification of the interferon response through IFNAR likely contributes to reovirus superinfection exclusion in vitro.

Figure 4-1. Signaling through IFNAR mediates reovirus superinfection exclusion. Wild- type or IFNAR KO SVECs were primarily infected with MOI = 10 PFU / cell with T1L, BC T3D^I, or media only (mock) for 24 h before adsorption with WT T3D^I. Superinfecting WT virus RNA concentration was quantified 48 h post primary infection by RT-qPCR. n = 3 plaque-purified clones from one independent experiment. Statistical significance

determined by one-way ANOVA with Tukey’s multiple comparisons test (* = p < 0.05;

**** = p < 0.0001).

4.3.2. Type I and type III interferon signaling does suppress reovirus reassortment frequency during coinfection in vivo.

Type I interferon restricts reovirus replication at secondary sites of replication, including the heart and brain (Phillips et al., 2020). However, type III interferon is the primary host defense against reovirus infection in the intestine (Peterson et al., 2019; Baldridge et al.,

2017). To determine whether interferon signaling influences reovirus reassortment in vivo, I quantified reassortment frequency from intestinal homogenates of 3-day old wild- type, IFNAR KO, and interferon-lamda receptor (IFNLR) KO B6 mice following a 48 h coinfection with WT and BC T3D^I reoviruses. Reassortment proceeded efficiently in mice of all genetic backgrounds, with reassortant viruses making up between 20-40% of viral progeny from each group (Fig. 4-2A-C). Additionally, WT and BC viruses were similarly represented in coinfection progeny, suggesting that there were no major fitness differences for the two viruses in vivo. Thus, type I interferon is a driver of reovirus superinfection exclusion in vitro, but type I and type III interferon signaling do not influence reassortment frequency in the intestine during coinfection.

Figure 4-2. Reassortment occurs frequently during coinfection in mice, irrespective of genotype. 3-day old wild-type B6 (A), IFNAR KO (B), and IFNLR KO (C) mice were

perorally inoculated with 1 x 10⁷ PFU WT and BC. At 48 h post-inoculation, mice were euthanized and reassortment frequency was quantified using HRM. Honeycomb plots indicate genotypes of individual coinfection progeny viruses. Each row represents an independently isolated clone. Each column represents the indicated genome segment.

Black indicates WT genome segments, and red indicates BC genome segments. n = 7 mice with 5 progeny clones analyzed per experiment. Each block of 5 progeny clones is derived from the same mouse.

4.3.3. Reovirus reassortants were not detected during superinfection in vivo.

Administration of IFN-β prior to infection potently limits reovirus replication (Jacobs and Ferguson, 1991). As such, it is possible that preexisting interferon responses are required to limit reovirus superinfection and reassortment. Therefore, I sought to determine whether type I and type III interferon signaling might influence reassortment frequency during superinfection in vivo. To do this, 3-day old wild-type, IFNAR KO, and IFNLR KO mice were first infected with BC reovirus for 24 h before superinfection with the WT virus. 24 h after superinfection, mice were sacrificed, intestines were

homogenized, and reassortment frequency was quantified from homogenized intestines by HRM. The BC primary infecting virus was much more highly represented in progeny viruses, constituting 94%, 100%, and 86% of all segments for progeny viruses from the wild-type, IFNAR KO, and IFNLR KO mice, respectively (Fig. 4-3A-C). Following this superinfection time course, no reassortant viruses were detected in any mice,

irrespective of genetic background (Fig. 4-3A-C), in contrast to what was observed during simultaneous coinfection (Fig. 4-2A-C). Thus, despite the presence of both viruses in the superinfection progeny from wild-type and IFNLR KO mice, no reassortment was detected. Taken together, these findings suggest that signaling through type I and type III IFN receptors is not responsible for the lack of reassortment detected during superinfection in vivo.

Figure 4-3. Reassortment does not occur following a 24 h superinfection time delay in mice, irrespective of genotype. 3-day old wild-type B6 (A), IFNAR KO (B), and IFNLR KO (C) mice were perorally inoculated with 1 x 10⁷ PFU BC. 24 h post-inoculation, mice were superinfected with 1 x 10⁷ PFU WT. At 48 h post-inoculation with the BC primary infecting virus, mice were euthanized and reassortment frequency was quantified using HRM. Honeycomb plots indicate genotypes of individual coinfection progeny viruses.

Each row represents an independently isolated clone. Each column represents the indicated genome segment. Black indicates WT genome segments, and red indicates BC genome segments. n = 5-7 mice with 5-7 progeny clones analyzed per experiment, as indicated. Each block of 5 progeny clones is derived from the same mouse.