Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
Microbiology & Immunology
Non Technical Summary
Animal herpesvirus including Bovine, Equine (BHV-1 and EHV-1, respectively) and Suid Herpesvirus -1 (also known as Pseudorabies virus (PRV) can cause pervasive and serious illnesses and severe economic losses. All three viruses have a similar capacity to infect sensory neurons and enter a latent form of infection. This latency, and the resulting reactivation that leads to overt disease, complicate efforts to control and limit the damage of viral infection. We study the neuronal transmission of PRV to understand the mechanisms of viral and host restrictions to viral spread. Importantly, we have observed that neuronal spread that follows reactivation involves a small number of viral particles transmitted to mucosal tissues. This limited number of particles has important effects on recognition by host innate immune signaling as well as potential ramifications on disease severity.We have hypothesized the nature of the restriction on neuronal spread is mediated by a process known as Superinfection Exclusion. For many species of virus, the first virion to infect a cell will exclude any subsequent virion entry, preventing that cell from becoming "superinfected". This process, called superinfection exclusion (SIE) is important because many aspects of viral pathology and transmission are affected by the number of virions that infect a single cell. In this proposal, we aim to discover the mechanism of an early form of PRV SIE and the transcriptional responses that govern exclusion in the nervous system. Currently, there is a critical knowledge gap regarding the mechanism and effectors of early exclusion preventing experimental manipulation of SIE. The ability to specifically target and manipulate SIE is necessary to study how SIE affects neuronal pathogenesis and spread in vitro and in vivo. The experiments in this proposal aim to bridge this gap by identifying how early PRV SIE blocks infection, and the specific cellular and viral genes that contributes to early SIE.This proposal will identify the mechanism of early PRV SIE and produce the tools necessary to alter levels of exclusion through the manipulation of gene expression. Once these tools and targets have been obtained, we can quickly move into applicable in vitro and in vivo studies that will evaluate the effect of early PRV SIE on neuronal transmission and viral pathogenesis. Understanding how PRV induces early SIE will help us to understand the transmission of the other animal herpesviruses within the nervous system and potentially be exploited by new antiviral therapies directed at treating and preventing herpesvirus associated diseases.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Several members of viral family alpha-herpesvirinae are pathogens that have a wide and pervasive impact on animal health, agricultural practices and economic productivity. Viruses in this group include Equine Herpesvirus-1 (EHV-1), Bovine Herpesvirus-1 (BoHV-1) and Suid Herpesvirus-1, also known as Aujesky's disease or Pseudorabies virus (PRV). All three viruses share the hallmarks of neurological infections, long periods of latency that complicate vaccination and control efforts, and the capacity to elicit severe disease [1,2]. Despite years of research, we understand surprisingly little about the pathways that regulate neuronal transmission of viral infection. We have previously found that neuronal spread of infection involves limited numbers of infectious particles transmitted between cells [3,4]. These limitations have uncharacterized effects on how these viruses interact with host immune responses and adapt to evolutionary pressures imposed by vaccination. The transmission of infectious viral particles during neuronal infection has important implications on viral activation of innate immune responses and evolution. Transmission can be quantified through experimental models of homologous coinfection; when multiple infectious particles of the same virus enter a single cell. Increasing homologous coinfection enhances PRV viral replication, increases the severity of disease and promotes the fitness of viral populations. Restrictions on homologous coinfection have been identified through our work on intercellular spread of PRV infection. One restriction we have observed is a time dependent process of cellular superinfection exclusion; when a second virus is prevented from infecting a cell (Criddle et al., 2016). A second restriction to coinfection occurs during viral spread, where neurons transmit a single infectious viral particle to spread infection (Taylor et al., 2012). The mechanisms that mediate the two restrictions to coinfection are currently unknown, but may be mediated by innate antiviral responses activated by IFN signaling. We hypothesize that host interferon signaling regulates the restrictions to PRV coinfection during viral transmission between mucosal epithelium and neurons. Characterizing the effects of Interferon signaling and Superinfection exclusion on the neuronal transmission of PRV will facilitate new methods to restrict infection and potentially prevent the outgrowth of vaccine evasion mutants.Our major goal is to identify the molecular factors that regulate PRV infection and spread within an infected host. We aim to answer the critical questions of how exclusion affects virions and what genes are responsible. To this end, we propose experiments that will quantify PRV virion entry, assess the impact of IFN activation and signaling on SIE and replication and characterize the transcriptional responses that correlate with SIE and IFN induction. Preliminary data presented in this proposal suggests that SIE blocks viral entry. Additionally, our preliminary work points to differential effect of antiviral transcription factors needed for IFN signaling. Finally, we present preliminary evidence that differential transcriptional responses correlate with SIE induction.SO1. Quantifying viral entry during early SIE.We hypothesize that early infection of a cell by an PRV virion blocks the entry of superinfecting virions. To test this hypothesis, we will use two assays with differently labeled virions to quantify virion fusion and capsid uptake--two steps in the process of virion entry required for viral infection. We will measure viral fusion during direct inoculation of cells not previously infected, as well as in cells already infected by unlabeled virus. This will be done by detecting beta-lactamase (Bla) fused to an essential virion component, which will be delivered to the cytoplasm of the target cell. In parallel, capsid uptake will be quantified by monitoring fluorescently labeled capsids that accumulate within target cells. Superinfecting virion fusion and capsid uptake will also be measured during PRV and ICP4-null viral infection in order to compare entry during conditions where exclusion is not implemented. Together, these experiments will determine the extent to which virion entry is blocked. Identifying the step that is interrupted during viral infection will facilitate further experimentation to determine the mechanism of PRV SIE. Further work will evaluate the pervasiveness of entry blocks during SIE for BHV and EHV infections.SO2: Cellular Antiviral signaling and viral superinfection exclusion. IFN dependent antiviral responses are associated with mechanisms of superinfection exclusion for other viruses. We hypothesize that IFN signaling mediates PRV superinfection exclusion to limit coinfection in epithelial cells. We will leverage primary fibroblast cell cultures from transgenic mice lacking IFN receptors that mediate Type I, Type II or Type III IFN responses. We will test these IFN-signaling deficient cells for the capacity to implement PRV superinfection exclusion. Additionally, we will examine the effect of viral mutants lacking genes that function as IFN-antagonists on superinfection exclusion. Our preliminary results suggest Type I IFN-signaling antagonizes superinfection exclusion, allowing for increased coinfection to occur. Further experiments will evaluate the role Type II and III IFN signaling and antiviral antagonism of these pathways on PRV, BHV-1 and EHV-1 superinfection.SO3: Identification of transcriptional responses required to establish SIE.We have found that transcriptional regulation is important to implement SIE. PRV mutants lacking the immediate-early viral transcription factor ICP4 are unable to establish SIE. Additionally, a cell line with a mutation in the RNA polymerase II co-factor TATA-Box binding protein (TBP) do not establish SIE. Prior work has shown that ICP4 and TBP directly interact to transcriptionally regulate gene expression during PRV infection. We hypothesize that ICP4 and TBP transcriptionally regulate genes needed to establish SIE. To test this hypothesis, we will utilize RNA sequencing methods to identify viral and cellular transcripts that are differentially regulated during conditions of SIE. RNA libraries will be generated for wild-type and ICP4-null PRV infections in both wild-type and TBP mutant cells. Comparative analysis of individual RNA transcript levels will lead to a list of genes that are increased under conditions that establish SIE. Identification of transcriptionally upregulated genes will lead to the viral and cellular mediators of SIE.Summary: This proposal will characterize a novel restriction on the neuronal spread of PRV infection. These findings will allow improved modeling of intra-host spread and identify points for potential manipulation for improved antiviral therapeutics. Additionally, these findings can be extrapolated to our understanding of EHV-1 and BHV-1 infections. Novel mechanisms of disease control and treatment will only improve dairy and beef production and other ungulate species.
Project Methods
Outcomes: The long-term outcomes of this work will include: 1) a better understanding of how viral and cellular responses alters coinfection of spread of alphaherpesviruses both within cells and during neuronal infection, 2) the identification of novel pathways that can be potentially exploited to limit viral replication and spread in the absence of adaptive immune responses, and 3) characterization of the molecular pathways that need to be exploited to improve vaccine efficacy and limit the outgrowth of vaccine evasion mutants. Through this work we have the potential to understand unknown aspects of alphaherpesvirus infection that can be exploited to improve agricultural outputs from cattle operations in Montana as well as guard against possible scenarios if/when current vaccine and epidemiological controls lose their effectiveness.SO 1. Quantifying viral entry during early SIERationale. The experiments proposed here will quantitate PRV infection at both virion fusion and capsid entry during SIE.Methods: Directly labeled virions to measure capsid entry into cells. We will utilize a previously characterized and published recombinant PRV expressing mRFP-VP26 fusion protein (PRV 180) [67,68]. The mRFP fusion labels virions with red fluorescence, allowing direct visualization of capsid assemblies under microscopy and flow cytometry applications.Virion fusion will be assayed using a recombinant PRV strain that incorporates the enzyme beta-lactamase [65,66] (Bla) fused to a virion component protein VP22 (VP22-Bla). As virions fuse with the plasma membrane, the VP22-Bla protein is deposited into the cell's cytoplasm. The extent of virion fusion with target membranes is measured by the extent of beta-lactamase cleavage of a fluorescent substrate.To quantify how SIE effects virion fusion or capsid uptake, Bla-VP22 or mRFP-VP26 containing virions will be inoculated under conditions of SIE induction. Cells will be infected with either wild-type PRV or IE180-null viruses. Additionally, either the mRFP-VP26 PRV recombinant or the VP14-Bla PRV recombinant will be applied simultaneously (T0) or under conditions of delayed inoculation (T3). Cells are first inoculated with the wild-type virus (T0). Quantification of plasma membrane fusion by virions incorporating Bla will be analyzed by flow cytometry after labeling by and subsequent cleavage of CCF2AM. Quantification of virion entry by mRFP-VP26 labeled capsids will be directly analyzed by flow cytometry one hour after inoculation.Expected Outcomes: Our preliminary data suggests a reduction in virion fusion during SIE. These results are consistent with the model that SIE is blocking a step of viral entry at or before virion fusion. If SIE were interfering with a later step of virion entry, then the Bla assay will show no differences between co-inoculation and delayed inoculation infections. If the lack of ICP4 expression directly correlates with a lack of SIE we would expect to see enhanced capsid uptake and virion fusion when PRV IE180-null is used as a primary inoculum.SO 2: Evaluating the importance of IFN signaling for PRV superinfection exclusion.We will test the role of IFN signaling on PRV coinfection and superinfection exclusion.Methods: To establish the conditions of PRV superinfection exclusion we will inoculate two fluorescent protein expressing viruses at different times after initial infection. Cells are infected with two recombinants of PRV Becker (wild-type virus), one expressing a Cyan FP (Turquoise2) and the other expressing Yellow FP (eYFP). The viruses are either applied together for simultaneous coinfection (T0) or the CFP virus is inoculated and coinfection with the YFP virus delayed until later times post infection (2, 3, and 4 hours post initial infection (hpi). Infections progress to fulminant FP expression prior to fluorescent microscopy or flow cytometric analysis. The loss of YFP expression correlates with an absence of YFP virus production. We will use this core design to implement two different types of experiments.Experiment #1 - Coinfection of IFN-signaling knockout cell cultures. To address the role of IFN signaling in the implementation of PRV superinfection exclusion, we will use cells harboring gene deletions for IFN receptors. We will acquire fibroblast cell cultures from knockout mice. Initially, we will target Type I, II, and III IFNs by working with knockout mice deleted for specific receptors, including: Type I IFNAR1-/-, Type II IFNGR1-/- and Type III IL28R-/- (IFNLR).Experiment #2 - Coinfection with IFN-antagonism null PRV mutants. To test the effect of PRV antagonism of IFN signaling and cellular antiviral effectors, coinfections will be performed with PRV mutants lacking one of 3 viral genes; ICP0, US3 and ICP22. Each of these genes differentially antagonizes IFN signaling and the establishment of the cellular antiviral defenses.Expected Outcome and Alternative approach: Interactions between IFN signaling pathways is an added complication to the interpretation of our experimental approach. The activation of different IFN pathways synergize or antagonize responses, producing altered host responses [42,69,70]. To address this issue would require production of combinatorial deletions of host IFN receptors. We will work with the expertise of colleagues here at Montana State University to use use the CRISPR/Cas9 gene editing system to disrupt or delete genes in porcine fibroblast or immortalized porcine cells (i.e. PK15 cells) and to produce pairwise deletion of cellular IFN receptors. In a similar manner, once an IFN signaling pathway has been identified, deletion of individual effector genes can be made with Cas9. These are two strategies that will be considered for development in future funding proposals.SO 3. Identification of transcriptional responses required to establish SIE.Rationale: Early PRV SIE requires a transcriptional response during early events after virion entry. To begin evaluating the role of transcriptional regulation during PRV SIE, we needed to determine if altered viral transcription influenced SIE.Method: We will employ RNA sequencing methods to evaluate transcriptional differences in viral and cellular genes early during infection. A comparative analysis of transcriptional differences between infection conditions will identify genes associated with SIE. We will compare RNA transcripts of ICP4-null PRV infected and wild-type PRV infected mouse fibroblasts. If additional resources are available, we will then compare RNA transcripts from wild-type or DN -TBP mouse fibroblasts infected with wild-type PRV. The different infection conditions will facilitate identification and comparative analysis of transcripts that are involved in SIE. Our transcriptomic analysis will require three methodologies: acquiring high quality RNA from infection conditions with and without exclusion, bioinformatic analysis of RNAseq data for statistically significant differences, and validation of gene targets by qRT-PCR.Expected Outcomes: RNA sequencing will produce a large amount of data, but comparative analysis of sequencing data will produce a limited number of potential gene targets. Using the previously described infection conditions that do not support SIE (ICP4-null virus, DN-TBP cells) will effectively identify the genes needed for SIE. Our strategy of comparing two conditions that are deficient in SIE will allow elimination of the majority of cellular transcripts that are unchanged or uninvolved in SIE. Our statistical analysis can also be validated by looking for genes that have been previously identified by RNA-seq to be upregulated during PRV infection [83].