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ABSTRACTIn recent years, an increasing number of emerging and remerging virus outbreaks have occurred and the rapid development of vaccines against these viruses has been crucial. Controlling the replication of premature termination codon (PTC)-containing viruses is a promising approach to generate live but replication-defective viruses that can be used for potent vaccines. Here, we used anticodon-engineered transfer RNAs (ACE-tRNAs) as powerful precision switches to control the replication of PTC-containing viruses. We showed that ACE-tRNAs display higher potency of reading through PTCs than genetic code expansion (GCE) technology. Interestingly, ACE-tRNA has a site preference that may influence its read-through efficacy. We further attempted to use ACE-tRNAs as a novel viral vaccine platform. Using a human immunodeficiency virus type 1 (HIV-1) pseudotyped virus as an RNA virus model, we found that ACE-tRNAs display high potency for read-through viral PTCs and precisely control their production. Pseudorabies virus (PRV), a herpesvirus, was used as a DNA virus model. We found that ACE-tRNAs display high potency for reading through viral PTCs and precisely controlling PTC-containing virus replication. In addition, PTC-engineered PRV completely attenuated and lost virulence in mice in vivo, and immunization with PRV containing a PTC elicited a robust immune response and provided complete protection against wild-type PRV challenge. Overall, replication-controllable PTC-containing viruses based on ACE-tRNAs provide a new strategy to rapidly attenuate virus infection and prime robust immune responses. This technology can be used as a platform for rapidly developing viral vaccines in the future.

Despite many efforts and diverse approaches, developing an effective herpesvirus vaccine remains a great challenge. Traditional inactivated and live-attenuated vaccines always raise efficacy or safety concerns. This study used Pseudorabies virus (PRV), a swine herpes virus, as a model. We attempted to develop a live but replication-incompetent PRV by genetic code expansion (GCE) technology. Premature termination codon (PTC) harboring PRV was successfully rescued in the presence of orthogonal system MbpylRS/tRNAPyl pair and unnatural amino acids (UAA). However, UAA incorporating efficacy seemed extremely low in our engineered PRV PTC virus. Furthermore, we failed to establish a stable transgenic cell line containing orthogonal translation machinery for PTC virus replication, and we demonstrated that orthogonal tRNAPyl is a key limiting factor. This study is the first to demonstrate that orthogonal translation system-mediated amber codon suppression strategy could precisely control PRV-PTC engineered virus replication. To our knowledge, this is the first reported PTC herpesvirus generated by GCE technology. Our work provides a proof-of-concept for generating UAAs-controlled PRV-PTC virus, which can be used as a safe and effective vaccine.

The transcription factor NF-κB plays a critical role in diverse biological processes. The NF-κB pathway can be activated by incoming pathogens and then stimulates both innate and adaptive immunity. However, many viruses have evolved corresponding strategies to balance NF-κB activation to benefit their replication. Pseudorabies virus (PRV) is an economically important pathogen that belongs to the alphaherpesvirus group. There is little information about PRV infection and NF-κB regulation. This study demonstrates for the first time that the UL24 protein could abrogate tumor necrosis factor alpha (TNF-α)-mediated NF-κB activation. An overexpression assay indicated that UL24 inhibits this pathway at or downstream of P65. Furthermore, co-immunoprecipitation analysis demonstrated that UL24 selectively interacts with P65. We demonstrated that UL24 could significantly degrade P65 by the proteasome pathway. For the first time, PRV UL24 was shown to play an important role in NF-κB evasion during PRV infection. This study expands our understanding that PRV can utilize its encoded protein UL24 to evade NF-κB signaling.

A Pseudorabies virus (PRV) variant has emerged in China since 2011 that is not protected by commercial vaccines, and has not been well studied. The PRV genome is large and difficult to manipulate, but it is feasible to use clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. However, identification of single guide RNA (sgRNA) through screening is critical to the CRISPR/Cas9 system, and is traditionally time and labor intensive, and not suitable for rapid and high throughput screening of effective PRV sgRNAs. In this study, we developed a recombinant PRV strain expressing firefly luciferase and enhanced green fluorescent protein (EGFP) as a reporter virus for PRV-specific sgRNA screens and rapid evaluation of antiviral compounds. Luciferase activity was apparent as soon as 4 h after infection and was stably expressed through 10 passages. In a proof of the principle screen, we were able to identify several PRV specific sgRNAs and confirmed that they inhibited PRV replication using traditional methods. Using the reporter virus, we also identified PRV variants lacking US3, US2, and US9 gene function, and showed anti-PRV activity for chloroquine. Our results suggest that the reporter PRV strain will be a useful tool for basic virology studies, and for developing PRV control and prevention measures.

Despite many efforts and diverse approaches, developing an effective herpesvirus vaccine remains a great challenge. Traditional inactivated and live-attenuated vaccines always raise efficacy or safety concerns. This study used Pseudorabies virus (PRV), a swine herpes virus, as a model. We attempted to develop a live but replication-incompetent PRV by genetic code expansion (GCE) technology. Premature termination codon (PTC) harboring PRV was successfully rescued in the presence of orthogonal system MbpylRS/tRNAPyl pair and unnatural amino acids (UAA). However, UAA incorporating efficacy seemed extremely low in our engineered PRV PTC virus. Furthermore, we failed to establish a stable transgenic cell line containing orthogonal translation machinery for PTC virus replication, and we demonstrated that orthogonal tRNAPyl is a key limiting factor. This study is the first to demonstrate that orthogonal translation system-mediated amber codon suppression strategy could precisely control PRV-PTC engineered virus replication. To our knowledge, this is the first reported PTC herpesvirus generated by GCE technology. Our work provides a proof-of-concept for generating UAAs-controlled PRV-PTC virus, which can be used as a safe and effective vaccine. [ABSTRACT FROM AUTHOR]

Bacterial artificial chromosomes (BACs) are powerful tools for the manipulation of the large genomes of DNA viruses, such as herpesviruses. However, the methods currently used to construct the recombinant viruses, an important intermediate link in the generation of BACs, involve the laborious process of multiple plaque purifications. Moreover, some fastidious viruses may be lost or damaged during these processes, making it impossible to generate BACs from these large-genome DNA viruses. Here, we introduce the CRISPR/Cas9 as a site-specific gene knock-in instrument that promotes the homologs recombination of a linearized transfer vector and the Pseudorabies virus genome through double incisions. The efficiency of recombination is as high as 86%. To our knowledge, this is the highest efficiency ever reported for Pseudorabies virus recombination. We also demonstrate that the positions and distances of the CRISPR/Cas9 single guide RNAs from the homology arms correlate with the efficiency of homologous recombination. Our work show a simple and fast cloning method of BACs with large genome inserted by greatly enhancing the HR efficiencies through CRISPR/Cas9-mediated homology-directed repair mechanism, and this method could be of helpful for manipulating large DNA viruses, and will provide a successful model for insertion of large DNA fragments into other viruses. [ABSTRACT FROM AUTHOR]

The transcription factor NF-κB plays a critical role in diverse biological processes. The NF-κB pathway can be activated by incoming pathogens and then stimulates both innate and adaptive immunity. However, many viruses have evolved corresponding strategies to balance NF-κB activation to benefit their replication. Pseudorabies virus (PRV) is an economically important pathogen that belongs to the alphaherpesvirus group. There is little information about PRV infection and NF-κB regulation. This study demonstrates for the first time that the UL24 protein could abrogate tumor necrosis factor alpha (TNF-α)-mediated NF-κB activation. An overexpression assay indicated that UL24 inhibits this pathway at or downstream of P65. Furthermore, co-immunoprecipitation analysis demonstrated that UL24 selectively interacts with P65. We demonstrated that UL24 could significantly degrade P65 by the proteasome pathway. For the first time, PRV UL24 was shown to play an important role in NF-κB evasion during PRV infection. This study expands our understanding that PRV can utilize its encoded protein UL24 to evade NF-κB signaling. [ABSTRACT FROM AUTHOR]

Currently, pseudorabies virus (PRV) variant strains are outbreaking in China; these variants belong to genotype II PRV. The traditional Bartha-K61 vaccine has failed to provide complete protection against the emergent variant strains. Therefore, rapid attenuation of current epidemic strains is needed for effective PRV control. In this study, we report a rapid method for editing the PRV genome using the CRISPR-Cas9 system. We developed a triple gE/gI/TK gene-inactivated HeN1 PRV strain, because mice were more susceptible to PRV infection, we then evaluated the attenuation of PRV in the mice and demonstrated that modified PRV was fully attenuated. Furthermore, the attenuated strain also induced immune protection in response to a parental PRV challenge. Overall, we showed that PRVs can be rapidly attenuated using CRISPR-Cas9 technology, which will be critical for PRV control, especially when new variant PRV strains emerge. [ABSTRACT FROM AUTHOR]

Pseudorabies virus (PRV), which is extremely infectious and can infect numerous mammals, has a risk of spillover into humans. Virus–host interactions determine viral entry and spreading. Here, we showed that neuropilin-1 (NRP1) significantly potentiates PRV infection. Mechanistically, NRP1 promoted PRV attachment and entry, and enhanced cell-to-cell fusion mediated by viral glycoprotein B (gB), gD, gH, and gL. Furthermore, through in vitro coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays, NRP1 was found to physically interact with gB, gD, and gH, and these interactions were C-end Rule (CendR) motif independent, in contrast to currently known viruses. Remarkably, we illustrated that the viral protein gB promotes NRP1 degradation via a lysosome-dependent pathway. We further demonstrate that gB promotes NRP1 degradation in a furin-cleavage-dependent manner. Interestingly, in this study, we generated gB furin cleavage site (FCS)-knockout PRV (Dfurin PRV) and evaluated its pathogenesis; in vivo, we found that Dfurin PRV virulence was significantly attenuated in mice. Together, our findings demonstrated that NRP1 is an important host factor for PRV and that NRP1 may be a potential target for antiviral intervention. IMPORTANCE Recent studies have shown accelerated PRV cross-species spillover and that PRV poses a potential threat to humans. PRV infection in humans always manifests as a high fever, tonic-clonic seizures, and encephalitis. Therefore, understanding the interaction between PRV and host factors may contribute to the development of new antiviral strategies against PRV. NRP1 has been demonstrated to be a receptor for several viruses that harbor CendR, including SARS-CoV-2. However, the relationships between NRP1 and PRV are poorly understood. Here, we found that NRP1 significantly potentiated PRV infection by promoting PRV attachment and enhanced cell-to-cell fusion. For the first time, we demonstrated that gB promotes NRP1 degradation via a lysosome-dependent pathway. Last, in vivo, Dfurin PRV virulence was significantly attenuated in mice. Therefore, NRP1 is an important host factor for PRV, and NRP1 may be a potential target for antiviral drug development. [ABSTRACT FROM AUTHOR]

Pseudorabies virus (PRV), the causative agent of Aujeszky׳s disease, has gained increased attention in China in recent years as a result of the outbreak of emergent pseudorabies. Several genomic and partial sequences are available for Chinese emergent and European–American strains of PRV, but limited sequence data exist for the earlier Chinese strains. In this study, we determined the complete genomic sequence of one earlier Chinese strain SC and one emergent strain HLJ8. Compared with other known sequences, we demonstrated that PRV strains from distinct geographical regions displayed divergent evolution. Additionally, we report for the first time, a recombination event between PRV strains, and show that strain SC is a recombinant of an endemic Chinese strain and a Bartha-vaccine-like strain. These results contribute to our understanding of PRV evolution. [ABSTRACT FROM AUTHOR]