Porcine deltacoronavirus (PDCoV) is a newly emerging threat to the global porcine industry. PDCoV has been successfully isolated using various medium additives including trypsin, and although we know it is important for viral replication, the mechanism has not been fully elucidated. Here, we systematically investigated the role of trypsin in PDCoV replication including cell entry, cell-to-cell membrane fusion and virus release. Using pseudovirus entry assays, we demonstrated that PDCoV entry is not trypsin dependent. Furthermore, unlike porcine epidemic diarrhea virus (PEDV), in which trypsin is important for the release of virus from infected cells, PDCoV release was not affected by trypsin. We also demonstrated that trypsin promotes PDCoV replication by enhancing cell-to-cell membrane fusion. Most importantly, our study illustrates two distinct spreading patterns from infected cells to uninfected cells during PDCoV transmission, and the role of trypsin in PDCoV replication in cells with different virus spreading types. Overall, these results clarify that trypsin promotes PDCoV replication by mediating cell-to-cell fusion transmission but is not crucial for viral entry. This knowledge can potentially contribute to improvement of virus production efficiency in culture, not only for vaccine preparation but also to develop antiviral treatments.

Porcine deltacoronavirus (PDCoV) is a recently discovered coronavirus that poses a potential threat to the global swine industry. Although we know that aminopeptidase N (APN) is important for PDCoV replication, it is unclear whether it is the primary functional receptor, and the mechanism by which it promotes viral replication is not fully understood. Here, we systematically investigated the role of porcine APN (pAPN) during PDCoV infection of non-susceptible cells, including in viral attachment and internalization. Using a viral entry assay, we found that PDCoV can enter non-susceptible cells but then fails to initiate efficient replication. pAPN and PDCoV virions clearly co-localized with the endocytotic markers RAB5, RAB7, and LAMP1, suggesting that pAPN mediates PDCoV entry by an endocytotic pathway. Most importantly, our study shows that regardless of which receptor PDCoV engages, only entry by an endocytotic route ultimately leads to efficient viral replication. This knowledge should contribute to the development of efficient antiviral treatments, which are especially useful in preventing cross-species transmission. IMPORTANCE PDCoV is a pathogen with potential for transmission across diverse species, although the mechanism of such host-switching events (from swine to other species) is poorly understood. Here, we show that PDCoV enters non-susceptible cells, but without efficient replication. We also investigated the key role played by aminopeptidase N in mediating PDCoV entry via an endocytotic pathway. Our results demonstrate that viral entry via endocytosis is a major determinant of efficient PDCoV replication. This knowledge provides a basis for future studies of the cross-species transmissibility of PDCoV and the development of appropriate anti-viral drugs.

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.

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1The outbreak of the severe acute respiratory syndrome coronavirus 2 2 (SARS-CoV-2) poses a huge threat to many countries around the world. However, 3 where is it origin and which animals are sensitive to cross-species transmission is 4 unclear. The interaction of virus and cell receptor is a key determinant of host range 5 for the novel coronavirus. Angiotensin-converting enzyme 2 (ACE2) is demonstrated 6 as the primary entry receptor for SARS-CoV-2. In this study, we evaluated the 7 SARS-CoV-2 entry mediated by ACE2 of 11 different species of animals, and 8 discovered that ACE2 of Rhinolophus sinicus (Chinese horseshoe bat), Felis catus 9 (domestic cat), Canis lupus familiaris (dog), Sus scrofa (pig), Capra hircus (goat) and 10 especially Manis javanica (Malayan pangolin) were able to render SARS-CoV-2 entry 11 in non-susceptible cells. This is the first report that ACE2 of Pangolin could mediate 12 SARS-CoV-2 entry which increases the presume that SARS-CoV-2 may have a 13 pangolin origin. However, none of the ACE2 proteins from Rhinolophus 14 ferrumequinum (greater horseshoe bat), Gallus gallus (chicken), Notechis scutatus 15 (mainland tiger snake), Mus musculus (house mouse) rendered SARS-CoV-2 entry. 16 Specifically, a natural isoform of Macaca mulatta (Rhesus monkey) ACE2 with a 17 mutation of Y217N was resistance to infection, which rises the possible impact of this 18 type of ACE2 during monkey studies of SARS-CoV-2. Overall, these results clarify 19 that SARS-CoV-2 could engage receptors of multiple species of animals and it is a 20 perplexed work to track SARS-CoV-2 origin and its intermediate hosts. 21 22 IMPORTANCE 1In this study, we illustrated that SARS-CoV-2 is able to engage receptors of 2 multiple species of animals. This indicated that it may be a perplexed work to track 3 SARS-CoV-2 origin and discover its intermediate hosts. This feature of virus is 4 considered to potentiate its diverse cross-species transmissibility. Of note, here is the 5 first report that ACE2 of Pangolin could mediate SARS-CoV-2 entry which increases 6 the possibility that SARS-CoV-2 may have a pangolin origin. And we also 7 demonstrated that not all species of bat were sensitive to SARS-CoV-2 infection. At 8 last, it is also important to detect the expression ratio of the Y217N ACE2 to the 9 prototype in Rhesus monkeys to be recruited for studies on SARS-CoV-2 infection.

equip eukaryotic cells with dual orthogonal expanded genetic codes that enable the specific reprogramming of distinct translational machineries with single-residue precision. [14] Tharp et al. demonstrated that UAU can be reassigned along with UAG or UAA to encode two distinct ncAAs in the same protein. [15] In addition, the same ncAA has been inserted into the same protein at several TAG sites by deleting RF1 in E. coli or engineering eukaryotic release factor 1 (eRF1) in mammalian cells. [16][17][18][19] However, the site-specific incorporation of three distinct ncAAs into a single mammalian protein remains a challenge.Effective and high-fidelity incorporation of three distinct ncAAs into a single mammalian protein will greatly enhance the investigation of protein structures and functions, [10,20,21] including the site-specific mimicry of posttranslational modifications and in situ labeling for multicolor imaging, among other applications. [22][23][24][25] To achieve this incorporation, ncAAs must be delivered by bio-orthogonal aaRS/ tRNA pairs that do not cross-react with each other or their counterparts in host cells. [9,26] The suppression systems driving the incorporation of three distinct ncAAs can cross-react at three different levels. First, an aaRS might charge the substrate ncAA for another aaRS. Second, an aaRS might charge a noncognate tRNA. Finally, a suppressor tRNA might recognize a noncognate nonsense codon. Four distinct aaRS/tRNA pairs have previously been used to expand the genetic code of mammalian cells including a tyrosyl (EcTyr) and leucyl (EcLeu) pair derived from E. coli in addition to an M. mazei pyrrolysyl (MmPyl) pair and an M. barkeri pyrrolysyl (MbPyl) pair. [10,21,22] In addition, the PylRS from Methanomethylophilus alvus has been used extensively in recent years to install UAAs into proteins in eukaryotes. [12] However, the MmPyl and MbPyl pairs cannot be used together because they are both derived from Methanosarcina and share similar synthetase structures. Thus, it is possible, in principle, to combine the EcTyr and EcLeu pairs with either the MmPyl or MbPyl pairs to incorporate three distinct ncAAs into a single mammalian protein.In this study, a systematic approach was used to select the optimal combination of three orthogonal ncAA incorporation systems based on read-through efficiency and orthogonality. Specifically, EcLeuRS/tRNA CUA , MmPylRS/tRNA UCA , and EcTyrRS/tRNA UUA were used to simultaneously incorporate three distinct ncAAs into mammalian proteins by suppressing Site-specific incorporation of distinct noncanonical amino acids (ncAAs) into proteins via genetic code expansion in mammalian cells represents a new avenue for protein engineering. Reassigning three TAGs with the same ncAA in mammalian cells has previously been achieved using translational machinery. However, simultaneous recoding of three nonsense codons with distinct ncAAs in mammalian cells remains a challenge due to low incorporation efficiencies. Here, three optimized aaRS/tRNA pairs (i.e., the E. col...

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The frequent emergence of drug resistance during the treatment of influenza A virus (IAV) infections highlights a need for effective antiviral countermeasures. Here, we present an antiviral method that utilizes unnatural amino acid-engineered drug-resistant (UAA-DR) virus. The engineered virus is generated through genetic code expansion to combat emerging drug-resistant viruses. The UAA-DR virus has unnatural amino acids incorporated into its drug-resistant protein and its polymerase complex for replication control. The engineered virus can undergo genomic segment reassortment with normal virus and produce sterilized progenies due to artificial amber codons in the viral genome. We validate in vitro that UAA-DR can provide a broad-spectrum antiviral strategy for several H1N1 strains, different DR-IAV strains, multidrug-resistant (MDR) strains, and even antigenically distant influenza strains (e.g., H3N2). Moreover, a minimum dose of neuraminidase (NA) inhibitors for influenza virus can further enhance the sterilizing effect when combating inhibitor-resistant strains, partly due to the promoted superinfection of unnatural amino acid-modified virus in cellular and animal models. We also exploited the engineered virus to achieve adjustable efficacy after external UAA administration, for mitigating DR virus infection on transgenic mice harboring the PylRS-tRNAPyl CUA pair, and to have substantial elements of the genetic code expansion technology, which further demonstrated the safety and feasibility of the strategy. We anticipate that the use of the UAA-engineered DR virion, which is a novel antiviral agent, could be extended to combat emerging drug-resistant influenza virus and other segmented RNA viruses.