Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies.

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

RIG-I triggers a signaling-abortive anti-SARS-CoV-2 defense in human lung cells.

Efficient immune responses against viral infection are determined by sufficient activation of nucleic acid sensor-mediated innate immunity1,2. Coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains an ongoing global pandemic. It is an urgent challenge to clarify the innate recognition mechanism to control this virus. Here we show that retinoic acid-inducible gene-I (RIG-I) sufficiently restrains SARS-CoV-2 replication in human lung cells in a type I/III interferon (IFN)-independent manner. RIG-I recognizes the 3' untranslated region of the SARS-CoV-2 RNA genome via the helicase domains, but not the C-terminal domain. This new mode of RIG-I recognition does not stimulate its ATPase, thereby aborting the activation of the conventional mitochondrial antiviral-signaling protein-dependent pathways, which is in accordance with lack of cytokine induction. Nevertheless, the interaction of RIG-I with the viral genome directly abrogates viral RNA-dependent RNA polymerase mediation of the first step of replication. Consistently, genetic ablation of RIG-I allows lung cells to produce viral particles that expressed the viral spike protein. By contrast, the anti-SARS-CoV-2 activity was restored by all-trans retinoic acid treatment through upregulation of RIG-I protein expression in primary lung cells derived from patients with chronic obstructive pulmonary disease. Thus, our findings demonstrate the distinctive role of RIG-I as a restraining factor in the early phase of SARS-CoV-2 infection in human lung cells.

Common low complexity regions for SARS-CoV-2 and human proteomes as potential multidirectional risk factor in vaccine development.

BACKGROUND: The rapid spread of the COVID-19 demands immediate response from the scientific communities. Appropriate countermeasures mean thoughtful and educated choice of viral targets (epitopes). There are several articles that discuss such choices in the SARS-CoV-2 proteome, other focus on phylogenetic traits and history of the Coronaviridae genome/proteome. However none consider viral protein low complexity regions (LCRs). Recently we created the first methods that are able to compare such fragments. RESULTS: We show that five low complexity regions (LCRs) in three proteins (nsp3, S and N) encoded by the SARS-CoV-2 genome are highly similar to regions from human proteome. As many as 21 predicted T-cell epitopes and 27 predicted B-cell epitopes overlap with the five SARS-CoV-2 LCRs similar to human proteins. Interestingly, replication proteins encoded in the central part of viral RNA are devoid of LCRs. CONCLUSIONS: Similarity of SARS-CoV-2 LCRs to human proteins may have implications on the ability of the virus to counteract immune defenses. The vaccine targeted LCRs may potentially be ineffective or alternatively lead to autoimmune diseases development. These findings are crucial to the process of selection of new epitopes for drugs or vaccines which should omit such regions.

Viral Subpopulation Screening Guides in Designing a High Interferon-Inducing Live Attenuated Influenza Vaccine by Targeting Rare Mutations in NS1 and PB2 Proteins.

Influenza A viruses continue to circulate among wild birds and poultry worldwide, posing constant pandemic threats to humans. Effective control of emerging influenza viruses requires new broadly protective vaccines. Live attenuated influenza vaccines with truncations in nonstructural protein 1 (NS1) have shown broad protective efficacies in birds and mammals, which correlate with the ability to induce elevated interferon responses in the vaccinated hosts. Given the extreme diversity of influenza virus populations, we asked if we could improve an NS1-truncated live attenuated influenza vaccine developed for poultry (PC4) by selecting viral subpopulations with enhanced interferon-inducing capacities. Here, we deconstructed a de novo population of PC4 through plaque isolation, created a large library of clones, and assessed their interferon-inducing phenotypes. While most of the clones displayed the parental interferon-inducing phenotype in cell culture, few clones showed enhanced interferon-inducing phenotypes in cell culture and chickens. The enhanced interferon-inducing phenotypes were linked to either a deletion in NS1 (NS1Δ76-86) or a substitution in polymerase basic 2 protein (PB2-D309N). The NS1Δ76-86 deletion disrupted the putative eukaryotic translation initiation factor 4GI-binding domain and promoted the synthesis of biologically active interferons. The PB2-D309N substitution enhanced the early transcription of interferon mRNA, revealing a novel role for the 309D residue in suppression of interferon responses. We combined these mutations to engineer a novel vaccine candidate that induced additive amounts of interferons and stimulated protective immunity in chickens. Therefore, viral subpopulation screening approaches can guide the design of live vaccines with strong immunostimulatory properties.IMPORTANCE Effectiveness of NS1-truncated live attenuated influenza vaccines relies heavily on their ability to induce elevated interferon responses in vaccinated hosts. Influenza viruses contain diverse particle subpopulations with distinct phenotypes. We show that live influenza vaccines can contain underappreciated subpopulations with enhanced interferon-inducing phenotypes. The genomic traits of such virus subpopulations can be used to further improve the efficacy of the current live vaccines.

Outbreak Severity of Highly Pathogenic Avian Influenza A(H5N8) Viruses Is Inversely Correlated to Polymerase Complex Activity and Interferon Induction.

Highly pathogenic avian influenza A(H5N8) viruses first emerged in China in 2010 and in 2014 spread throughout Asia and to Europe and the United States via migrating birds. Influenza A(H5N8) viruses were first detected in the Netherlands in 2014 and caused five outbreaks in poultry farms but were infrequently detected in wild birds. In 2016, influenza A(H5N8) viruses were reintroduced into the Netherlands, resulting in eight poultry farm outbreaks. This outbreak resulted in numerous dead wild birds with severe pathology. Phylogenetic analysis showed that the polymerase genes of these viruses had undergone extensive reassortment between outbreaks. Here, we investigated the differences in virulence between the 2014-15 and the 2016-17 outbreaks by characterizing the polymerase complex of influenza A(H5N8) viruses from both outbreaks. We found that viruses from the 2014-15 outbreak had significantly higher polymerase complex activity in both human and avian cell lines than did those from the 2016-17 outbreak. No apparent differences in the balance between transcription and replication of the viral genome were observed. Interestingly, the 2014-15 polymerase complexes induced significantly higher levels of interferon beta (IFN-ß) than the polymerase complexes of the 2016-17 outbreak viruses, mediated via retinoic acid-inducible gene I (RIG-I). Inoculation of primary duck cells with recombinant influenza A(H5N8) viruses, including viruses with reassorted polymerase complexes, showed that the polymerase complexes from the 2014-15 outbreak induced higher levels of IFN-ß despite relatively minor differences in replication capacity. Together, these data suggest that despite the lower levels of polymerase activity, the higher 2016-17 influenza A(H5N8) virus virulence may be attributed to the lower level of activation of the innate immune system.IMPORTANCE Compared to the 2014-15 outbreak, the 2016-17 outbreak of influenza A(H5N8) viruses in the Netherlands and Europe was more virulent; the number of dead or diseased wild birds found and the severity of pathological changes were higher during the 2016-17 outbreak. The polymerase complex plays an important role in influenza virus virulence, and the gene segments of influenza A(H5N8) viruses reassorted extensively between the outbreaks. In this study, the 2014-15 polymerase complexes were found to be more active, which is counterintuitive with the observed higher virulence of the 2016-17 outbreak viruses. Interestingly, the 2014-15 polymerase complexes also induced higher levels of IFN-ß. These findings suggest that the higher virulence of influenza A(H5N8) viruses from the 2016-17 outbreak may be related to the lower induction of IFN-ß. An attenuated interferon response could lead to increased dissemination, pathology, and mortality, as observed in (wild) birds infected during the 2016-2017 outbreak.

Type I interferon antagonistic properties of influenza B virus polymerase proteins.

The innate immune system, in particular the type I interferon (IFN) response, is a powerful defence against virus infections. In turn, many if not all viruses have evolved various means to circumvent, resist, or counteract this host response to ensure efficient replication and propagation. Influenza viruses are no exception to this rule, and several viral proteins have been described to possess IFN-antagonistic functions. Although the viral nonstructural protein 1 appears to be a major antagonist in influenza A and B viruses (IAV and IBV), we have previously shown that a specific motif in the IAV polymerase proteins exerts an IFN-suppressive function very early in infection. The question remained whether a similar function would also exist in IBV polymerases. Here, we show that indeed a specific amino acid position (A523) of the PB1 protein in the IBV polymerase complex confers IFN-antagonistic properties. Mutation of this position leads to enhanced activation of the IFN-mediated signalling pathway after infection and subsequent reduction of virus titres. This indicates that inhibition of innate immune responses is a conserved activity shared by polymerase proteins of IAV and IBV.

Characterization of a novel bat-HKU2-like swine enteric alphacoronavirus (SeACoV) infection in cultured cells and development of a SeACoV infectious clone.

Swine enteric alphacoronavirus (SeACoV), also known as swine acute diarrhea syndrome coronavirus (SADS-CoV), belongs to the species Rhinolophus bat coronavirus HKU2. Herein, we report on the primary characterization of SeACoV in vitro. Four antibodies against the SeACoV spike, membrane, nucleocapsid and nonstructural protein 3 capable of reacting with viral antigens in SeACoV-infected Vero cells were generated. We established a DNA-launched SeACoV infectious clone based on the cell adapted passage-10 virus and rescued the recombinant virus with a unique genetic marker in cultured cells. Six subgenomic mRNAs containing the leader-body junction sites, including a bicistronic mRNA encoding the accessory NS7a and NS7b genes, were experimentally identified in SeACoV-infected cells. Cellular ultrastructural changes induced by SeACoV infection were visualized by electron microscopy. The availability of the SeACoV infectious clone and a panel of antibodies against different viral proteins will facilitate further studies on understanding the molecular mechanisms of SeACoV replication and pathogenesis.

Porcine deltacoronavirus nsp15 antagonizes interferon-ß production independently of its endoribonuclease activity.

Porcine deltacoronavirus (PDCoV) is an emerging swine coronavirus causing diarrhea and intestinal damage in nursing piglets. Previous work showed that PDCoV infection inhibits type I interferon (IFN) production. To further identify and characterize the PDCoV-encoded IFN antagonists will broaden our understanding of its pathogenesis. Nonstructural protein 15 (nsp15) encodes an endoribonuclease that is highly conserved among vertebrate nidoviruses (coronaviruses and arteriviruses) and plays a critical role in viral replication and transcription. Here, we found that PDCoV nsp15 significantly inhibits Sendai virus (SEV)-induced IFN-ß production. PDCoV nsp15 disrupts the phosphorylation and nuclear translocation of NF-κB p65 subunit, but not antagonizes the activation of transcription factor IRF3. Interestingly, site-directed mutagenesis found that PDCoV nsp15 mutants (H129A, H234A, K269A) lacking endoribonuclease activity also suppress SEV-induced IFN-ß production and NF-κB activation, suggesting that the endoribonuclease activity is not required for its ability to antagonize IFN-ß production. Taken together, our results demonstrate that PDCoV nsp15 is an IFN antagonist and it inhibits interferon-ß production via an endoribonuclease activity-independent mechanism.

Engineering partial resistance to cucumber mosaic virus in tobacco using intrabodies specific for the viral polymerase.

A single-chain variable antibody fragment (scFv) library tested against the non-structural NSP5 protein of human rotavirus A was screened by a yeast two-hybrid system against three proteins derived from the RNA-dependent RNA polymerase (RdRp) of cucumber mosaic virus (CMV), with the aim of blocking their function and preventing viral infection once expressed in planta. The constructs tested were (i) '2a' consisting of the full-length 2a gene (839 amino acids, aa), (ii) 'Motifs' covering the conserved RdRp motifs (IV-VII) (132 aa) and (iii) 'GDD' located within the conserved RdRp motif VI (GDD, 22 aa). In yeast two-hybrid (Y2H) selection assays the '2a' and 'Motifs' constructs interacted with 96 and 25 library constructs, respectively, while the 'GDD' construct caused transactivation. Y2H-interacting scFvs were analyzed in vivo for their interaction with the 2a and Motifs proteins in a mammalian transient expression system. Eighteen tobacco lines stably transformed with four selected scFvs were produced and screened for resistance against two different CMV isolates. Different levels of resistance and rate of recovery were observed with CMV of both groups I and II, particularly in lines expressing intrabodies against the full-length 2a protein. This work describes for the first time the use of intrabodies against the RdRp of CMV to obtain plants that reduce infection of a pandemic virus, showing that the selected scFvs can modulate virus infection and induce premature recovery in tobacco plants.

Functional Evolution of the 2009 Pandemic H1N1 Influenza Virus NS1 and PA in Humans.

In 2009, a pandemic H1N1 influenza A virus (IAV) (pH1N1) emerged in the human population from swine causing a pandemic. Importantly, this virus is still circulating in humans seasonally. To analyze the evolution of pH1N1 in humans, we sequenced viral genes encoding proteins inhibiting general gene expression (nonstructural protein 1 [NS1] and PA-X) from circulating seasonal viruses and compared them to the viruses isolated at the origin of the pandemic. Recent pH1N1 viruses contain amino acid changes in the NS1 protein (E55K, L90I, I123V, E125D, K131E, and N205S), as previously described (A. M. Clark, A. Nogales, L. Martinez-Sobrido, D. J. Topham, and M. L. DeDiego, J Virol 91:e00721-17, 2017, https://doi.org/10.1128/JVI.00721-17), and amino acid changes in the PA-X protein (V100I, N204S, R221Q, and L229S). These amino acid differences between early and more recent pH1N1 isolates are responsible for increased NS1-mediated inhibition of host gene expression and decreased PA-X-mediated shutoff, including innate immune response genes. In addition, currently circulating pH1N1 viruses have acquired amino acid changes in the PA protein (V100I, P224S, N321K, I330V, and R362K). A recombinant pH1N1 virus containing PA, PA-X, and NS1 genes from currently circulating viruses is fitter in replication in cultured cells and in mice and is slightly more pathogenic than the original ancestor pH1N1 virus. These results demonstrate the need to monitor the evolution of pH1N1 in humans for mutations in the viral genome that could result in enhanced virulence. Importantly, these results further support our previous findings suggesting that inhibition of global gene expression mediated by NS1 and PA-X proteins is subject to a balance which can determine virus pathogenesis and fitness.IMPORTANCE IAVs emerge in humans from animal reservoirs, causing unpredictable pandemics. One of these pandemics was caused by an H1N1 virus in 2009, and this virus is still circulating seasonally. To analyze host-virus adaptations likely affecting influenza virus pathogenesis, protein amino acid sequences from viruses circulating at the beginning of the pandemic and those circulating currently were compared. Currently circulating viruses have incorporated amino acid changes in two viral proteins (NS1 and PA-X), affecting innate immune responses, and in the PA gene. These amino acid differences led to increased NS1-mediated and decreased PA-X-mediated inhibition of host gene expression. A recombinant pH1N1 virus containing PA, PA-X, and NS1 genes from recently circulating viruses is fitter in replication in tissue culture cells and in mice, and the virus is more pathogenic in vivo Importantly, these results suggest that a balance in the abilities of NS1 and PA-X to induce host shutoff is beneficial for IAVs.

Establishment of a platform for molecular and immunological characterization of the RNA-dependent-RNA-polymerase NS5B of an Egyptian HCV isolate.

The present work aimed at establishing a platform to enable frequent characterization of the HCV RNA-dependent-RNA-polymerase from Egyptian clinical isolates. Subjecting amplified HCV-NS5B coding gene from Egyptian patient's serum to sequencing, multiple alignment, and phylogenetic analysis confirmed its subtype 4a origin. Nucleotide sequence analysis revealed presence of an additional start codon at the beginning of the NS5B gene. Peptide sequence alignment demonstrated presence of unique amino acid residues in our 4a-NS5B sequence distinct from the JFH-1-NS5B sequence as well as unique amino acids compared to other genotypes. The distinct molecular structure of the herein characterized 4a-NS5B from the 2a-JFH-1-NS5B was further demonstrated both in the built 3D models and the Ramachandran plots corresponding to each structure. Both the unique amino acid residues and 3D structure of the 4a-NS5B may influence both genotype 4a replication rate and response to therapy in comparison to other genotypes. Many resistance mutations to polymerase inhibitors were found both in ours and other genotypes' sequences. The presence of the required amino acid motifs for the RNA dependent RNA polymerase activity encouraged to clone the NS5B570-encoding sequence downstream CMV promotor in a mammalian expression vector. Such construct was used for both prokaryotic expression in bacteria and for DNA immunization. Successful mammalian expression and induction of specific immune response were demonstrated by ELISA and Western blotting. The potential of both the raised antibodies and the expressed NS5B to differentiate between HCV-infected and control human sera were demonstrated which reflect their diagnostic value.

Which Plant Proteins Are Involved in Antiviral Defense? Review on In Vivo and In Vitro Activities of Selected Plant Proteins against Viruses.

Plants have evolved a variety of defense mechanisms to tackle virus attack. Endogenous plant proteins can function as virus suppressors. Different types of proteins mediate defense responses against plant viruses. Pathogenesis-related (PR) proteins are activated upon pathogen infections or in different stress situations and their production is one of many components in plant defense. Ribosome-inactivating proteins (RIPs) suppress translation by enzymatically damaging ribosomes and they have been found to have antiviral activity. RNA-binding proteins (RBPs) bind to target RNAs via specialized RNA-binding domain and can directly or indirectly function in plant defense system against RNA viruses. Proteins involved in silencing machinery, namely Dicer-like (DCL) proteins, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDRs) confer innate antiviral defense in plants as they are able to degrade foreign RNA of viral origin. This review aims to provide a comprehensive and up-to-date picture of plant proteins participating in antiviral defense. As a result we discuss proteins conferring plant antiviral resistance and their potential future applications in different fields of life including agriculture and medicine.

Optimized clade 2.3.2.1c H5N1 recombinant-vaccine strains against highly pathogenic avian influenza.

A/Puerto Rico/8/34 (PR8)-derived recombinant viruses have been used for seasonal flu vaccines; however, they are insufficient for vaccines against some human-fatal H5N1 highly pathogenic avian influenza (HPAI) viruses (HPAIV) due to low productivity. Additionally, the polymerase basic 2 (PB2) protein, an important mammalian-pathogenicity determinant, of PR8 possesses several mammalian-pathogenic mutations. We previously reported two avian PB2 genes (01310 and 0028) related to efficient replication in embryonated chicken eggs (ECEs) and nonpathogenicity in BALB/c mice. In this study, we generated PR8-derived H5N1 recombinant viruses harboring hemagglutinin (attenuated) and neuraminidase genes of a clade 2.3.2.1c H5N1 HPAIV (K10-483), as well as the 01310 or 0028 PB2 genes, and investigated their replication and immunogenicity. Compared with a control virus harboring six internal PR8 genes (rK10-483), the recombinant viruses possessing the 01310 and 0028 PB2 genes showed significantly higher replication efficiency in ECEs and higher antibody titers in chickens. In contrast to rK10-483, none of the viruses replicated in BALB/c mice, and all showed low titers in Madin-Darby canine kidney cells. Additionally, the recombinant viruses did not induce a neutralization antibody but elicited decreased protective immune responses against K10-483 in mice. Thus, the highly replicative and mammalian nonpathogenic recombinant H5N1 strains might be promising vaccine candidates against HPAI in poultry.

A Novel Porcine Circovirus Distantly Related to Known Circoviruses Is Associated with Porcine Dermatitis and Nephropathy Syndrome and Reproductive Failure.

Porcine circovirus-associated disease (PCVAD) is clinically manifested by postweaning multisystemic wasting syndrome (PMWS), respiratory and enteric disease, reproductive failure, and porcine dermatitis and nephropathy syndrome (PDNS). Porcine circovirus 2 (PCV2) is an essential component of PCVAD, although an etiologic role in PDNS is not well established. Here, a novel circovirus, designated porcine circovirus 3 (PCV3), was identified in sows that died acutely with PDNS-like clinical signs. The capsid and replicase proteins of PCV3 are only 37% and 55% identical to PCV2 and bat circoviruses, respectively. Aborted fetuses from sows with PDNS contained high levels of PCV3 (7.57 × 107 genome copies/ml), and no other viruses were detected by PCR and metagenomic sequencing. Immunohistochemistry (IHC) analysis of sow tissue samples identified PCV3 antigen in skin, kidney, lung, and lymph node samples localized in typical PDNS lesions, including necrotizing vasculitis, glomerulonephritis, granulomatous lymphadenitis, and bronchointerstitial pneumonia. Further study of archived PDNS tissue samples that were negative for PCV2 by IHC analysis identified 45 of 48 that were PCV3 positive by quantitative PCR (qPCR), with 60% of a subset also testing positive for PCV3 by IHC analysis. Analysis by qPCR of 271 porcine respiratory disease diagnostic submission samples identified 34 PCV3-positive cases (12.5%), and enzyme-linked immunosorbent assay detection of anti-PCV3 capsid antibodies in serum samples found that 46 (55%) of 83 samples tested were positive. These results suggest that PCV3 commonly circulates within U.S. swine and may play an etiologic role in reproductive failure and PDNS. Because of the high economic impact of PCV2, this novel circovirus warrants further studies to elucidate its significance and role in PCVAD. IMPORTANCE: While porcine circovirus 2 (PCV2) was first identified in sporadic cases of postweaning multisystemic wasting syndrome in Canada in the early 1990s, an epidemic of severe systemic disease due to PCV2 spread worldwide in the ensuing decade. Despite being effectively controlled by commercial vaccines, PCV2 remains one of the most economically significant viruses of swine. Here, a novel porcine circovirus (PCV3) that is distantly related to known circoviruses was identified in sows with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. PCV2, which has previously been associated with these clinical presentations, was not identified. High levels of PCV3 nucleic acid were observed in aborted fetuses by quantitative PCR, and PCV3 antigen was localized in histologic lesions typical of PDNS in sows by immunohistochemistry (IHC) analysis. PCV3 was also identified in archival PDNS diagnostic samples that previously tested negative for PCV2 by IHC analysis. The emergence of PCV3 warrants further investigation.

Polymerase Mechanism-Based Method of Viral Attenuation.

Vaccines remain the most effective way of preventing infection and spread of infectious diseases. These prophylactics have been used for centuries but still to this day only three main design strategies exist: (1) live attenuated virus (LAV) vaccines, (2) killed or inactivated virus vaccines, (3) and subunit vaccines of the three, the most efficacious vaccines remain LAVs. LAVs replicate in relevant tissues, elicit strong cellular and humoral responses, and often confer lifelong immunity. While this vaccine strategy has produced the majority of successful vaccines in use today, there are also important safety concerns to consider with this approach. In the past, the development of LAVs has been empirical. Blind passage of viruses in various cell types results in the accumulation of multiple attenuating mutations leaving the molecular mechanisms of attenuation unknown. Also, due to the high error rate of RNA viruses and selective pressures of the host environment, these LAVs, derived from such viruses, can potentially revert back to wild-type virulence. This not only puts the vaccinee at risk, but if shed can put those that are unvaccinated at risk as well. While these vaccines have been successful there still remains a need for a rational design strategy by which to create additional LAVs.One approach for rational vaccine design involves increasing the fidelity of the viral RdRp. Increased fidelity decreases the viral mutational frequency thereby reducing the genetic variation the virus needs in order to evade the host imposed bottlenecks to infection. While polymerase mutants exist which decrease viral mutation frequency the mutations are not in conserved regions of the polymerase, which doesn't lend itself toward using a common mutant approach toward developing a universal vaccine strategy for all RNA viruses. We have identified a conserved lysine residue in the active site of the PV RdRp that acts as a general acid during nucleotide incorporation. Mutation from a lysine to an arginine results in a high fidelity polymerase that replicates slowly thus creating an attenuated virus that is genetically stable and less likely to revert to a wild-type phenotype. This chapter provides detailed methods in which to identify the conserved lysine residue and evaluating fidelity and attenuation in cell culture (in vitro) and in the PV transgenic murine model (in vivo).

Antiviral Protection via RdRP-Mediated Stable Activation of Innate Immunity.

For many emerging and re-emerging infectious diseases, definitive solutions via sterilizing adaptive immunity may require years or decades to develop, if they are even possible. The innate immune system offers alternative mechanisms that do not require antigen-specific recognition or a priori knowledge of the causative agent. However, it is unclear whether effective stable innate immune system activation can be achieved without triggering harmful autoimmunity or other chronic inflammatory sequelae. Here, we show that transgenic expression of a picornavirus RNA-dependent RNA polymerase (RdRP), in the absence of other viral proteins, can profoundly reconfigure mammalian innate antiviral immunity by exposing the normally membrane-sequestered RdRP activity to sustained innate immune detection. RdRP-transgenic mice have life-long, quantitatively dramatic upregulation of 80 interferon-stimulated genes (ISGs) and show profound resistance to normally lethal viral challenge. Multiple crosses with defined knockout mice (Rag1, Mda5, Mavs, Ifnar1, Ifngr1, and Tlr3) established that the mechanism operates via MDA5 and MAVS and is fully independent of the adaptive immune system. Human cell models recapitulated the key features with striking fidelity, with the RdRP inducing an analogous ISG network and a strict block to HIV-1 infection. This RdRP-mediated antiviral mechanism does not depend on secondary structure within the RdRP mRNA but operates at the protein level and requires RdRP catalysis. Importantly, despite lifelong massive ISG elevations, RdRP mice are entirely healthy, with normal longevity. Our data reveal that a powerfully augmented MDA5-mediated activation state can be a well-tolerated mammalian innate immune system configuration. These results provide a foundation for augmenting innate immunity to achieve broad-spectrum antiviral protection.

HP-PRRSV is attenuated by de-optimization of codon pair bias in its RNA-dependent RNA polymerase nsp9 gene.

There is an urgent need to develop new vaccines against highly pathogenic PRRS virus (HP-PRRSV) variant in China. The actual use of each codon pairs is more or less frequent than that of the statistical prediction and codon pair bias (CPB) usage affects gene translation. We "shuffled" the existing codons in HP-PRRSV genes GP5, M, nsp2 and nsp9, so that the CPB of these genes could be more negative. De-optimization of nsp9, the RNA-dependent RNA polymerase, significantly decreased PRRSV replication in porcine alveolar macrophages (PAMs). In vitro study showed that HV-nsp9(min) and HV-nsp29(min) were remarkably attenuated in PAMs, and inoculation of pigs with 2 ml⁎10(5.0) TCID50/ml of HV-nsp9(min) or HV-nsp29(min) did not cause PRRS. Importantly, pigs immunized with HV-nsp29(min) were fully protected against different HP-PRRSV strains׳ lethal challenges. Our results imply that the CPB de-optimized HV-nsp29(min) has the potential to be used as a live vaccine candidate against HP-PRRSV.

[Effect of Point Mutations in the Polymerase Genes of the Influenza A/PR/8/34 (H1N1) Virus on the Immune Response in a Mouse Model].

The vaccine strains for live attenuated influenza vaccines (LAIVs) have cold-adapted, temperature-sensitive, and attenuated phenotypes, which are guaranteed by the presence of specific mutations from the master donor virus in their internal genes. In this study, we used mutant viruses of the pathogenic A/Puerto Rico/8/34 (H1N1) that contained ts-mutations in PB1 (K265N, V591I), PB2 (V478L), and PA (L28P, V341L) genes along and/or in different combinations to evaluate the impact of these mutations in the immune responses. Sequential addition of tested mutations resulted in the stepwise decrease in virus-specific serum and, to a lesser extent, mucosal antibody levels. We demonstrated strong positive correlation between virus attenuation (virus titer in lung) and antibody titers. The ts-mutations in PB1, PB2, and PA genes are mostly involved in the modulation of the humoral immunity, but also have a moderate effect on the cellular adaptive immune response.

Characterizing the roles of Cryphonectria parasitica RNA-dependent RNA polymerase-like genes in antiviral defense, viral recombination and transposon transcript accumulation.

An inducible RNA-silencing pathway, involving a single Dicer protein, DCL2, and a single Argonaute protein, AGL2, was recently shown to serve as an effective antiviral defense response in the chestnut blight fungus Cryphonectria parasitica. Eukaryotic RNA-dependent RNA polymerases (RdRPs) are frequently involved in transcriptional and posttranscriptional gene silencing and antiviral defense. We report here the identification and characterization of four RdRP genes (rdr1-4) in the C. parasitica genome. Sequence relationships with other eukaryotic RdRPs indicated that RDR1 and RDR2 were closely related to QDE-1, an RdRP involved in RNA silencing ("quelling") in Neurospora crassa, whereas RDR3 was more closely related to the meiotic silencing gene SAD-1 in N. crassa. The RdRP domain of RDR4, related to N. crassa RRP-3 of unknown function, was truncated and showed evidence of alternative splicing. Similar to reports for dcl2 and agl2, the expression levels for rdr3 and rdr4 increased after hypovirus CHV-1/EP713 infection, while expression levels of rdr1 and rdr2 were unchanged. The virus-responsive induction patterns for rdr3 and rdr4 were altered in the Δdcl2 and Δagl2 strains, suggesting some level of interaction between rdr3 and rdr4 and the dcl2/agl2 silencing pathway. Single rdr gene knockouts Δrdr1-4, double knockouts Δrdr1/2, Δrdr2/3, Δrdr1/3, and a triple knockout, Δrdr1/2/3, were generated and evaluated for effects on fungal phenotype, the antiviral defense response, viral RNA recombination activity and transposon expression. None of the single or multiple rdr knockout strains displayed any phenotypic differences from the parental strains with or without viral infection or any significant changes in viral RNA accumulation or recombination activity or transposon RNA accumulation, indicating no detectable contribution by the C. parasitica rdr genes to these processes.