RESUMO
Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality rates in newborn piglets, leading to massive losses to the swine industry worldwide during recent epidemics. Intense research efforts are now focusing on defining viral characteristics that confer a growth advantage, pathogenicity, or cell adaptability in order to better understand the PEDV life cycle and identify suitable targets for antiviral or vaccine development. Here, we report a unique phenomenon of PEDV nucleocapsid (N) cleavage by the PEDV-encoded 3C-like protease (3Cpro) during infection. The identification of the 3Cpro cleavage site at the C terminus of N supported previous observations that PEDV 3Cpro showed a substrate requirement slightly different from that of severe acute respiratory syndrome coronavirus (SARS-CoV) 3Cpro and revealed a greater flexibility in its substrate recognition site. This cleavage motif is present in the majority of cell culture-adapted PEDV strains but is missing in emerging field isolates. Remarkably, reverse-genetics-derived cell culture-adapted PEDVAVCT12 harboring uncleavable N displayed growth retardation in Vero E6-APN cells compared to the wild-type virus. These observations altogether shed new light on the investigation and characterization of the PEDV nucleocapsid protein and its possible link to cell culture adaptation. IMPORTANCE: Recurrent PEDV outbreaks have resulted in enormous economic losses to swine industries worldwide. To gain the upper hand in combating this disease, it is necessary to understand how this virus replicates and evades host immunity. Characterization of viral proteins provides important clues to mechanisms by which viruses survive and spread. Here, we characterized an intriguing phenomenon in which the nucleocapsids of some PEDV strains are proteolytically processed by the virally encoded main protease. Growth retardation in recombinant PEDV carrying uncleavable N suggests a replication advantage provided by the cleavage event, at least in the cell culture system. These findings may direct us to a more complete understanding of PEDV replication and pathogenicity.
Assuntos
Cisteína Endopeptidases/metabolismo , Nucleocapsídeo/metabolismo , Vírus da Diarreia Epidêmica Suína/fisiologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Técnicas de Cultura de Células , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Genoma Viral , Nucleocapsídeo/química , Proteólise , Suínos , Doenças dos Suínos/virologia , Células VeroRESUMO
The coronavirus spike protein and the influenza virus hemagglutinin are class I viral membrane fusion proteins. While the two proteins display strong structural conservation and the mechanisms underlying membrane fusion are similar, they share no sequence similarity. Whether they are functionally interchangeable is currently unknown. In this study, we constructed scIAV-S, a single-cycle influenza A virus pseudotyped with the spike protein of porcine epidemic diarrhea virus (PEDV), and demonstrated that this virus could infect cultured cells and trigger massive syncytium formation. Treatment with endocytosis inhibitors did not affect syncytium formation by infected cells. Moreover, the infectivity of scIAV-S was associated with the degree of cell adaptation of PEDV-S. Intriguingly, scIAV-S lacking functional neuraminidase (NA) exhibited substantially higher infectivity, suggesting a pivotal role of the sialic acid in the binding/entry of PEDV. Together, scIAV-S offers a robust platform for the investigation of the entry mechanism of PEDV or, possibly, of other coronaviruses.
Assuntos
Infecções por Coronavirus/veterinária , Vírus da Influenza A/genética , Vírus da Diarreia Epidêmica Suína/genética , Glicoproteína da Espícula de Coronavírus/genética , Doenças dos Suínos/virologia , Animais , Linhagem Celular , Infecções por Coronavirus/virologia , Vírus da Influenza A/metabolismo , Vírus da Diarreia Epidêmica Suína/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , SuínosRESUMO
Porcine epidemic diarrhoea virus (PEDV) causes acute diarrhoea and dehydration in swine of all ages, with significant mortality in neonatal pigs. The recent rise of PEDV outbreaks in Asia and North America warrants an urgent search for effective vaccines. However, PEDV vaccine research has been hampered by difficulties in isolating and propagating the virus in mammalian cells, thereby complicating the recovery of infectious PEDV using a full-length infectious clone. Here, we engineered VeroE6 cells to stably express porcine aminopeptidase N (pAPN) and used them as a platform to obtain a high-growth variant of PEDV, termed PEDVAVCT12. Subsequently, the full-length cDNA clone was constructed by assembling contiguous cDNA fragments encompassing the complete genome of PEDVAVCT12 in a bacterial artificial chromosome. Infectious PEDV could be recovered, and the rescued virus displayed phenotypic properties identical to the parental virus. Interestingly, we found that PEDVAVCT12 contained a C-terminal deletion of the spike gene, resulting in disruption of the ORF3 start codon. When a functional ORF3 gene was restored, the recombinant virus could not be rescued, suggesting that ORF3 could suppress PEDV replication in vitro. In addition, a high-growth and genetically stable recombinant PEDV expressing a foreign protein could be rescued by replacing the ORF3 gene with the mCherry gene. Together, the results of this study provide a means to generate genetically defined PEDV as a promising vaccine candidate.
Assuntos
Infecções por Coronavirus/veterinária , DNA Complementar/genética , DNA Viral/genética , Diarreia/veterinária , Vírus da Diarreia Epidêmica Suína/genética , Doenças dos Suínos/virologia , Animais , Sequência de Bases , Chlorocebus aethiops , Infecções por Coronavirus/virologia , DNA Complementar/metabolismo , DNA Viral/metabolismo , Diarreia/virologia , Genoma Viral , Dados de Sequência Molecular , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/fisiologia , Suínos , Células VeroRESUMO
Upon co-infection with influenza B virus (FluB), influenza A virus (FluA) replication is substantially impaired. Previously, we have shown that the nucleoprotein of FluB (BNP) can inhibit FluA polymerase machinery, retarding the growth of FluA. However, the molecular mechanism underlying this inhibitory action awaited further investigation. Here, we provide evidence that BNP hinders the proper formation of FluA polymerase complex by competitively binding to the nucleoprotein of FluA. To exert this inhibitory effect, BNP must be localized in the nucleus. The interaction does not require the presence of the viral RNA but needs an intact BNP RNA-binding motif. The results highlight the novel role of BNP as an anti-influenza A viral agent and provide insights into the mechanism of intertypic interference.
Assuntos
Vírus da Influenza A/fisiologia , Vírus da Influenza B/fisiologia , Influenza Humana/virologia , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Animais , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Coinfecção/metabolismo , Coinfecção/virologia , Cães , Células HEK293 , Humanos , Influenza Humana/metabolismo , Células Madin Darby de Rim Canino , Proteínas do Nucleocapsídeo , RNA Viral/metabolismoRESUMO
While viral inhibition by tethering of budding virions to host cell membranes has been focused upon as one of the main functions of BST-2/tetherin, BST-2 is thought to possess other functions as well. Overexpression of BST-2 was found here to down-regulate transient protein expression. Removal of the N- and C-terminal regions of BST-2, previously described to be involved in signal transduction, reduced the impact of BST-2. These results suggest that BST-2-mediated signaling may play a role in regulating the levels of transiently expressed proteins, highlighting a new function for BST-2 that may also have implications for viral inhibition.
Assuntos
Antígenos CD/fisiologia , Regulação para Baixo , Animais , Antígenos CD/genética , Linhagem Celular , Cães , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/fisiologia , Células HEK293 , Humanos , RNA Mensageiro/genética , Transdução de SinaisRESUMO
African swine fever virus (ASFV) is a large, double-stranded DNA virus that causes a fatal, contagious disease specifically in pigs. However, prevention and control of ASFV outbreaks have been hampered by the lack of an effective vaccine or antiviral treatment for ASFV. Although ASFV has been reported to adapt to a variety of continuous cell lines, the phenotypic and genetic changes associated with ASFV adaptation to MA-104 cells remain poorly understood. Here, we adapted ASFV field isolates to efficiently propagate through serial viral passages in MA-104 cells. The adapted ASFV strain developed a pronounced cytopathic effect and robust infection in MA-104 cells. Interestingly, the adapted variant maintained its tropism in primary porcine kidney macrophages. Whole genome analysis of the adapted virus revealed unique gene deletions in the left and right variable regions of the viral genome compared to other previously reported cell culture-adapted ASFV strains. Notably, gene duplications at the 5' and 3' ends of the viral genome were in reverse complementary alignment with their paralogs. Single point mutations in protein-coding genes and intergenic regions were also observed in the viral genome. Collectively, our results shed light on the significance of these unique genetic changes during adaptation, which facilitate the growth of ASFV in MA-104 cells.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Doenças dos Suínos , Suínos , Animais , Genoma Viral , Deleção de Genes , Surtos de Doenças , Doenças dos Suínos/epidemiologiaRESUMO
Alveolar macrophages are tissue-resident immune cells that protect epithelial cells in the alveoli from invasion by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, the interaction between macrophages and SARS-CoV-2 is inevitable. However, little is known about the role of macrophages in SARS-CoV-2 infection. Here, we generated macrophages from human induced pluripotent stem cells (hiPSCs) to investigate the susceptibility of hiPSC-derived macrophages (iMΦ) to the authentic SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variants as well as their gene expression profiles of proinflammatory cytokines during infection. With undetectable angiotensin-converting enzyme 2 (ACE2) mRNA and protein expression, iMΦ were susceptible to productive infection with the Delta variant, whereas infection of iMΦ with the Omicron variant was abortive. Interestingly, Delta induced cell-cell fusion or syncytia formation in iMΦ, which was not observed in Omicron-infected cells. However, iMΦ expressed moderate levels of proinflammatory cytokine genes in response to SARS-CoV-2 infection, in contrast to strong upregulation of these cytokine genes in response to polarization by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Overall, our findings indicate that the SARS-CoV-2 Delta variant can replicate and cause syncytia formation in macrophages, suggesting that the Delta variant can enter cells with undetectable ACE2 levels and exhibit greater fusogenicity.
Assuntos
COVID-19 , Células Gigantes , Células-Tronco Pluripotentes Induzidas , Humanos , Enzima de Conversão de Angiotensina 2/genética , COVID-19/virologia , Citocinas/genética , Macrófagos , SARS-CoV-2/genéticaRESUMO
The emergence and rapid evolution of SARS-CoV-2 variants have posed a major challenge to the global efforts to control the COVID -19 pandemic. In this study, we investigated the potential of two SARS-CoV-2 variants, BA.2 and BA.5, to evade neutralization by a human monoclonal antibody targeting the virus's spike RBD (mAb 1D1). By subjecting the viruses to serial propagation in the presence of the antibody, we found that BA.2 exhibited poor growth, whereas BA.5 regained robust growth with significantly higher kinetics than the parental virus. Genetic analysis identified a single mutation, A475V, in the spike protein of BA.5 that substantially reduced the neutralizing activities of monoclonal antibodies and convalescent sera. In addition, the A475V mutation alone in BA.2 moderately reduced the neutralizing activity but completely abolished the neutralizing effect of mAb 1D1 when F486V or L452R were also present. Our results shed light on the possible evolutionary development of SARS-CoV-2 variants under selection pressure by monoclonal antibodies and have implications for the development of effective antibody therapies and vaccines against the virus.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Anticorpos Monoclonais/uso terapêutico , Soroterapia para COVID-19RESUMO
Coronaviruses isolated from bats and pangolins are closely related to SARS-CoV-2, the causative agent of COVID-19. These so-called sarbecoviruses are thought to pose an acute pandemic threat. As SARS-CoV-2 infection and vaccination have become more widespread, it is not known whether neutralizing antibodies to SARS-CoV-2 can cross-neutralize coronaviruses transmitted by bats or pangolins. In this study, we analyzed antibody-mediated neutralization with serum samples from COVID-19 patients (n = 31) and those immunized with inactivated SARS-CoV-2 vaccines (n = 20) against lentivirus-based pseudo-viruses carrying the spike derived from ancestral SARS-CoV-2, bat (RaTG13 or RshSTT182), or pangolin coronaviruses (PCoV-GD). While SARS-CoV-2, PCoV-GD, and RshSTT182 spikes could promote cell-cell fusion in VeroE6 cells, the RaTG13 spike did not. RaTG13, on the other hand, was able to induce cell-cell fusion in cells overexpressing ACE2. Dramatic differences in neutralization activity were observed, with the highest level observed for RaTG13, which was even significantly higher than SARS-CoV-2, PCoV-GD, and RshSTT182 pseudo-viruses. Interestingly, pseudo-viruses containing the chimeric protein in which the receptor-binding domain (RBD) of PCoV-GD spike was replaced by that of RaTG13 could be strongly neutralized, whereas those carrying RaTG13 with the RBD of PCoV-GD were significantly less neutralized. Because the high neutralizing activity against RaTG13 appears to correlate with its low affinity for binding to the human ACE2 receptor, our data presented here might shed light on how pre-existing immunity to SARS-CoV-2 might contribute to protection against related sarbecoviruses with potential spillover to the human host.
Assuntos
COVID-19 , Quirópteros , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Pangolins , SARS-CoV-2/genética , Glicoproteína da Espícula de CoronavírusRESUMO
Virus-like particles (VLPs) are highly immunogenic and versatile subunit vaccines composed of multimeric viral proteins that mimic the whole virus but lack genetic material. Due to the lack of infectivity, VLPs are being developed as safe and effective vaccines against various infectious diseases. In this study, we generated a chimeric VLP-based COVID-19 vaccine stably produced by HEK293T cells. The chimeric VLPs contain the influenza virus A matrix (M1) proteins and the SARS-CoV-2 Wuhan strain spike (S) proteins with a deletion of the polybasic furin cleavage motif and a replacement of the transmembrane and cytoplasmic tail with that of the influenza virus hemagglutinin (HA). These resulting chimeric S-M1 VLPs, displaying S and M1, were observed to be enveloped particles that are heterogeneous in shape and size. The intramuscular vaccination of BALB/c mice in a prime-boost regimen elicited high titers of S-specific IgG and neutralizing antibodies. After immunization and a challenge with SARS-CoV-2 in K18-hACE2 mice, the S-M1 VLP vaccination resulted in a drastic reduction in viremia, as well as a decreased viral load in the lungs and improved survival rates compared to the control mice. Balanced Th1 and Th2 responses of activated S-specific T-cells were observed. Moderate degrees of inflammation and viral RNA in the lungs and brains were observed in the vaccinated group; however, brain lesion scores were less than in the PBS control. Overall, we demonstrate the immunogenicity of a chimeric VLP-based COVID-19 vaccine which confers strong protection against SARS-CoV-2 viremia in mice.
RESUMO
In response to the SARS-CoV-2 Delta variant, which partially escaped the vaccine-induced immunity provided by two doses of vaccination with CoronaVac (Sinovac), the National Vaccine Committee recommended the heterologous CoronaVac-ChAdOx1 (Oxford−AstraZeneca), a prime−boost vaccine regimen. This pilot study aimed to describe the immunogenicity and adverse events of the heterologous CoronaVac-ChAdOx1 regimen, in comparison with homologous CoronaVac, and homologous ChAdOx1. Between May and August 2021, we recruited a total of 354 participants from four vaccination groups: the CoronaVac-ChAdOx1 vaccinee (n = 155), the homologous CoronaVac vaccinee (n = 32), the homologous ChAdOx1 vaccinee (n = 47), and control group of COVID-19 patients (n = 120). Immunogenicity was evaluated by measuring the level of IgG antibodies against the receptor-binding domain (anti-SRBD) of the SARS-CoV-2 spike protein S1 subunit and the level of neutralizing antibodies (NAbs) against variants of concern (VOCs) using the plaque reduction neutralization test (PRNT) and pseudovirus neutralization test (pVNT). The safety profile was recorded by interviewing at the 1-month visit after vaccination. The anti-SRBD level after the second booster dose of the CoronaVac-ChAdOx1 group at 2 weeks was higher than 4 weeks. At 4 weeks after the second booster dose, the anti-SRBD level in the CoronaVac-ChAdOx1 group was significantly higher than either homologous CoronaVac, the homologous ChAdOx1 group, and Control group (p < 0.001). In the CoronaVac-ChAdOx1 group, the PRNT50 level against the wild-type (434.5 BAU/mL) was the highest; followed by Alpha variant (80.4), Delta variant (67.4), and Beta variant (19.8). The PVNT50 level was also found to be at its highest against the wild-type (432.1); followed by Delta variants (178.3), Alpha variants (163.9), and Beta variant (42.2), respectively. The AEs in the CoronaVac-ChAdOx1 group were well tolerated and generally unremarkable. The CoronaVac-ChAdOx1 heterologous regimen induced higher immunogenicity and a tolerable safety profile. In a situation when only CoronaVac-ChAdOx1 vaccines are available, they should be considered for use in responding to the Delta variant.
RESUMO
Sequence analysis of the nucleoprotein (NP) of swine-origin influenza virus H1N1 (S-OIV) reveals a number of atypical characteristics including an early start codon and a highly conserved, non-aromatic residue at position 313. Using an in vitro viral polymerase reconstitution assay, we found that the polymerase complex containing the NP of S-OIV (NP(S-OIV)) yielded substantially lower activity than those assayed with NP derived from other influenza virus strains. Moreover, alteration of the early start codon or introduction of an aromatic residue at position 313 (V313Y) did not increase but instead exacerbated the poor polymerase activity. Interestingly, when NP(S-OIV) was allowed to compete with that of a mouse-adapted influenza virus (A/PR/8/34) to form progeny virions, only progeny bearing NP(S-OIV) were produced, despite the low polymerase activity associated with NP(S-OIV). Our results indicated that NP(S-OIV) requires both the early start codon and the V313 residue for its optimal function. These characteristics are required for a strong compatibility between the S-OIV polymerase subunits and its indigenous NP over that of other strains, which might explain why productive reassortment between S-OIV and seasonal influenza viruses has yet to occur in nature.
Assuntos
Códon de Iniciação , Vírus da Influenza A Subtipo H1N1/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Vírus Reordenados/genética , Ribonucleoproteínas/metabolismo , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Animais , Western Blotting , Linhagem Celular , Cães , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/epidemiologia , Influenza Humana/virologia , Mutação , Proteínas do Nucleocapsídeo , Pandemias , Vírus Reordenados/fisiologia , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais/metabolismoRESUMO
The use of virus-vectored platforms has increasingly gained attention in vaccine development as a means for delivering antigenic genes of interest into target hosts. Here, we describe a single-cycle influenza virus-based SARS-CoV-2 vaccine designated as scPR8-RBD-M2. The vaccine utilizes the chimeric gene encoding 2A peptide-based bicistronic protein cassette of the SARS-CoV-2 receptor-binding domain (RBD) and influenza matrix 2 (M2) protein. The C-terminus of the RBD was designed to link with the cytoplasmic domain of the influenza virus hemagglutinin (HA) to anchor the RBD on the surface of producing cells and virus envelope. The chimeric RBD-M2 gene was incorporated in place of the HA open-reading frame (ORF) between the 3' and 5' UTR of HA gene for the virus rescue in MDCK cells stably expressing HA. The virus was also constructed with the disrupted M2 ORF in segment seven to ensure that M2 from the RBD-M2 was utilized. The chimeric gene was intact and strongly expressed in infected cells upon several passages, suggesting that the antigen was stably maintained in the vaccine candidate. Mice inoculated with scPR8-RBD-M2 via two alternative prime-boost regimens (intranasal-intranasal or intranasal-intramuscular routes) elicited robust mucosal and systemic humoral immune responses and cell-mediated immunity. Notably, we demonstrated that immunized mouse sera exhibited neutralizing activity against pseudotyped viruses bearing SARS-CoV-2 spikes from various variants, albeit with varying potency. Our study warrants further development of a replication-deficient influenza virus as a promising SARS-CoV-2 vaccine candidate.
RESUMO
While porcine epidemic diarrhea virus (PEDV) infects and replicates in enterocytes lining villi of neonatal piglets with high efficiency, naturally isolated variants typically grow poorly in established cell lines, unless adapted by multiple passages. Cells infected with most cell-adapted PEDVs usually displayed large syncytia, a process triggered by the spike protein (S). To identify amino acids responsible for S-mediated syncytium formation, we constructed and characterized chimeric S proteins of the cell-adapted variant, YN144, in which the receptor binding domain (RBD) and S1/S2 cleavage site were replaced with those of a poorly culturable field isolate (G2). We demonstrated that the RBD, not the S1/S2 cleavage site, is critical for syncytium formation mediated by chimeric S proteins. Further mutational analyses revealed that a single mutation at the amino acid residue position 672 (V672F) could enable the chimeric S with the entire RBD derived from the G2 strain to trigger large syncytia. Moreover, recombinant PEDV viruses bearing S of the G2 strain with the single V672F substitution could induce extensive syncytium formation and replicate efficiently in VeroE6 cells stably expressing porcine aminopeptidase N (VeroE6-APN). Interestingly, we also demonstrated that while the V672F mutation is critical for the syncytium formation in VeroE6-APN cells, it exerts a minimal effect in Huh-7 cells, thereby suggesting the difference in receptor preference of PEDV among host cells.
Assuntos
Mutação , Vírus da Diarreia Epidêmica Suína/genética , Glicoproteína da Espícula de Coronavírus/genética , Replicação Viral/genética , Substituição de Aminoácidos , Animais , Fusão Celular , Chlorocebus aethiops , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Suínos , Doenças dos Suínos/virologia , Células VeroRESUMO
The M2 protein (AM2 and BM2) of influenza A and B viruses function as a proton channel essential for viral replication. They also carry a cytoplasmic tail whose functions are not fully delineated. It is currently unknown whether these proteins could be replaced functionally in a viral context. Here, we generated single-cycle influenza A viruses (scIAV-ΔHA) carrying various M2-2A-mCherry constructs in the segment 4 (HA) and evaluated their growth in complementing cells. Intriguingly, the scIAV-ΔHA carrying AM2 and that bearing BM2 grew comparably well in MDCK-HA cells. Furthermore, while the virus carrying chimeric B-AM2 in which the BM2 transmembrane fused with the AM2 cytoplasmic tail produced robust infection, the one bearing the AM2 transmembrane fused with the BM2 cytoplasmic tail (A-BM2) exhibited severely impaired growth. Altogether, we demonstrate that AM2 and BM2 are functionally interchangeable and underscore the role of compatibility between transmembrane and cytoplasmic tail of the M2 protein.
Assuntos
Vírus da Influenza A/fisiologia , Vírus da Influenza B/fisiologia , Proteínas da Matriz Viral/metabolismo , Replicação Viral , Animais , Linhagem Celular , Células Cultivadas , Genes Reporter , Teste de Complementação Genética , Humanos , Fenótipo , Recombinação Genética , Proteínas da Matriz Viral/genéticaRESUMO
Vesicular stomatitis virus (VSV) is highly immunogenic and able to stimulate both innate and adaptive immune responses. However, its ability to induce adverse effects has held back the use of VSV as a potential vaccine vector. In this study we developed VSV-ΔP, a safe yet potent replication-defective recombinant VSV in which the phosphoprotein (P) gene was deleted. VSV-ΔP replicated only in supporting cells expressing P (BHK-P cells) and at levels more than 2 logs lower than VSV. In vivo studies indicated that the moderate replication of VSV-ΔP in vitro was associated with the attenuation of this virus in the mouse model, whereas mice intracranially injected with VSV succumbed to neurotoxicity. Furthermore, we constructed VSV and VSV-ΔP expressing a variety of antigens including hemagglutinin-neuraminidase (HN) from Newcastle disease virus (NDV), hemagglutinin (HA) from either a 2009 H1N1 pandemic influenza virus (pdm/09) or the avian H7N9. VSV and VSV-ΔP incorporated the foreign antigens on their surface resulting in induction of robust neutralizing antibody, serum IgG, and hemagglutination inhibition (HAI) titers against their corresponding viruses. These results indicated that VSV with P gene deletion was attenuated in vitro and in vivo, and possibly expressed the foreign antigen on its surface. Therefore, the P gene-deletion strategy may offer a potentially useful and safer approach for attenuating negative-sense RNA viruses which use phosphoprotein as a cofactor for viral replication.
Assuntos
Vetores Genéticos/genética , Vesiculovirus/genética , Vacinas Virais/uso terapêutico , Replicação Viral/genética , Animais , Regulação Viral da Expressão Gênica/genética , Vetores Genéticos/efeitos adversos , Vetores Genéticos/uso terapêutico , Hemaglutininas/genética , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Camundongos , Vírus da Doença de Newcastle/genética , Fosfoproteínas/genética , Deleção de Sequência/genética , Vesiculovirus/patogenicidade , Vacinas Virais/genéticaRESUMO
The nucleoprotein of influenza B virus (BNP) shares several characteristics with its influenza A virus counterpart (ANP), including localization in the host's nucleus. However, while the nuclear localization signal(s) (NLS) of ANP are well characterized, little is known about those of BNP. In this study, we showed that the fusion protein bearing the BNP N-terminus fused with GFP (N70-GFP) is exclusively nuclear, and identified a highly conserved KRXR motif spanning residues 44-47 as a putative NLS. In addition, we demonstrated that residues 3-15 of BNP, though not an NLS, are also crucial for nuclear import. Results from mutational analyses of N70-GFP and the full-length BNP suggest that this region may be required for protection of the N-terminus from proteolytic cleavage. Altogether, we propose that the N-terminal region of BNP contains the NLS and cleavage-protection motif, which together drive its nuclear localization.
Assuntos
Transporte Ativo do Núcleo Celular , Vírus da Influenza B/genética , Sinais de Localização Nuclear , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Animais , Fusão Gênica Artificial , Linhagem Celular , Núcleo Celular/química , Análise Mutacional de DNA , Cães , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Vírus da Influenza B/fisiologia , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genéticaRESUMO
Pandemic influenza A/H1N1 (2009) and avian influenza A/H5N1 neuraminidase (NA) differ at two critical residues, positions 149 and 347. Recombinant influenza A viruses were constructed in which these two residues in pandemic influenza A/H1N1 (2009) NA were changed to the corresponding amino acids of avian influenza A/H5N1 NA, and vice versa. Recombinant viruses bearing N1 NA with the oseltamivir resistance mutation H274Y in combination with mutations at residues 149 and 347 were also constructed. Recombinant viruses grew normally in allantoic fluid and were subsequently studied for viral infectivity (TCID50), substrate binding (Km) and sensitivity to oseltamivir (Ki). The data demonstrated that infectivity of mutant viruses in Madin Darby canine kidney cells was comparable to, or even greater than, the infectivity of the parental viruses harboring wild-type N1 NA. Furthermore, mutations at NA residues 149 and 347 altered Km and Ki values, and thus modulated oseltamivir sensitivity. Although these mutants have yet to be observed among natural isolates, the minimal costs to the growth of recombinant viruses indicate their possible viability. Reassortment between pandemic influenza A/H1N1 (2009) and avian influenza A/H5N1 viruses may therefore generate new influenza A viruses with increased infectivity and oseltamivir resistance, and continued surveillance will be crucial for public health preparedness.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/genética , Neuraminidase/genética , Oseltamivir/farmacologia , Proteínas Virais/genética , Fatores de Virulência/genética , Substituição de Aminoácidos , Animais , Linhagem Celular , Cães , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/patogenicidade , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/crescimento & desenvolvimento , Virus da Influenza A Subtipo H5N1/patogenicidade , Mutagênese Sítio-Dirigida , Neuraminidase/metabolismo , Proteínas Virais/metabolismo , Virulência , Fatores de Virulência/metabolismoRESUMO
Given that co-infection of cells with equivalent titers of influenza A and B viruses (FluA and FluB) has been shown to result in suppression of FluA growth, it is possible that FluB-specific proteins might hinder FluA polymerase activity and replication. We addressed this possibility by individually determining the effect of each gene of FluB on the FluA polymerase assay and found that the nucleoprotein of FluB (NP(FluB)) inhibits polymerase activity of FluA in a dose-dependent manner. Mutational analyses of NP(FluB) suggest that functional NP(FluB) is necessary for this inhibition. Slower growth of FluA was also observed in MDCK cells stably expressing NP(FluB). Further analysis of NP(FluB) indicated that it does not affect nuclear import of NP(FluA). Taken together, these findings suggest a novel role of NP(FluB) in inhibiting replication of FluA, providing more insights into the mechanism of interference between FluA and FluB and the lack of reassortants between them.
Assuntos
Vírus da Influenza A/fisiologia , Vírus da Influenza B/fisiologia , Nucleoproteínas/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas do Core Viral/metabolismo , Interferência Viral , Replicação Viral , Animais , Linhagem Celular , Análise Mutacional de DNA , Cães , Vírus da Influenza A/genética , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza B/genética , Nucleoproteínas/genética , RNA Polimerase Dependente de RNA/genética , Proteínas do Core Viral/genéticaRESUMO
Despite several lines of evidence suggesting possible mechanisms by which the influenza virus polymerase complex, comprising PB2, PB1 and PA, work in concert during virus replication, exactly how they function is not entirely understood. The N terminal region of the PA subunit has been shown to play a key role in various functions through a number of conserved amino acid residues. However, little is known about the role of amino acids reported to be unique for a virus strain. Here, we investigated the functional implication of an amino acid (S186) present uniquely in the N terminus of the PA subunit of the pandemic H1N1 influenza virus and determined the effect of its mutation in terms of polymerase activity as well as virus growth. Using chimeric constructs of PA derived from A/PR/8/34 (H1N1) (PR8) and the swine-origin influenza virus (S-OIV) H1N1, we found that, when complexed with PB2 and PB1 of PR8, the chimeric PA protein containing the N terminus of S-OIV (1-213) with the remaining region from PR8 showed significantly reduced polymerase activity. Recombinant viruses harboring the chimeric PA also grew poorly in MDCK cells and embryonated eggs. Likewise, the chimeric PA in which the N terminus of PA of PR8 (1-213) was assembled with the remaining region of PA of S-OIV showed a similar phenotype when complexed with PB2 and PB1 of S-OIV. Interestingly, when S186 in the N terminus was altered to the residue common in most strains of influenza virus (G186), the chimeric as well as wild-type PA of S-OIV showed severely impaired polymerase activity when assayed with PB2 and PB1 of S-OIV. Collectively, this finding suggests that S186 at the N terminal region of PA of S-OIV is necessary for the protein to function optimally.