Transcriptor High Fidelity Cdna Synthesis Kit Pdf Download
BACH1 expression was regulated by using lentiviral vector infection, and the efficiency was tested using qRT-PCR and Western blot analysis (Additional file 2: Fig. S2). The CCK-8 assay revealed that BACH1-knockdown significantly impaired the proliferation of hDPSCs (Fig. 4a) while BACH1-overexpression increased the proliferation (Additional file 2: Fig. S3a). The EdU assay, one used to identify cells undergoing DNA synthesis, revealed remarkably fewer proliferating (EdU-positive) cells in the LV-shBACH1 group than that of the control group (Fig. 4b). As cells that have DNA replication are in the S phase of the cell cycle, the results of the EdU labelling assay were in agreement with the cell cycle test. The BACH1-knockdown group showed a lower proportion of S and G2/M phases and a higher proportion of G0/G1 phases implying cell cycle arrest (Fig. 4c).
transcriptor high fidelity cdna synthesis kit pdf download
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Viral diversity has major implications on pathogenesis, drug resistance, and vaccine development. Since next-generation sequencing (NGS) platforms are widely available, virus populations can be studied much faster compared to the classical methodology of single genome sequencing. However, these technologies require rigorous estimation of error rates and identification of error sources, especially when viral haplotypes are quantified (reviewed in ). For instance, several studies have investigated the accuracy of the pyrosequencing technology, and it is well known that homopolymeric regions are the main source of insertion-deletion (indel) errors , . Moreover, the PCR polymerase can also contribute to this effect . PCR artifacts are well known and addressed by optimizing PCR conditions and using high fidelity DNA polymerases . Recently, primer identifiers have been described to circumvent some of the remaining PCR artifacts .
Ribavirin (1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-triazole-3-carboxamide), a purine analogue, inhibits replication of RNA viruses by different mechanisms, including the depletion of the cellular GTP pool by competing with cellular inosine monophosphate for inosine monophosphate dehydrogenase, interfering with RNA capping, inhibiting RdRP or by acting as a mutagen leading to lethal mutagenesis5,6,7. As a mutagen, ribavirin has been used to select high-fidelity variants of positive-sense, single-stranded RNA viruses of the Piconaviridae and Togaviridae families8,9,10,11 with similar RdRP structures12. In contrast, IAV possesses a heterotrimeric polymerase complex formed by the PB2, PB1 and PA proteins with the RdRP catalytic function residing in the PB1 protein13. The molecular workings of its polymerase, including fidelity, are poorly understood, and it is not known if ribavirin can induce mutagenesis in the IAV genome. Favipiravir (T-705), a novel antiviral drug and a purine analogue with a broad antiviral polymerase activity, has recently been shown to induce mutagenesis in the influenza genome14.
Here, to gain insights into the biological significance of IAV mutation frequency and viral genetic diversity on viral pathogenesis, we generate influenza RdRP fidelity variants by the serial passage of a human seasonal H3N2 influenza virus (A/Wuhan/359/95; Wuhan95) in the presence of ribavirin. We confirm that ribavirin functions as a mutagen for IAV by increasing G-to-A and C-to-T mutations in vitro. PB1-V43I mutation is identified to increase polymerase fidelity in recombinant Wuhan95 virus as well as in a highly pathogenic H5N1 virus (A/Vietnam/1203/04; VN04). The recombinant VN04 virus with the PB1-V43I mutation replicates to comparable titres as the wild-type counterpart in vitro or in the mouse lungs, but has reduced population diversity at day 3 post inoculation. Such reduced genetic diversity at an early time point post infection is associated with a reduced lethality and neurovirulence. Our results identify a single V43I mutation in PB1 protein that affects viral genetic diversity and provide the first experimental evidence of the role of genetic diversity in IAV pathogenicity.
The error-prone RdRP assures adaptation and survival of RNA viruses under different selection pressures. Using an IAV RdRP fidelity variant selected with ribavirin, we provide direct experimental evidence that supports the biological significance of viral genetic diversity on IAV pathogenesis. We demonstrate that a single V43I mutation in PB1 protein confers resistance to ribavirin and increased selectivity to nucleoside, leading to a RdRP with increased fidelity and a reduction in population genetic diversity of both seasonal H3N2 (Wuhan95) and highly pathogenic H5N1 (VN04) viruses. The increased RdRP fidelity similarly reduced VN04 virus population genetic diversity in the mouse lungs at day 3 post inoculation without affecting lung virus titres. Such a reduction in virus population genetic diversity attenuated viral lethality by tenfold with decreased viral neurovirulence. We consider that such a difference in diversity might have affected viral pathogenicity at several levels. First, a more diverse viral population would increase the survival possibility by having subpopulations that may escape neutralizing antibodies or specific T-cell response, adapt better to the host replication machinery or expand tissue tropism. The lung titres between mice inoculated with the VN1203 wild-type or the PB1-V43I-mutant viruses were comparable suggesting that the difference in lethality was not due to differences in the replication capability between these two viruses. It was noted that the PB1-V43I VN04 virus showed reduced neurotropism while compared with the VN1203 wild-type virus. Such a difference was previously demonstrated by Pfeiffer et al.19 and Vignizzi et al.20 that different virus population may act synergistically and contribute neurovirulence of poliovirus. Furthermore, with the Wuhan wild-type and PB1-V43I viruses, we demonstrated that they differed in the ability in generating monoclonal antibody escape mutants (Supplementary Fig. 5). Further studies would be needed to elucidate the precise mechanism leading to differences in lethality observed with the VN1203 wild-type and PB1-V43I mutants. However, these results provide experimental evidence supporting the role of viral genetic diversity in viral pathogenicity. The reported IAV fidelity variant can be further applied to study viral population dynamics under different selection pressures within and between hosts.
The ability to manipulate the RdRP mutational rate has so far been restricted to the non-segmented, positive-sense, single-stranded RNA viruses of the Piconaviridae and Togaviridae families, both with similar polymerase structures. Studies on the high-fidelity variants of poliovirus showed reduced viral pathogenicity in vivo as a result of reduced ability to adapt to the in vivo environment, including the inability to acquire a reversion mutation (back from the introduced attenuating mutation) at permissive temperature19 or reduced neutrotropism in mice20. High-fidelity chikungunya virus, an arbovirus that requires transmission between an invertebrate (mosquito) and a vertebrate host, was shown to replicate to lower viral titres than that of the wild-type virus in both hosts9. Interestingly, the viral fitness (replication efficiency) of the wild-type and the high-fidelity viruses in vitro was often comparable or only moderately affected9,19,20. One the other hand, RdRP with increased mutational frequency (low-fidelity RdRP) would also lead to reduced pathogenicity in vivo, as the Coxsackievirus B3 fidelity variant with increased mutation frequency was observed to be attenuated in vivo24. Overall, our data and that from others in different virus models support the idea that the RNA viruses replicate within a finely tuned and narrow error threshold to achieve optimal survival, especially under in vivo conditions. Attenuation of virus virulence in vivo without significantly affecting viral replication in vitro would be a desirable property to be included in the future development of live-attenuated vaccines25.
Structural insights are available for multiple domains derived from the PB2 and PA proteins, but there is limited structural insight available for the PB1 protein that possess the catalytic function for RdRP26. So far, it has been demonstrated that the nucleoside analogue favipiravir can induce mutagenesis in the genome of IAV14. However, resistance to favipiravir as a mutagen had not been reported hitherto, raising doubts on whether it is technically feasible to obtain a resistant variant to mutagen for IAV RdRP, which has a heterotrimer structure. Through serial passaging of the Wuhan95 virus in the presence of ribavirin, a total of 11 mutations were identified in the PB2, PB1, PA and NP proteins. We have selected the PB1-V43I mutation for further characterization because of its most significantly decreased sensitivity to ribavirin in the mini-genome system. However, we cannot exclude the possibility that some other mutations, alone or in combination, may also alter IAV RdRP fidelity. This was shown to be the case with enterovirus, where both the G64S and A372V mutations were reported to confer the high-fidelity phenotype10. The V43I mutation lies within the putative viral RNA-binding domain in the N terminus of PB1 and is generally quite conserved. A blast search among available influenza PB1 sequences at the NCBI Influenza Virus Source identified a total of five isolates possessing the PB1-V43I mutation. Three out of the five identified sequences are avian origins (two highly pathogenic H5N1 from chicken and Muscovy duck and one H3 influenza virus from ruddy turnstone), and the other two are swine (H1N1) origin. The role of this mutation remains to be determined in the adaptation of IAV to different animal species.
Overall, a single PB1-V43I mutation was identified to confer resistance to ribavirin and altered selectivity to guanosine for the polymerase complex derived from both human seasonal H3N2 and the avian-origin H5N1 highly pathogenic influenza viruses. Our results suggest that the PB1-V43I mutation reduced PB1-binding affinity for ribavirin and confer to increased selectivity for NTP, which likely serves as the mechanism for the altered RdRP fidelity and reduced mutational frequency. Applying a fidelity variant with reduced mutational frequency, we provide the first experimental evidence for the role of viral genetic diversity in IAV pathogenesis. This model can be applied to the repertoire of well-developed IAV models to further investigate the role of genetic diversity and population dynamics on influenza pathogenesis and transmission. 350c69d7ab