RESUMO
Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.
Assuntos
Citidina , Vírus da Hepatite B , RNA Viral , Transcrição Reversa , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , RNA Viral/genética , RNA Viral/metabolismo , Citidina/análogos & derivados , Citidina/metabolismo , Citidina/genética , Humanos , Transcrição Reversa/genética , Metilação , Replicação Viral/genética , Epigênese Genética , Vírion/metabolismo , Vírion/genética , TranscriptomaRESUMO
Surface modification of a polymer substrate with a mercapto functionality is crucial in many applications such as flexible circuitry and point-of-care biosensors. We present here a novel bifunctional molecular adhesive, 3-mercaptopropylsilatrane (MPS), as an interface between polymer and metal surfaces. Under ambient conditions, surface modification of polymer surfaces with a mercapto functionality can be achieved with low concentration (0.46 mM) of MPS in aqueous solvent (50% ethanol) in a short time (<30 min). Three popular polymers for optical sensors, polycarbonate, polyethylene terephthalate, and poly(methyl methacrylate), were employed as substrates, and MPS films formed on these substrates were examined and compared with that on a glass substrate. The films were characterized by UV-vis spectroscopy, water contact angle, X-ray photoelectron spectroscopy, and atomic force microscopy. MPS was also used as a bifunctional linker for the construction of a gold nanoparticle (AuNP) sub-monolayer on these polymer surfaces. Under optimized preparation conditions, the absorbance and full width at half-maximum of the plasmon band are comparable to those of a AuNP-modified glass substrate. Hence, MPS may have a potential to be a key component in polymer substrate-based localized surface plasmon resonance sensors. A self-catalytic surface reaction mechanism is also proposed to account for the results. As compared to a glass surface with a high number of silanol groups, the successful formation of an MPS film on polymer surfaces with relatively few reactive sites is probably due to the lateral polymerization of MPS starting from a condensed MPS molecule on a reactive site of a polymer surface.