| Summary: | 碩士 === 長庚大學 === 生物醫學研究所 === 99 === Influenza virus remains one of major pathogens that cause large morbidity and mortality in the world every year. Although there are two efficient neuraminidase inhibitors, oseltamivir and zanamivir, available for clinical treatment, the emergence of resistant influenza virus has prompted to find alternative antiviral agents. In our study, we have screened several novel compounds of which BPR3P0128 exhibits a powerful anti-influenza virus activity, EC50 0.02M. BPR3P0128 can not only reduce the M1 viral RNA synthesis but also block the cytopathic effect induced by influenza A or B in MDCK cells with neutralization assay. Besides it was approved to block virus cap-binding activity of PB2, an important component of influenza virus RNPs that assists virus RNA transcription and replication. Thus, we try to identify several host factors implicated in the RNP complex using MALDI-TOF mass analysis. Of these candidate proteins, heat shock protein family, HSP60, HSP70 and HSP90, were found. According to previous researches, hsp70 and hsp90 were identified as cellular interacting partners of the influenza virus ribonucleoprotein (RNP) complex, and may regulate viral replication, however, the function of HSP60 remains elusive. Thus, we want to investigate the biological significance of Hsp60 in influenza virus. We found that Hsp60 has direct interaction with viral protein PB2 using co-immunoprecipitation assay. Besides, silencing of hsp60 could induce the viral protein NS1 expression and virus yield. In previously studies, hsp60 was also approved to regulate the immune responses during pathogen invasion. We used RT-PCR to detect the expression level of IL-6 and IL-8 underlying virus infection in silenced and non-silenced cells, respectively. We found that mRNA of both cytokines was reduced in the hsp60-silenced cells. In conclusion, we supposed that the inhibition mechanism of 3P may rely on the interruption of binding between HSP60 and PB2 and more detail studies were still ongoing. As BPR3P0128 shows no tendency to induce resistant virus variant, it may be suitable as anti-influenza virus agent.
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