RESUMEN
Ribonucleotide reductases (RRs or RNRs) catalyze the reduction of the OH group on the 2nd carbon of ribose, reducing four ribonucleotides (NTPs) to the corresponding deoxyribonucleotides (dNTPs) to promote DNA synthesis. Large DNA viruses, such as herpesviruses and poxviruses, could benefit their replication through increasing dNTPs via expression of viral RRs. Little is known regarding the relationship between cellular RRs and RNA viruses. Mammalian RRs contain two subunits of ribonucleotide reductase M1 polypeptide (RRM1) and two subunits of ribonucleotide reductase M2 polypeptide (RRM2). In this study, expression of cellular RRMs, including RRM1 and RRM2, is found to be down-regulated in hepatitis C virus (HCV)-infected Huh7.5 cells and Huh7 cells with HCV subgenomic RNAs (HCVr). As expected, the NTP/dNTP ratio is elevated in HCVr cells. Compared with that of the control Huh7 cells with sh-scramble, the NTP/dNTP ratio of the RRM-knockdown cells is elevated. Knockdown of RRM1 or RRM2 increases HCV replication in HCV replicon cells. Moreover, inhibitors to RRMs, including Didox, Trimidox and hydroxyurea, enhance HCV replication. Among various HCV viral proteins, the NS5A and/or NS3/4A proteins suppress the expression of RRMs. When these are taken together, the results suggest that HCV down-regulates the expression of RRMs in cultured cells to promote its replication.
RESUMEN
The nonstructural protein 1 (NS1) of influenza A virus (IAV) possesses multiple functions, such as the inhibition of the host antiviral immune responses, to facilitate viral infection. To search for cellular proteins interacting with the IAV NS1 protein, the yeast two-hybrid system was adopted. Proteasome family member PSMB4 (proteasome subunit beta type 4) was found to interact with the NS1 protein in this screening experiment. The binding domains of these two proteins were also determined using this system. The physical interactions between the NS1 and cellular PSMB4 proteins were further confirmed by co-immunoprecipitation assay and confocal microscopy in mammalian cells. Neither transiently nor stably expressed NS1 protein affected the PSMB4 expression in cells. In contrast, PSMB4 reduced the NS1 protein expression level, especially in the presence of MG132. As expected, the functions of the NS1 protein, such as inhibition of interferon activity and enhancement of transient gene expression, were suppressed by PSMB4. PSMB4 knockdown enhances IAV replication, while its overexpression attenuates IAV replication. Thus, the results of this study suggest that the cellular PSMB4 protein interacts with and possibly facilitates the degradation of the NS1 protein, which in turn suppresses IAV replication.
