Ribonucleotide reductases (RNRs) are crucial to all living cells, since they provide deoxyribonucleotides (dNTPs) for DNA synthesis and repair. In Mycobacterium tuberculosis, a class Ib RNR comprising nrdE-and nrdF2-encoded subunits is essential for growth in vitro. Interestingly, the genome of this obligate human pathogen also contains the nrdF1 (Rv1981c) and nrdB (Rv0233) genes, encoding an alternate class Ib RNR small (R2) subunit and a putative class Ic RNR R2 subunit, respectively. However, the role(s) of these subunits in dNTP provision during M. tuberculosis pathogenesis is unknown. In this study, we demonstrate that nrdF1 and nrdB are dispensable for the growth and survival of M. tuberculosis after exposure to various stresses in vitro and, further, that neither gene is required for growth and survival in mice. These observations argue against a specialist role for the alternate R2 subunits under the conditions tested. Through the construction of nrdR-deficient mutants of M. tuberculosis and Mycobacterium smegmatis, we establish that the genes encoding the essential class Ib RNR subunits are specifically regulated by an NrdR-type repressor. Moreover, a strain of M. smegmatis mc 2 155 lacking the 56-kb chromosomal region, which includes duplicates of nrdHIE and nrdF2, and a mutant retaining only one copy of nrdF2 are shown to be hypersensitive to the class I RNR inhibitor hydroxyurea as a result of depleted levels of the target. Together, our observations identify a potential vulnerability in dNTP provision in mycobacteria and thereby offer a compelling rationale for pursuing the class Ib RNR as a target for drug discovery.

Research on applying RNA interference (RNAi) to counter HBV replication has led to identification of potential therapeutic sequences. However, before clinical application liver-specific expression and efficient delivery of these sequences remain an important objective. We recently reported short-term inhibition of HBV replication in vivo by using helper dependent adenoviral vectors (HD Ads) expressing anti-HBV sequences from a constitutively active cytomegalovirus (CMV) promoter. To develop the use of liver-specific transcription regulatory elements we investigated the utility of the murine transthyretin (MTTR) promoter for expression of anti-HBV primary microRNAs (pri-miRs). HD Ads containing MTTR promoter effected superior expression of anti-HBV pri-miRs in mice compared to HD Ads containing the CMV promoter. MTTR-containing HD Ads resulted in HBV replication knockdown of up to 94% in mice. HD Ads expressing trimeric anti-HBV pri-miRs silenced HBV replication for 5 weeks. We previously showed that the product of the codelivered lacZ gene induces an immune response, and the duration of HBV silencing in vivo is likely to be attenuated by this effect. Nevertheless, expression of anti-HBV pri-miRs from MTTR promoter is well suited to countering HBV replication and development of HD Ads through attenuation of their immunostimulatory effects should advance their clinical utility.

Adenoviruses are very powerful hepatotropic vectors. To make adenoviruses more effective for clinical use, polymer conjugation and deletion of viral vector sequences have been used successfully. However, further modifications to attenuate immunostimulation as well as improvements in large-scale production are necessary before the therapeutic potential of adenovirus-mediated delivery of RNAi activators is realized.

HD Ads have potential utility for delivery of therapeutic HBV-silencing sequences and alterations of these vectors to attenuate their immune responses may further improve their efficacy.

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