RNA interference (RNAi) is a diverse, conserved regulatory mechanism in eukaryotic cells, which silences the target gene expression in a homology-dependent manner. Although it has been well documented that RNAi is an antiviral mechanism in plants and insects, it is still unclear whether RNAi naturally limits viral infections in vertebrates. Viruses are masters of adopting strategies to subvert cellular defense mechanisms. Not only can viruses use elaborate strategies to suppress the effects of defensive RNAi, but they can also redirect or interfere with cellular functions orchestrated by endogenous small RNAs. In our work we have focused on studying the relationship of human adenovirus type 5 (Ad5) infection and the RNAi/miRNA pathways. We show that Ad5 infection inhibits RNAi by blocking the activity of Dicer and the RNA-induced silencing complex (RISC). For Dicer inhibition, the virus-associated RNAs, VA RNAI and VA RNAII bind Dicer through their terminal stems and are cleaved by Dicer into functional small RNAs that are incorporated into active RISC. Furthermore, by cloning small RNAs, we found that approximately 80% of Ago2-containing RISC immunopurified from late infected cells was associated with VA RNA-derived small RNAs (mivaRNAs). Interestingly, the small RNAs derived from VA RNAII, the minor VA RNA species, appear to be the major mivaRNAs occupying RISC and associate with polyribosomes, which indicates their potential roles as miRNAs regulating translation of cellular mRNAs. During our previous work, we observed that the strand bias of VA RNAI derived small RNA (mivaRI) incorporating into active RISC varied in the different viable Ad5 mutant viruses infected cells. It has been reported that Ad5 VA RNAI had two transcription initiation sites, which produced two clusters of VA RNAI with 3 nt difference at their 5’ end. Our data show that this heterogeneity resulted in a dramatic difference in mivaRI guide strand selection. Collectively, our data contributes to understanding the interplay between virus and host. This study would be beneficial in designing optimal adenovirus vectors for therapeutic RNAi application.

Human adenovirus (HAdV) is a common pathogen causing a broad spectrum of diseases. HAdV encodes the pVII protein, which is involved in nuclear delivery, protection and expression of viral DNA. To suppress the cellular interferon (IFN) and RNA interference (RNAi) systems, HAdVs encode non-coding virus-associated (VA) RNAs. In this thesis we have investigated the functional significance of the pVII protein and VA RNAI in HAdV-5 infected cells. We report that the propeptide module is the destabilizing element targeting the precursor pVII protein for proteasomal degradation. We also found that the Cul3-based E3 ubiquitin ligase complex alter the precursor pVII protein stability via binding to the propeptide sequence. In addition, we show that inhibition of the Cul3 protein reduces HAdV-5 E1A gene expression. Collectively, our results suggest a novel function for the pVII propeptide module and involvement of Cul3 in viral E1A gene expression. Our studies show that the cellular E3 ubiquitin ligase MKRN1 is a novel pVII interacting protein in HAdV-5 infected cells. MKRN1 expression reduced the pVII protein accumulation in virus-infected cells and affected infectious virus formation. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the prolonged HAdV-5 infection. Furthermore, the precursor pVII protein enhanced MKRN1 self-ubiquitination, suggesting the direct involvement of pVII in the initiation of MKRN1 degradation. Hence, we propose that the MKRN1 is a novel antiviral protein and that HAdV-5 infection counteracts its antiviral activity. In papers III and IV, we tested the ability of various plant and animal virus encoded RNAi/miRNA and IFN suppressor proteins to functionally substitute for the HAdV-5 VA RNAI. Our results revealed that the Vaccinia virus E3L protein was able to partially substitute for the HAdV-5 VA RNAI functions in virus-infected cells. Interestingly, the E3L protein rescued the translational defect but did not stimulate viral capsid mRNA accumulation observed with VA RNA. Additionally, we show that the HAdV-4 and HAdV-37 VA RNAI are more effective in virus replication compared to HAdV-5 and HAdV-12 VA RNAI. In paper IV, we employed a novel triplex-specific probing assay, based on the intercalating and cleaving agent benzoquinoquinaxline 1,10-phenanthroline (BQQ-OP), to unravel triplex structure formation in 
VA RNAI. The BQQ-OP cleavage of HAdV-4 VA RNAI indicates that a potential 
triplex is formed involving the highly conserved stem 4 of the central domain and side 
stem 7. Further, the integrity of HAdV-4 VA RNAI stem 7 contributes to the virus growth in vivo.