Abstract
Human adenoviruses (HAds) encode for one or two highly abundant virus-associated RNAs, designated VA RNAI and VA RNAII, which fold into stable hairpin structures resembling miRNA precursors. Here we show that the terminal stem of the VA RNAs originating from Ad4, Ad5, Ad11 and Ad37, all undergo Dicer dependent processing into virus-specific miRNAs (so-called mivaRNAs). We further show that the mivaRNA duplex is subjected to a highly asymmetric RISC loading with the 3′-strand from all VA RNAs being the favored strand, except for the Ad37 VA RNAII, where the 5′-mivaRNAII strand was preferentially assembled into RISC. Although the mivaRNA seed sequences are not fully conserved between the HAds a bioinformatics prediction approach suggests that a large fraction of the VA RNAII-, but not the VA RNAI-derived mivaRNAs still are able to target the same cellular genes. Using small RNA deep sequencing we demonstrate that the Dicer processing event in the terminal stem of the VA RNAs is not unique and generates 3′-mivaRNAs with a slight variation of the position of the 5′ terminal nucleotide in the RISC loaded guide strand. Also, we show that all analyzed VA RNAs, except Ad37 VA RNAI and Ad5 VA RNAII, utilize an alternative upstream A start site in addition to the classical +1 G start site. Further, the 5′-mivaRNAs with an A start appears to be preferentially incorporated into RISC. Although the majority of mivaRNA research has been done using Ad5 as the model system our analysis demonstrates that the mivaRNAs expressed in Ad11- and Ad37-infected cells are the most abundant mivaRNAs associated with Ago2-containing RISC. Collectively, our results show an unexpected variability in Dicer processing of the VA RNAs and a serotype-specific loading of mivaRNAs into Ago2-based RISC.
Highlights
Human adenoviruses (HAd) are non-enveloped DNA viruses with a linear double-stranded DNA genome of about 30 000–40 000 base pairs
Whereas a mutant in VA RNAII expression grows essentially as wild type [9] a double mutant in VA RNAI and VA RNAII shows an approximately 60-fold reduction in virus growth compared to a 10-fold reduction in a VA RNAI only mutant virus [10]. Taken together these results suggest that VA RNAII can enhance virus growth with a significantly lower efficiency compared to VA RNAI
The data show that the virus-associated RNA (VA RNA) from all tested serotypes are processed by the Dicer enzyme (Figure 3) into small RNAs that resemble in size mature cellular miRNAs
Summary
Human adenoviruses (HAd) are non-enveloped DNA viruses with a linear double-stranded DNA genome of about 30 000–40 000 base pairs. More than 60 types of HAds have been described so far. They are classified into seven distinct subgroups A to G based on immunological, biological and biochemical characteristics [1]. Human adenoviruses can infect a wide range of cell types, a property making adenovirus one of the most prominent viral infectious agents in mammalian cells. HAds cause a broad spectrum of acute and chronic infections, such as respiratory tract infections and diverse ocular and gastrointestinal diseases. Viruses belonging to subgroups A and F infect mainly the gastrointestinal tract whereas viruses from subgroups B, C and E infect preferentially the respiratory tract. Subgroup D viruses, which is the largest subgroup of viruses, have a broad spectra of tropism including ocular and respiratory tract infections [4]
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