Arenaviral NPs are the newest members of the 3′-5′ DEDDH exoribonuclease family and their RNase activities play an unprecedented role in inhibiting type I IFN production (8
). We have shown that NP exoribonuclease preferentially degrades dsRNA templates in vitro
(), which is consistent with the known role of triphosphate dsRNA as the pathogen-associated molecular pattern RNA that triggers the type I IFN production (27
). We therefore propose a novel immune evasion mechanism in which NP suppresses the IFN induction by selectively degrading these immune-stimulatory RNAs. In this study, we provide atomic views of the active form of this unique exoribonuclease enzyme of the LASV NP protein in the process of trimming the 3′ end of its preferred dsRNA substrate.
While our manuscript was in preparation, Hastie and colleagues (12
) published the structure of a catalytically inactive version of LASV NP-C in complex with a dsRNA at 2.9 Å. At this resolution, it was not possible to visualize the sequence of the dsRNA and water molecules in the active site as well as to identify all possible side chain interactions. As such, only two residues (Gly-392 and Tyr-429) were identified to be essential for binding to and processing dsRNA substrate. Our studies with a catalytically active enzyme have revealed several mechanistic insights into this unique NP exoribonuclease activity. First of all, the NP catalytic cavity binds the 4-bp RNA duplex and directly positions the 3′-end of the cleaving strand in the catalytically active site (). The cleaving strand of the RNA substrate directly or indirectly interacts with mostly conserved residues (Gly-392, Glu-391, Asp-466, Arg-492, Gln-462, and Ser-430) of the NP protein, whereas the complementary strand interacts with the non-conserved residues (Tyr-429, Arg-393, Gln-425, Asp-426, Gln-422, and Asp-465) (), perhaps allowing for the flexibility of NP binding the different dsRNA substrates. Mutational analyses suggest that the conserved residues are indeed important for the NP exoribonuclease activity, whereas the non-conserved residues can tolerate alanine substitutions without affecting the enzymatic and biological functions (). Therefore, the strict requirement of several residues in positioning the cleaving strand and the flexibility in binding the complementary strand provide not only the structural conservation that is important for this unique dsRNA-selective enzymatic mechanism but also the necessary flexibility needed to process any forms of dsRNA substrates that can potentially trigger IFN production. It is important to note that in addition to the five catalytic residues, the Arg-492 residue is indispensable for exoribonuclease activity. We propose that Arg-492, in addition to positioning the 3′ ribonucleotide, may be directly involved in the cleavage step.
Furthermore, the 3′ end ribonucleotide in the NP-C·dsRNA complex structure remained in a double-stranded form, although directly presented to the catalytic residues for catalysis. In contrast, other known 3′-5′ exonucleases such as Trex1, Trex2, and Klenow fragment contained essentially single-stranded nucleic acid in their catalytic pockets, even though Trex1 showed a preference for the dsDNA substrate (29
) and Klenow was co-crystalized with a hairpin (ds) DNA (23
). We obtained a trapped intermediate product complex of LASV NP-C in the process of cleaving dsRNA that reveals a unique mechanism by which NP exoribonuclease activity cleaves the 3′ ribonucleotide prior to separating the double-stranded form. Most known 3′-5′ exoribonucleases rely on ATP-dependent helicase activity to unwind RNA duplexes to promote exonucleolytic activity, with the exception of E. coli
RNase R (30
We have also conducted a time course soaking experiment to explore the kinetic process of the LASV NP exoribonuclease function ( and and supplemental Figs. S4 and S6
). Taken these data together, we propose that the LASV NP exoribonuclease processes dsRNA substrate in a four-step mechanism (1
). NP positions the 3′ end ribonucleotide into the active site, through the dsRNA-binding interface (2
). NP then uses the 5 catalytic residues (Asp-389, Glu-391, Asp-466, Asp-533, and His-528) and Arg-492 to break the phosphodiester bond of the 3′ ribonucleotide from the cleaving strand (in its double-stranded form), via a well known two-metal mechanism (3
). The 3′ end ribonucleotide is then dissociated from the base pair formation with the complementary strand, possibly through interactions with residues Tyr-429, Ser-430, Asp-426, and Arg-393 (4
). The cleaved nucleotide monophosphate is released from the active site.
FIGURE 7. Conformational changes observed for the 5′-ppp dsRNA and metal ions relative to the five catalytic residues over the time course of the soaking experiment. These molecules before and 1, 1.5, and 5 min after soaking are shown in red, green, blue (more ...)
Our current study also challenges a long-standing belief that TCRV NP is unique among the arenaviral NPs in its lack of IFN suppressive function. A previous study by Martínez-Sobrido et al.
) has shown that the NP proteins from various arenaviruses of both Old World and New World groups, including LASV, lymphocytic choriomeningitis virus (LCMV), Whitewater Arroyo virus (WWAV), Pichinde virus (PICV), Junin virus (JUNV), Machupo virus (MACV), and Latino virus (LATV), can effectively inhibit IFN production, with a notable exception of TCRV, the only known arenavirus isolated from bats (1
). Nevertheless, we have unequivocally shown here that TCRV NP resembles LASV NP in tertiary structure, exoribonuclease activity in vitro
, as well as in its ability to suppress Sendai virus-induced IFN production in cell culture (). We have also produced preliminary data to show that cells infected with Tacaribe virus produce similarly low levels of type I IFNs as seen with those infected with LCMV and PICV (data not shown). The NP gene used in the current study was provided by J. Nunberg, University of Montana, and was also amplified from Tacaribe virus purchased from ATCC (strain 11573). It is not clear what source of TCRV NP gene was used in the original report, however (7
). Therefore, the discrepancy between the findings by Martínez-Sobrido (7
) and ours needs to be investigated further. Regardless, we have provided in the current study compelling structural, enzymatic, and biological evidence to demonstrate that TCRV NP has 3′-5′ exoribonuclease activity and IFN suppressive ability as those of the LASV NP. We therefore believe that the NP RNase activity-mediated inhibition of the IFN production via degradation of the immune-stimulatory RNAs is a common mechanism among all known arenaviruses.
In summary, we have provided the structural basis for a distinctive mechanism of a new member of 3′-5′ DEDDH exoribonuclease family, LASV NP, in binding and cleaving a dsRNA substrate, and have shown that suppression of IFN production via NP RNase activity by degrading immune-stimulatory RNAs represents a common mechanism of arenaviruses to mediate innate immune evasion. These new discoveries may help stimulate the development of specific therapeutics against these important viral pathogens that can cause high mortality and morbidity in humans.