The ribonuclease Dicer is a multidomain enzyme that plays a fundamental role in the biogenesis of small regulatory RNAs (srRNAs), which control gene expression by targeting complementary transcripts and inducing their cleavage or repressing their translation. Recent studies of Dicer’s domains have permitted to propose their roles in srRNA biogenesis. For all of Dicer’s domains except one, called DUF283 (domain of unknown function), their involvement in RNA substrate recognition, binding or cleavage has been postulated. For DUF283, the interaction with Dicer’s protein partners has been the only function suggested thus far. In this report, we demonstrate that the isolated DUF283 domain from human Dicer is capable of binding single-stranded nucleic acids in vitro. We also show that DUF283 can act as a nucleic acid annealer that accelerates base-pairing between complementary RNA/DNA molecules in vitro. We further demonstrate an annealing activity of full length human Dicer. The overall results suggest that Dicer, presumably through its DUF283 domain, might facilitate hybridization between short RNAs and their targets. The presented findings reveal the complex nature of Dicer, whose functions may extend beyond the biogenesis of srRNAs.

Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by a variety of proteins are being elucidated, less is known about non-protein factors, e.g. RNA molecules, that may influence this enzyme's activity. Therefore, we decided to investigate the question of whether the RNA molecules can function not only as Dicer substrates but also as its regulators. Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript, we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs that are longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding to this enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by participating in regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.

Tick-borne encephalitis virus (TBEV), of the genus Flavivirus, is a causative agent of severe encephalitis in endemic regions of northern Asia and central and northern Europe. Interferon induced transmembrane proteins (IFITMs) are restriction factors that inhibit the replication cycles of numerous viruses, including flaviviruses such as the West Nile virus, dengue virus, and Zika virus. Here, we demonstrate the role of IFITM1, IFITM2, and IFITM3 in the inhibition of TBEV infection and in protection against virus-induced cell death. We show the most significant role being that of IFITM3, including the dissection of its functional motifs by mutagenesis. Furthermore, through the use of CRISPR–Cas9-generated IFITM1/3-knockout monoclonal cell lines, we confirm the role and additive action of endogenous IFITMs in TBEV suppression. However, the results of co-culture assays suggest that TBEV might partially escape IFN- and IFITM-mediated suppression during high-density co-culture infection when the virus enters naïve cells directly from infected donor cells. Thus, cell-to-cell spread may constitute a strategy for virus escape from innate host defenses. Importance: TBEV infection may result in encephalitis, chronic illness or death. TBEV is endemic in northern Asia and Europe; however, due to climate change, new endemic centers arise. Although effective TBEV vaccines have been approved, vaccination coverage is low, and, due to the lack of specific therapeutics, infected individuals depend on their immune responses to control the infection. The IFITM proteins are components of the innate antiviral defenses that suppress cell entry of many viral pathogens. However, no studies regarding the role of IFITM proteins in the TBEV infection have been published so far. Understanding of antiviral innate immune responses is crucial for future development of antiviral strategies. Here, we show the important role of IFITM proteins in the inhibition of TBEV infection and virus-mediated cell death. However, our data suggest that TBEV cell-to-cell spread may be less prone to both IFN- and IFITM-mediated suppression, potentially facilitating escape from IFITM-mediated immunity.

MicroRNAs (miRNAs) have been identified as a class of crucial regulators of virus-host crosstalk, modulating such processes as viral replication, antiviral immune response, viral latency, and pathogenesis. Pseudorabies virus (PRV), a model for the study of alphaherpesvirus biology, codes for 11 distinct miRNAs mapped to the ~4.6 kb intron of Large Latency Transcript (LLT). Recent studies have revealed the role of clusters consisting of nine and eleven miRNA genes in the replication and virulence of PRV. The function of separate miRNA species in regulating PRV biology has not been thoroughly investigated. To analyze the regulatory potential of three PRV miRNAs located in the frontal cluster of the LLT intron, we generated a research model based on the constitutive expression of viral miRNAs in swine testis cells (ST_LLT [1–3] cell line). Using a cell culture system providing a stable production of individual miRNAs at high levels, we demonstrated that the LLT [1–3] miRNA cluster significantly downregulated IE180, EP0, and gE at the early stages of PRV infection. It was further determined that LLT [1–3] miRNAs could regulate the infection process, leading to a slight distortion in transmission and proliferation ability. Collectively, our findings indicate the potential of LLT [1–3] miRNAs to retard the host responses by reducing viral antigenic load and suppressing the expansion of progeny viruses at the early stages of infection.