Methylation is one of the critical modifications that regulates numerous biological processes. Guanine capping and methylation at the 7th position (m7G) have been shown to mature mRNA for increased RNA stability and translational efficiency. The m7G capped cap0 RNA remains immature and requires additional methylation at the first nucleotide (N1-2\'-O-Me), designated as cap1, to achieve full maturation. This cap1 RNA with N1-2\'-O-Me prevents its recognition by innate immune sensors as non-self. Viruses have also evolved various strategies to produce self-like capped RNAs with the N1-2\'-O-Me that potentially evades the antiviral response and establishes an efficient replication. In this review, we focus on the importance of the presence of N1-2\'-O-Me in viral RNAs and discuss the potential for drug development by targeting host and viral N1-2\'-O-methyltransferases.

Most cytoplasmic-replicating negative-strand RNA viruses (NSVs) initiate genome transcription by cap snatching. The source of host mRNAs from which the cytoplasmic NSVs snatch capped-RNA leader sequences has remained elusive. Earlier reports have pointed towards cytoplasmic-RNA processing bodies (P body, PB), although several questions have remained unsolved. Here, the nucleocapsid (N) protein of plant- and animal-infecting members of the order Bunyavirales, in casu Tomato spotted wilt virus (TSWV), Rice stripe virus (RSV), Sin nombre virus (SNV), Crimean-Congo hemorrhagic fever virus (CCHFV) and Schmallenberg virus (SBV) have been expressed and localized in cells of their respective plant and animal hosts. All N proteins localized to PBs as well as stress granules (SGs), but extensively to docking stages of PB and SG. TSWV and RSV N proteins also co-localized with Ran GTPase-activating protein 2 (RanGAP2), a nucleo-cytoplasmic shuttling factor, in the perinuclear region, and partly in the nucleus when co-expressed with its WPP domain containing a nuclear-localization signal. Upon silencing of PB and SG components individually or concomitantly, replication levels of a TSWV minireplicon, as measured by the expression of a GFP reporter gene, ranged from a 30% reduction to a four-fold increase. Upon the silencing of RanGAP homologs in planta, replication of the TSWV minireplicon was reduced by 75%. During in vivo cap-donor competition experiments, TSWV used transcripts destined to PB and SG, but also functional transcripts engaged in translation. Altogether, the results implicate a more complex situation in which, besides PB, additional cytoplasmic sources are used during transcription/cap snatching of cytoplasmic-replicating and segmented NSVs.

Influenza virus transcription is catalyzed by the viral RNA-polymerase (FluPol) through a cap-snatching activity. The snatching of the cap of cellular mRNA by FluPol is preceded by its binding to the flexible C-terminal domain (CTD) of the RPB1 subunit of RNA-polymerase II (Pol II). To better understand how FluPol brings the 3\'-end of the genomic RNAs in close proximity to the host-derived primer, we hypothesized that FluPol may recognize additional Pol II subunits/domains to ensure cap-snatching. Using binary complementation assays between the Pol II and influenza A FluPol subunits and their structural domains, we revealed an interaction between the N-third domain of PB2 and RPB4. This interaction was confirmed by a co-immunoprecipitation assay and was found to occur with the homologous domains of influenza B and C FluPols. The N-half domain of RPB4 was found to be critical in this interaction. Punctual mutants generated at conserved positions between influenza A, B, and C FluPols in the N-third domain of PB2 exhibited strong transcriptional activity defects. These results suggest that FluPol interacts with several domains of Pol II (the CTD to bind Pol II), initiating host transcription and a second transcription on RPB4 to locate FluPol at the proximity of the 5\'-end of nascent host mRNA.

Viruses with negative-strand RNA genomes (NSVs) include many highly pathogenic and economically devastating disease-causing agents of humans, livestock, and plants-highlighted by recent Ebola and measles virus epidemics, and continuously circulating influenza virus. Because of their protein-coding orientation, NSVs face unique challenges for efficient gene expression and genome replication. To overcome these barriers, NSVs deliver a large and multifunctional RNA-dependent RNA polymerase into infected host cells. NSV-encoded polymerases contain all the enzymatic activities required for transcription and replication of their genome-including RNA synthesis and mRNA capping. Here, we review the structures and functions of NSV polymerases with a focus on key domains responsible for viral replication and gene expression. We highlight shared and unique features among polymerases of NSVs from the Mononegavirales, Bunyavirales, and Articulavirales orders.

Influenza virus RNA-dependent RNA polymerase (vRdRp) does not have capping activity and relies on the capped RNAs produced by the host RNA polymerase II (RNAPII). The viral polymerases process the capped RNAs to produce short capped RNA fragments that are used as primers to initiate the transcription of viral mRNAs. This process, known as cap-snatching, can be targeted by antiviral therapeutics. Here, anthralin was identified as an inhibitor against influenza a virus (IAV) infection by targeting the cap-snatching activity of the viral polymerase. Anthralin, an FDA-approved drug used in the treatment of psoriasis, shows antiviral activity against IAV infection in vitro and in vivo. Importantly, anthralin significantly reduces weight loss, lung injury, and mortality caused by IAV infection in mice. The mechanism of action study revealed that anthralin inhibits the cap-binding function of PB2 subunit and endonuclease activity of PA. As a result, viral mRNA transcription is blocked, leading to the decreases in viral RNA replication and viral protein expression. In conclusion, anthralin has been demonstrated to have the potential of an alternative antiviral against influenza virus infection. Also, targeting the captive pocket structure that includes the N-terminus of PA endonuclease domain and the C-terminal of PB2 cap-binding domain of IAV RdRp may be an excellent strategy for developing anti-influenza drugs.

In common with all segmented negative-sense RNA viruses, bunyavirus transcripts contain heterologous sequences at their 5\' termini originating from capped host cell RNAs. These heterologous sequences are acquired by a so-called cap-snatching mechanism. Whereas for nuclear replicating influenza virus the source of capped primers as well as the cap-binding and endonuclease activities of the viral polymerase needed for cap snatching have been functionally and structurally well characterized, our knowledge on the expected counterparts of cytoplasmic replicating bunyaviruses is still limited and controversial. This review focuses on the cap-snatching mechanism of bunyaviruses in the light of recent structural and functional data.

Like their animal-infecting counterparts, plant bunyaviruses use capped RNA leaders cleaved from host cellular mRNAs to prime viral genome transcription in a process called cap-snatching, but in vivo systems to investigate the details of this process are lacking for them. Here, we report that Rice stripe tenuivirus (RSV) and Tomato spotted wilt tospovirus (TSWV) cleave capped RNA leaders from mRNAs transiently expressed by agroinfiltration, which makes it possible to artificially deliver defined cap donors to the two plant bunyaviruses with unprecedented convenience. With this system, some ideas regarding how plant bunyaviruses select and use capped RNA leaders can be tested easily. We were also able to obtain clear evidence that the capped RNA leaders selected by TSWV are generally longer than those by RSV. TSWV frequently uses the prime-and-realign mechanism in transcription primed by capped RNA leaders shorter than a certain length, like that has been demonstrated recently for RSV.

RNA processing and decay pathways have important impacts on RNA viruses, particularly animal-infecting bunyaviruses, which utilize a cap-snatching mechanism to translate their mRNAs. However, their effects on plant-infecting bunyaviruses have not been investigated. The roles of mRNA degradation and non-sense-mediated decay components, including DECAPPING 2 (DCP2), EXORIBONUCLEASE 4 (XRN4), ASYMMETRIC LEAVES2 (AS2) and UP-FRAMESHIFT 1 (UPF1) were investigated in infection of Arabidopsis thaliana by several RNA viruses, including the bunyavirus, tomato spotted wilt virus (TSWV). TSWV infection on mutants with decreased or increased RNA decapping ability resulted in increased and decreased susceptibility, respectively. By contrast, these mutations had the opposite, or no, effect on RNA viruses that use different mRNA capping strategies. Consistent with this, the RNA capping efficiency of TSWV mRNA was higher in a dcp2 mutant. Furthermore, the TSWV N protein partially colocalized with RNA processing body (PB) components and altering decapping activity by heat shock or coinfection with another virus resulted in corresponding changes in TSWV accumulation. The present results indicate that TSWV infection in plants depends on its ability to snatch caps from mRNAs destined for decapping in PBs and that genetic or environmental alteration of RNA processing dynamics can affect infection outcomes.

The influenza virus RNA-dependent RNA polymerase (RdRP) cleaves the 5\' end of nascent capped host RNAs and uses the capped RNA fragment to prime viral transcription in a mechanism called \'cap snatching\'. Cap snatching requires an intimate association between influenza RdRP and cellular RNA polymerase II (Pol II), which is the source of nascent capped host RNAs targeted by influenza virus. Recent structural studies have revealed how influenza RdRP binds to Pol II and how this binding promotes the initiation of viral transcription by influenza RdRP. In this review we focus on these recent insights into the mechanism of cap snatching by influenza virus and the impact of cap snatching on host gene expression during influenza virus infection.

The hantavirus RNA-dependent RNA polymerase (RdRp) snatches 5\' capped mRNA fragments from the host cell transcripts and uses them as primers to initiate transcription and replication of the viral genome in the cytoplasm of infected cells. Hantavirus nucleocapsid protein (N protein) binds to the 5\' caps of host cell mRNA and protects them from the attack of cellular decapping machinery. N protein rescues long capped mRNA fragments in cellular P bodies that are later processed by an unknown mechanism to generate 10- to 14-nucleotide-long capped RNA primers with a 3\' G residue. Hantavirus RdRp has an N-terminal endonuclease domain and a C-terminal uncharacterized domain that harbors a binding site for the N protein. The purified endonuclease domain of RdRp nonspecifically degraded RNA in vitro It is puzzling how such nonspecific endonuclease activity generates primers of appropriate length and specificity during cap snatching. We fused the N-terminal endonuclease domain with the C-terminal uncharacterized domain of the RdRp. The resulting NC mutant, with the assistance of N protein, generated capped primers of appropriate length and specificity from a test mRNA in cells. Bacterially expressed and purified NC mutant and N protein required further incubation with the lysates of human umbilical vein endothelial cells (HUVECs) for the specific endonucleolytic cleavage of a test mRNA to generate capped primers of appropriate length and defined 3\' terminus in vitro Our results suggest that an unknown host cell factor facilitates the interaction between N protein and NC mutant and brings the N protein-bound capped RNA fragments in close proximity to the endonuclease domain of the RdRp for specific cleavage at a precise length from the 5\' cap. These studies provide critical insights into the cap-snatching mechanism of cytoplasmic viruses and have revealed potential new targets for their therapeutic intervention.IMPORTANCE Humans acquire hantavirus infection by the inhalation of aerosolized excreta of infected rodent hosts. Hantavirus infections cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with mortality rates of 15% and 50%, respectively (1). Annually 150,000 to 200,000 cases of hantavirus infections are reported worldwide, for which there is no treatment at present. Cap snatching is an early event in the initiation of virus replication in infected hosts. Interruption in cap snatching will inhibit virus replication and will likely improve the prognosis of the hantavirus disease. Our studies provide mechanistic insight into the cap-snatching mechanism and demonstrate the requirement of a host cell factor for successful cap snatching. Identification of this host cell factor will reveal a novel therapeutic target for combating this viral illness.