Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5′-terminal regions of cap0-, cap1- and 5′ppp- mRNAs

Abstract

Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA’s 5′-terminal ‘cap’. The minimal ‘cap0’ consists of N7-methylguanosine linked to the first nucleotide via a 5′-5′ triphosphate (ppp) bridge. Cap0 is further modified by 2′-O-methylation of the next two riboses, yielding ‘cap1’ (m7GpppNmN) and ‘cap2’ (m7GpppNmNm). However, some viral RNAs lack 2′-O-methylation, whereas others contain only ppp- at their 5′-end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0- and 5′ppp- mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K1/2,app ∼9 to 23 nM). The 2′-O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K1/2,app ∼450 nM). The 5′-terminal regions of 5′ppp-mRNAs were recognized by IFIT5 (K1/2,app ∼400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5′-terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1- and 5′ppp- mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations.

Zika Virus Methyltransferase: Structure and Functions for Drug Design Perspectives

“…On the other hand, 2=-O-methylation of the cap is expected to mask the presence of exogenous viral RNAs from host cell sensors such as RIG-I and MDA5 (9)(10)(11), which induce the production of interferons. In addition, 2=-O-methylation of viral RNA is thought to prevent translation restriction by interferon-stimulated genes (ISGs) such as the IFIT-1 gene (12,13). Thus, molecules inhibiting ZIKV MTase activities are expected to translate into a strong antiviral effect (41).…”

“…The 2=-O-methylation of the cap structure was demonstrated to protect viral RNA from being recognized by host cell sensors such as RIG-I and MDA5 that stimulate the production of interferons (9)(10)(11). In turn, interferon-stimulated genes, such as IFIT-1, can detect miscapped RNA and restrict their translation into proteins (12,13). DENV mutant viruses lacking N-7-methyltransferase (MTase) activity show strong replication defects in infected cells, whereas those altering the 2=-O-MTase activity show only attenuated phenotypes (7,14), as most cell lines used in the laboratory for virus replication are deprived of the RIG-I/MDA5 antiviral pathway.…”

Identification of Interferon-Stimulated Gene Proteins That Inhibit Human Parainfluenza Virus Type 3

“…The four members of the human IFIT family regulate translation by differentially recognizing the 5= termini of their target RNA (28,48). The exact features in the RNA that they recognize is a matter of some debate, but the current consensus is that they bind to 2=-O-unmethylated 5= cap structure (cap 0) and/or 5=-triphosphates (5=-ppp) on the RNA, which are generally absent in cellular mRNA but found in transcripts of intracellular pathogens, such as viruses that lack cap methyltransferase activity and bacteria (62)(63)(64)(65)(66)(67). IFIT1, in particular, shows a high preference for mRNA with unmethylated cap (63-67), whereas IFIT5, which had no inhibitory effect on PIV3, prefers capless, 5=-ppp RNA (67).…”

Herpes Simplex Virus 1 Tegument Protein UL41 Counteracts IFIT3 Antiviral Innate Immunity

“…In this response, IFN-I, such as IFN-␣ and IFN-␤, are critical to innate immunity against viral infection and the modulation of adaptive immunity through the induction of a wide array of ISGs (2,48). Over the past decade, it has become clear that IFIT family proteins inhibit viral infection through suppressing translation initiation, binding to uncapped or incompletely capped viral RNA, or sequestering viral proteins or RNA in the cytoplasm (3,6,7,11,12,46,(49)(50)(51). However, the antiviral activity of individual IFIT family members remains to be defined and corroborated.…”

“…However, the ortholog of human IFIT5 is absent from mouse (6,9). Recent studies indicate that IFIT proteins play an important role in antiviral processes and restrict viral replication through altering protein synthesis, binding to viral RNAs, or interacting with structural or nonstructural viral proteins (8,10,11). IFIT3 was demonstrated to inhibit the replication of many DNA and RNA viruses, such as hepatitis B virus (HBV), human papillomavirus (HPV), hepatitis C virus (HCV), West Nile virus (WNV), porcine reproductive and respiratory syndrome virus (PRRSV), Dengue virus (DV), vesicular stomatitis virus (VSV), and others (6,(12)(13)(14)(15).…”