The Double-stranded RNA Binding Domain of the Vaccinia Virus E3L Protein Inhibits Both RNA- and DNA-induced Activation of Interferon β

Marq, Jean‐Baptiste; Hausmann, Stéphane; Luban, Jeremy; Kolakofsky, Daniel; Garcin, Dominique

doi:10.1074/jbc.m109.018895

Cited by 30 publications

(29 citation statements)

References 34 publications

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“…RIG-I can thus be activated by more than one type of dsRNA, and in more than one way. As mentioned above, poly(I-C), a dsRNA mimetic, strongly induces IFN via RIG-I in a 5Ј ppp-independent fashion in A549 cells (24), and this activation is not inhibited by 5Ј ppp dsRNAs with a single unpaired 5Ј ppp nt (Fig. 8).…”

Section: Discussionmentioning

confidence: 63%

Short Double-stranded RNAs with an Overhanging 5′ ppp-Nucleotide, as Found in Arenavirus Genomes, Act as RIG-I Decoys

Marq

Hausmann

Veillard

et al. 2011

Journal of Biological Chemistry

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Arenavirus RNA genomes are initiated by a "prime and realign" mechanism, such that the initiating GTP is found as a single unpaired (overhanging) nucleotide when the complementary genome ends anneal to form double-stranded (ds) RNA panhandle structures. dsRNAs modeled on these structures do not induce interferon (IFN), as opposed to bluntended 5 ppp dsRNA. This study examines whether these viral structures can also act as decoys, by trapping RIG-I in inactive dsRNA complexes. We examined the ability of various dsRNAs to activate the RIG-I ATPase (presumably a measure of helicase translocation on dsRNA) relative to their ability to induce IFN. We found that there is no simple relationship between these two properties, as if RIG-I can translocate on short dsRNAs without inducing IFN. Moreover, we found that 5 ppp dsRNAs with a single unpaired 5 ppp-nucleotide can in fact competitively inhibit the ability of blunt-ended 5 ppp dsRNAs to induce IFN when co-transfected into cells and that this inhibition is strongly dependent on the presence of the 5 ppp. In contrast, 5 ppp dsRNAs with a single unpaired 5 ppp-nucleotide does not inhibit poly(I-C)-induced IFN activation, which is independent of the presence of a 5 ppp group.

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Section: Discussionmentioning

confidence: 63%

Short Double-stranded RNAs with an Overhanging 5′ ppp-Nucleotide, as Found in Arenavirus Genomes, Act as RIG-I Decoys

Marq

Hausmann

Veillard

et al. 2011

Journal of Biological Chemistry

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“…However, a mutant RNase III, which was defective in catalytic activity but has intact dsRNA binding, showed a greatly weakened supplementary function (32). In addition, E3 protein has been shown to inhibit dsDNA-induced cytokine responses (33,34). Therefore, the mechanism of how vaccinia E3 protein functions to modulate dsRNA-activated cellular antiviral responses is undetermined.…”

Section: Figmentioning

confidence: 99%

Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA

Dueck

Chen

et al. 2015

J Virol

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Poxviridae is a diverse family of large dsDNA viruses (130 to 300 kb) which replicate exclusively in the cytoplasm of infected cells and exhibit a varied host range (1). Some members are specific to a single host, such as variola virus, the causative agent of smallpox, while others, such as vaccinia virus, display a broad host range. Poxviruses encode a number of immunomodulatory proteins, e.g., host range proteins (2). Orthopoxviruses encode a number of host range genes, such as SPI-1, K1L, C7L, p28/N1R, B5R, K3L, and E3L, the alteration or deletion of which restricts the virus replication in specific cell lines (3). These host range genes display varied restrictions and biological functions. For example, SPI-1 is required for vaccinia virus replication in PK15 and A549 cells and in mice (4, 5). K1L, an ankyrin repeat-containing protein which inhibits IkB-␣ degradation and interferon (IFN)-stimulated effectors, is required for replication in RK13 cells (6). A double deletion of C7L and K1L leads to restriction of the virus replication in PK1, RK13, and multiple human cell lines, while C7L deletion alone suppresses apoptosis and limits replication in hamster Dede cells (7). P28/N1R, an E3-RING finger ubiquitin ligase involved in protein degradation and apoptosis suppression, is required for replication in mouse macrophage (8, 9), while B5R, a membrane glycoprotein, activates Src, allowing growth in VERO, CEF, PK15, and quail (QT-6) cells (10).The most studied vaccinia host range gene is E3L. E3 protein is expressed early in the virus replication cycle and is known to suppress multiple innate immune pathways (3,11,12). E3 protein possesses biochemical capacities to bind Z-form DNA via the Z-DNA binding domain (ZBD) toward the N terminus and to bind to dsRNA through the C-terminal dsRNA binding domain (DRBD) (13,14). Since the best characterized biological functions of vaccinia virus E3 protein, such as host range function and inhibition of innate immune responses (e.g., cytokine expression, apoptosis and interferon-induced antiviral activity), all are mediated primarily via the DRBD region of E3 protein (15-17), generally it has been believed that E3 protein functions through seques-

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“…B, doubly purified 5Ј ppp-ssRNA1** (54Ј-mer) was annealed with an 18Ј-mer complementary to positions 1-18, as indicated on the left. Samples were then transfected into either wild-type A549 cells or A549 cells in which RIG-I was knocked down (KD) via miRNA expression (27), and their ability to activate the IFN␤ promoter was determined. Ctl shows the level of IFN␤ activation in untransfected cells.…”

Section: Discussionmentioning

confidence: 99%

Unpaired 5′ ppp-Nucleotides, as Found in Arenavirus Double-stranded RNA Panhandles, Are Not Recognized by RIG-I

Marq

Kolakofsky

Garcin

2010

Journal of Biological Chemistry

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Arenavirus and bunyavirus RNA genomes are unusual in that they are found in circular nucleocapsids, presumably due to the annealing of their complementary terminal sequences. Moreover, arenavirus genome synthesis initiates with GTP at position ؉2 of the template rather than at the precise 3 end (position ؉1). After formation of a dinucleotide, 5 pppGpC OH is then realigned on the template before this primer is extended. The net result of this "prime and realign" mechanism of genome initiation is that 5 pppG is found as an unpaired 5 nucleotide when the complementary genome ends anneal to form a doublestranded (dsRNA) panhandle. Using 5 pppRNA made in vitro and purified so that all dsRNA side products are absent, we have determined that both this 5 nucleotide overhang, as well as mismatches within the dsRNA (as found in some arenavirus genomes), clearly reduce the ability of these model dsRNAs to induce interferon upon transfection into cells. The presence of this unpaired 5 ppp-nucleotide is thus another way that some viruses appear to use to avoid detection by cytoplasmic pattern recognition receptors.Most viral RNA-dependent RNA polymerases (RdRP) 2 maintain genome length in a straightforward manner, by initiating genome (and antigenome) synthesis with an NTP opposite the precise 3Ј end of their antigenome (and genome) templates, and terminating synthesis after incorporating the last nucleotide opposite their 5Ј termini. Some segmented negative strand RNA viruses, however, have chosen a different approach. For example, when Tacaribe arenavirus (TCRV) genome and antigenome RNAs are annealed, this dsRNA contains a single unpaired 5Ј nucleotide overhang (pppG) rather than flush or blunt ends (1). The initiating pppG could not have been coded for at the precise template/antigenome 3Ј end, which is also G. Moreover, when TCRV RNA synthesis is reconstituted in vitro, certain di-and trinucleotides efficiently prime this synthesis, and GpC, the most efficient primer, led to transcripts whose 5Ј ends were also at position Ϫ1 (see Fig. 1) (2). As the sequence 3Ј OH GCG… is present at all arenavirus genome and antigenome 3Ј ends, we proposed that TCRV genomes were initiated by 5Ј pppGpC OH (an uncapped primer) formed at an internal position (in bold) of the 3Ј OH GCG… sequence (Fig. 1). This pppGpC OH could then realign on the template such that its C residue is lined up opposite the G at the precise template 3Ј end, and then extended for genome synthesis (Fig. 1). Support for this prime and realign mechanism was also obtained by studying Hantaan (bunya)virus (HTN), where both mRNA and genome synthesis are thought to involve priming and realigning (3) as well as mRNA synthesis of Germiston and Rift Valley fever virus, two other bunyaviruses (4). More recently, influenza A virus was also reported to use a prime and realign mechanism to initiate genome synthesis from antigenome templates (5). Thus all 3 families of segmented negative strand RNA viruses appear to use this unusual mechanism for initiating viral RNA synthesis...

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The Double-stranded RNA Binding Domain of the Vaccinia Virus E3L Protein Inhibits Both RNA- and DNA-induced Activation of Interferon β

Cited by 30 publications

References 34 publications

Short Double-stranded RNAs with an Overhanging 5′ ppp-Nucleotide, as Found in Arenavirus Genomes, Act as RIG-I Decoys

Short Double-stranded RNAs with an Overhanging 5′ ppp-Nucleotide, as Found in Arenavirus Genomes, Act as RIG-I Decoys

Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA

Unpaired 5′ ppp-Nucleotides, as Found in Arenavirus Double-stranded RNA Panhandles, Are Not Recognized by RIG-I

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