INTRODUCTION
Vaccinia virus (VV) encodes a variety of factors that function in the evasion of
host defenses. Included in this list are decoy receptors for various interferons as well as
inhibitors of interferon inducible antiviral enzymes (1, 29, 35). As a consequence, VV
has been shown to be resistant to the antiviral action of interferon in most cell lines (40).
VV has also been shown to rescue the replication of interferon sensitive viruses such as
vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) (37, 38).
The interferon resistance exhibited by VV is due to its ability to inhibit the
activation of interferon inducible antiviral enzymes like dsRNA dependent protein kinase
(PKR) and 2’5’ oligo adenylate synthetase (OAS) (5, 9, 34). Both enzymes are induced
by interferon in an inactive state, and require the presence of dsRNA for activation (7,
11). The activation of PKR leads to phosphorylation of the eukaryotic translation
initiation factor (eIF2) on its  subunit which results in the inhibition of translation
initiation in infected cells (30, 39). The activation of OAS in turn leads to activation of a
latent endoribonuclease (RNase L). Upon activation RNase L cleaves rRNA and mRNA
in the cell, thereby leading to an arrest of protein synthesis in the infected cell.
The E3L gene is one of the key interferon resistance genes encoded by VV that
confers a broad host range phenotype to VV in cells in culture (5). Recently the role of
E3L has been established as a virulence factor in VV pathogenesis in mice (6). The E3L
gene encodes a 25 KDa protein that contains a Z-DNA binding domain in its amino
terminus and a single copy of a dsRNA-binding motif (dsRBM) in its carboxy terminus
(6). The ability of the E3L gene product to bind to and sequester dsRNA (via its dsRBM)
is thought to be important for inhibiting the activation of the PKR and OAS enzymes (9,
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10, 34). As a result, there is no inhibition of protein synthesis in the infected cell and the
virus continues to replicate. The dsRBM of E3L is conserved among many other dsRNA
binding proteins, including PKR and an RNA-specific adenosine deaminase 1 (ADAR1)
(24, 27). NMR and crystallography studies of the dsRBM motifs from PKR and other
proteins (E.coli RNaseIII and Staufen) reveal the presence of a similar ---- protein
topology (14, 26). VV deleted of the E3L gene (VVE3L) can be functionally
complemented in cells in culture by expression of other heterologous dsRNA binding
proteins such as E.coli RNaseIII, reovirus 3 and the rotavirus NSP3 gene product (4, 18,
33). These proteins are thought to complement the function of the E3L gene product, at
least in part, by virtue of their ability to bind and sequester the dsRNA made during
vaccinia infection.
In this study we examined whether the human ADAR1 gene product could
functionally replace the vaccinia E3L gene product. ADAR1 is a cellular, interferon
inducible enzyme that belongs to a family of RNA editing enzymes that catalyze the C-6
deamination of adenosine (A) to yield inosine (I) in cellular pre-mRNAs as well as viral
RNAs (2, 27). Because inosines are interpreted as guanosines during translation editing
can frequently lead to codon changes in mRNA that in turn alters protein function (19,
32). ADARs are implicated in two different types of editing processes that require
double stranded regions in the RNA. First, A to I editing is found at multiple sites within
some viral RNAs, as seen in polyoma virus antisense RNA and measles virus RNA (3, 8,
16). The second type of editing is highly site specific and is limited to few adenosines in
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both viral RNAs and cellular pre-mRNAs. This includes hepatitis delta virus RNA and
glutamate receptor pre-mRNA in higher vertebrates (28, 32).
The interferon inducible form of ADAR1 consists of three distinct copies of the
conserved dsRNA binding motif (RI, RII and RIII) in the central region and two Z-DNA
binding domains in the amino terminus (12, 20, 21). The deaminase activity of ADAR1
is located in the carboxy terminus of the protein. ADAR1 and E3L are very similar in the
organization of their domains in that both proteins possess Z-DNA binding and dsRNAbinding motifs (22). Because of the high degree of homology between ADAR1 and E3L
proteins we asked the question whether ADAR1 could functionally replace the loss of
E3L. In order to investigate whether the deaminase function of ADAR1 was required to
complement the function of E3L two different forms of ADAR1 were expressed from the
E3L locus of VVE3L; wild type ADAR1, which is capable of both binding and editing
dsRNA, and a catalytically inactive mutant of ADAR1 (ADAR1cat-) which retains its
ability to bind to dsRNA but is incapable of carrying out its editing function because of
two point mutations (H910Q and E912A) in the catalytic domain (21). Our results
suggest that the restricted host range phenotype of VVE3L was partially restored by
wild type ADAR1. ADAR1cat- could rescue the phenotype of VVE3L in HeLa cells to a
less extent than wild type ADAR1. In vivo experiments have shown that wild type
ADAR1 could partially restore the non neurovirulent phenotype of VVE3L during
intracranial infection of mice. This rescue of neurovirulence of VVE3L by wild type
ADAR1 was stronger than that of ADAR1cat-. Thus, in addition to dsRNA binding, the
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ability of ADAR1 to catalyze the deamination of adenosines to inosines in dsRNA
appears to be necessary to partially restore the loss of E3L in VVE3L.