In vitro analysis of virus-associated RNA I (VAI RNA): inhibition of the double-stranded RNA-activated protein kinase PKR by VAI RNA mutants correlates with the in vivo phenotype and the structural integrity of the central domain

Abstract

Adenoviruses use the virus-encoded virus-associated RNA (VAI RNA) as a defense against cellular antiviral response by blocking the activation of the interferon-induced, double-stranded RNA-activated protein kinase PKR. The structure of VAI RNA consists of two long, imperfectly base-paired duplex regions connected by a complex short stem-loop at the center, referred to as the central domain. By using a series of adenovirus mutants with linker-scan mutations in the VAI RNA gene, we recently showed that the critical elements required for function in the VAI RNA molecule are in the central domain and that these same elements of the central domain are also involved in binding to PKR. In virus-infected cells, VAI RNA interacts with latent kinase, which is bound to ribosomes; this interaction takes place in a complex milieu. To more fully understand the relationship between structure and function and to determine whether the in vivo phenotype of these mutants can be reproduced in vitro, we have now analyzed these mutant VAI alleles for their ability to block the activation of a partially purified PKR from HeLa cells. We have also derived the structure of these mutants

Noncoding RNAs produced by oncogenic human herpesviruses

“…These and other similarities between the two sets of RNAs such as transcription by polymerase III and binding to the cellular La protein, led to the hypothesis that they may perform analogous functions in infected cells. The VA RNAs have been shown to be critical for adenovirus replication by rescuing cells from the shutdown of protein translation mediated by the cellular kinase PKR, which is induced by interferon and activated by double‐stranded RNAs produced during replication of many viruses (Hovanessian, 1989; Ghadge et al, 1994). Construction of adenovirus mutants in which VA RNAs were replaced by EBERs showed that EBERs could indeed functionally substitute for the VA RNAs (Bhat and Thimmappaya, 1983, 1985).…”

Effect of single-base substitutions in the central domain of virus-associated RNA I on its function

“…Plasmids containing wild-type (WT) or mutant VAI genes were transcribed in vitro by using T7 RNA polymerase as described previously (7) with an in vitro transcription kit (Riboprobe II core system; Promega catalog no. P2590).…”

“…The hexylamine agarose column fraction was preincubated at 30ЊC for 10 min with in vitro-transcribed VAI RNA prior to the addition of dsRNA and [␥-32 P]ATP. The phosphorylated proteins were analyzed on SDS-12.5% polyacrylamide gels (7).…”

“…In vitro inhibition of autophosphorylation of PKR by mutant VAI RNAs. We recently showed that the in vivo properties of the VAI mutants can be faithfully reproduced in vitro (7). In this study, a considerably purified PKR preparation from HeLa cells was preincubated with in vitro-transcribed WT or mutant VAI RNAs that were previously tested for their function in vivo in the context of the viral chromosome.…”

“…VAI RNA binds to and irreversibly inactivates PKR and thereby protects and maintains the eIF-2 activity (4,8,12,14,22). The structural requirements of VAI RNA for its function have been investigated by introducing deletions and linker-scan and base-compensatory substitutions into the VAI RNA and analyzing such mutants in vivo in virus infections and transient assays and also in vitro for their capacities to bind to and block the autophosphorylation activity of PKR (1,4,5,7,8,(19)(20)(21)24). These studies showed that mutant molecules would retain function as long as the integrity of the central domain was maintained.…”