Structural requirements of adenovirus VAI RNA for its translation enhancement function.

Abstract

Recently, by genetic and biochemical approaches, it has been shown that adenovirus VAI RNA is required for efficient translation of viral mRNAs at late times after infection. To understand the nucleotide sequences and the domains of the VAI RNA that are responsible for the role of VAI RNA in enhancement of translation, a mutational analysis of the VAI gene was undertaken. Deletion, substitution, and insertion mutations covering most of the nucleotide sequences of VAI RNA were introduced into the VAI gene at the plasmid level. These mutant genes were then reintroduced into the virus, and growth properties of the mutant viruses were studied. The majority of the mutants retained normal or nearly normal levels of biological function. Mutations in the region between +43 and +53 and between +107 and the 3' end of the gene resulted in a considerable loss of activity. These mutants, however, grew significantly better than did an adenovirus type 5 mutant lacking both functional VAI and VAII genes, indicating that they retain a portion of their activity. Because no one mutation was able to completely abolish the function, we suggest that the VAI RNA may have multiple functional sites for its translation modulation function. These multiple sites may be short oligonucleotide sequences that may interact with cellular or viral components or both during translation.Adenoviruses synthesize large amounts of two low-molecular-weight RNAs designated VAI and VAII (20,23). The genes coding for these two RNAs are located between 29.0 and 31.0 map units (m.u.) on the adenoviral chromosome. Nucleotide sequences of these RNAs have been determined (1,19,25). The virus-associated (VA) RNA genes are transcribed by RNA polymerase III (23,31). An intragenic transcription control region for the VAI gene has been identified, and it maps between +10 and +69 (4, 8, 12) (the numbering here and throughout this article is relative to the starting point of the VAI-G RNA). In the infected cells, a cellular phosphoprotein of 45,000 molecular weight has been shown to be associated with VA RNAs, and the nucleoprotein complex is recognized by the anti-La class of lupus sera (9,13,14 30.0 m.u.) cloned between the PvuII and EcoRI sites of pBR322. Plasmids were propagated in Escherichia coli HB101.Mutagenesis of VAI gene. Substitution and insertion mutations located in the 5' half of the gene were constructed with HindIII linkers as described in an earlier report (4). Deletion mutations in the 3' half of the molecule were obtained by the D-loop mutagenesis procedure (11,22). A 53-bp deletion in 187 on May 11, 2018 by guest

Adenovirus Virus-Associated RNA Is Processed to Functional Interfering RNAs Involved in Virus Production

“…Also, blocking of svaRNAs drastically affects AdWT production (Fig. 7B), and adenoviruses that express properly folded VAI RNA with 3Ј-end mutations show a decrease in viral replication (3). These data strongly suggest that svaRNAs use the silencing machinery to inhibit gene expression and therefore improve adenovirus viability.…”

“…However, substitutions of the sequences at the 3Ј termini of VAI were introduced into adenoviruses that express VAI molecules that are able to be properly folded and transported to the cytoplasm of infected cells. Even though these VAI 3Ј-end mutants should be functional in blocking PKR, the mutant viruses showed a 10-fold decrease in viral replication (3). This indicates that the 3Ј end of VAI could play a different role in adenovirus viability.…”

“…Adenovirus serotypes 2 and 5, like most human adenovirus serotypes, express two VA RNAs, VAI and VAII, whereas others carry a single VA RNA more related to VAI (32). While VAI is more abundant and has been better studied, little is known about VAII, which might carry out nonessential functions (27,48) and compensate in part for the lack of VAI in VAI-mutated adenovirus (3,4,48). The main function of VAI is the blockade of RNAdependent protein kinase (PKR), an interferon-inducible serine-threonine protein kinase activated in infected cells as part of the antiviral response.…”

Posttranscriptional regulation of hsp70 expression in human cells: effects of heat shock, inhibition of protein synthesis, and adenovirus infection on translation and mRNA stability.

“…Plasmid pHFPA-1 (29) (27), a human embryonic kidney cell line transformed by the pre-early regions of adenovirus 5, was grown in 100-mm dishes in Dulbecco modified Eagle medium containing 5% calf serum and heat shocked as described. Infections of cell line 293 cells with adenovirus 5 (wild type strain dl704 [12], a generous gift of B. Thimmappaya) were performed as described (70).…”

Structural features of adenovirus 2 virus-associated RNA required for binding to the protein kinase DAI

“…The RNAs were all purified by sequential electrophoresis through denaturing and non-denaturing gels to remove dsRNA contaminants (19). During purification it became apparent that some mutations affect the electrophoretic mobility of the RNA in denaturing gels as reported earlier (24,25,39,40). To correlate the gel mobility with the structures of the mutant VA RNA molecules and their binding to DAI in vitro, we systematically examined the mobility of the RNAs.…”