Growth-Restricted Dengue Virus Mutants Containing Deletions in the 5′ Noncoding Region of the RNA Genome

Abstract

The dengue type 4 virus (DEN4) RNA genome contains a 101-nt 5' noncoding (NC) sequence which is predicted to form a stable secondary structure. DEN4 cDNA from which infectious RNA can be transcribed was used to engineer deletions in the 5' NC region for functional analysis of RNA structure and for isolation of DEN4 mutants that could be evaluated as candidates for use in a live attenuated vaccine. Eleven distinct deletions in the region of the DEN4 genome between nts 18 and 98 were constructed; each mutation was predicted to alter or disrupt the local base-parings in the 5' NC RNA structure. An infectious virus was not recovered from the RNA transcripts of five of these deletion mutants. Significantly, four of the five apparently lethal deletions were located in a 5- to 6-nt base-paired region of a predicted long stem or adjacent to it. In contrast, with one exception, mutants which yielded infectious virus had deletions which were located in a loop or short stem region. The effect of the deletions on the efficiency of translation of viral RNA transcripts was examined in vitro. The RNA transcripts of deletion constructs which did not yield viable virus were translated at an efficiency ranging from 40 to 160% that of wild-type virus transcripts. The translation efficiency of infectious RNA transcripts also varied. Deletion mutants recovered from RNA transcripts that exhibited low to moderate efficiency of translation had a small plaque morphology and exhibited reduced growth in simian LLC-MK2 and mosquito C6/36 cells compared to the wild-type virus. Among the 11 mutant constructs, deletion of nts 82-87 caused the greatest reduction in translation efficiency. Nevertheless, an infectious virus was recovered from LLC-MK2 cells transfected with the RNA transcripts of mutant d(82-87). The progeny of this mutant produced small plaques on LLC-MK2 cells and grew to low titer in these cells. Unlike wild-type DEN4 or other DEN4 deletion mutants tested, mutant d(82-87) failed to produce plaques on C6/36 cells and was also replication-defective in Aedes aegypti and Aedes albopictus following intrathoracic inoculation.

Molecular Basis for Adaptation of a Chimeric Dengue Type‐4/Japanese Encephalitis Virus to Vero Cells

“…The conservation of such a structure has previously been interpreted to mean that this structure is essential to viral replication as well as expression of the RNA genome (7,15). In this region, compared with the corresponding region in the parental virus DEN4 814669 strain, three mutations were found in DEN4/JE.…”

Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication

“…Sequences of 5′NCR together with capsid protein gene and 3′NCR are required to structurally interact during virus replication (Alvarez et al, 2005;You et al, 2001). The functional significance of dengue 5′NCR is demonstrated by the restricted growth of DENV-4 deletion mutants (Cahour et al, 1995), attenuation effect of the DENV-2 PDK-53 vaccine candidate (Butrapet et al, 2000), and reduced virus replication of DENV-2 5′NCR mutants (Leardkamolkarn et al, 2010;Sirigulpanit et al, 2007).…”

cis-acting RNA elements required for replication of bovine viral diarrhea virus–hepatitis C virus 5′ nontranslated region chimeras

“…The Flavivirdae is an important family of human and animal RNA viral pathogens + Virus particles consist of a lipid bilayer envelope with embedded transmembrane glycoproteins surrounding a protein-RNA nucleocapsid+ Genome RNAs are single-stranded, of positive polarity, and function as the sole mRNA species for translation of a single long open reading frame (ORF)+ Mature viral proteins are produced by co-and posttranslational processing of the resulting polyprotein by the action of cellular and viral proteases+ Structural proteins destined for incorporation into virus particles are encoded in the N-terminal portion of the polyprotein with the nonstructural proteins which form components of the viral RNA replicase being encoded in the remainder+ RNA replication occurs via synthesis of a full-length negative-strand intermediate and is asymmetic, favoring synthesis of positive-strand RNAs+ Besides these common features, the three currently recognized genera of the Flaviviridae exhibit distinct differences in transmission, host range, and pathogenesis+ Members of the classical flavivirus genus, such as yellow fever virus and dengue virus, are typically transmitted to vertebrate hosts via arthropod vectors and cause acute self-limiting disease (Monath & Heinz, 1996)+ The pestiviruses, such as bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV), cause economically important livestock disease and are spread by direct contact or the fecal-oral route (Thiel et al+, 1996)+ The most recently characterized member of this family is the common and exclusively human pathogen, hepatitis C virus (HCV)+ HCV, the sole member of the hepacivirus genus, is transmitted by contaminated blood or blood products and is the most common agent of non-A, non-B hepatitis, affecting more that 1% of the population worldwide (Houghton, 1996)+ Unlike flavivirus and pestivirus infections, which are usually eliminated by host immune response, chronic HCV infections are common and can cause mild to severe liver disease including cancer+ Despite the importance of this virus family, little is known about the details of how RNA replication occurs+ For all three genera, full-length functional cDNA clones have been constructed and RNAs transcribed from these cDNA templates are infectious )+ For flaviviruses and pestiviruses, mutagenesis of these clones and efficient RNA transfection of permissive cell cultures provides a means of probing the role of cis RNA elements and viral proteins in replicase assembly and function+ Such analyses are not yet possible for HCV since this virus is unable to replicate efficiently in cell culture+ Like many other RNA viruses (Huang, 1997), the 59 and 39 terminal sequences of the Flaviviridae are thought to contain conserved cis-elements important for translation, RNA replication, and packaging (Bukh et al+, 1992;Deng & Brock, 1993;Cahour et al+, 1995;Kolykhalov et al+, 1996;Men et al+, 1996;Tanaka et al+, 1996;Mandl et al+, 1998)+ The 59 nontranslated region (NTR) functions initially at the level of translation+ Similar to most cellular mRNAs, flavivirus genome RNAs are translated in a cap-dependent manner+ These RNAs contain a 59 cap s...…”