Substitution or deletion mutations between nt 54 and 70 in the 5′ non-coding region of dengue type 2 virus produce variable effects on virus viability

Abstract

A C57U nucleotide mutation in a predicted RNA stem structure (nt 11-16/56-61) of the 59 non-coding region (59NCR) of dengue 2 (DEN-2) 16681 virus is partially attenuating, but unstable during serial passage of certain candidate DEN-2 PDK-53-based vaccine viruses containing this mutation. Here, 11 different mutations (one or more point substitution and/or deletion) between nt 54 and 70 in the 59NCR of the pD2/IC-30P-A (16681) infectious clone are described. Four mutants were infectious. Three mutants with single point substitutions replicated well in cell culture and exhibited variable neurovirulence in mice. Constructs containing multiple substitutions or any deletions failed to produce infectious viruses. Unexpectedly, a double C57U+G58C mutant replicated as efficiently as D2/IC-30P-A virus, and was more neurovirulent for newborn ICR mice. Thus, despite its predicted additional disruption of the RNA stem structure, the engineered contiguous secondary G58C mutation caused reversion of the partially attenuated phenotype caused by the 59NCR-C57U mutation.

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

“…In our system, the GFP expression level would reflect the efficiency of initial viral polyprotein translation and the viral RNA self-replication within the single-round of the transfected cells. Previously, we have shown the 5′-69 mutation of DENV-2 virus is partially attenuated for neurovirulence in newborn mice (Sirigulpanit et al, 2007). This A69U substitution was conserved among the wt DENV-2 strains of the American genotype causing dengue fever, but not dengue hemorrhagic fever, in humans (Leitmeyer et al, 1999).…”

“…For this purpose, we chose five 5′NCR mutations previously shown to have attenuation effects in mouse neurovirulence and 2 deletion mutants that were lethal for virus (Sirigulpanit et al, 2007;Leardkamolkarn et al, 2010) to be incorporated in the replicons (Table 1), and evaluated whether the mutation effects could be measured using the replicon reporter system in vitro. Positive GFP expressions were observed in cells transfected with all 4 types of Δ-DENV/GFP replicons containing 5′-55M, 5′-60M, 5′-14M, 5′-15M, 5′-57M, but the positive cell numbers were less than those of the Δ-DENV/GFP replicons with wt 16681 5′NCR at 48 h pt (Fig.…”

“…The DENV-2.5′-D57M and DENV-2.5′D5758M deletion mutants were included as negative control constructs, since our previous results demonstrated that these deletions were totally lethal for DENV-2 (Sirigulpanit et al, 2007).…”

“…Full-length DENV-2 cDNA plasmids containing various mutations in the 5′NCR (Table 1) were made previously (Sirigulpanit et al, 2007;Leardkamolkarn et al, 2010). A cDNA fragment with A69T substitution isolated from full-length DENV-2.5′-69M plasmid was cloned into the FL-DENV/GFP plasmid to replace the corresponding wt fragment.…”

“…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).…”