Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Nov;84(21):11407-17.
doi: 10.1128/JVI.01159-10. Epub 2010 Aug 18.

RNA structures required for production of subgenomic flavivirus RNA

Anneke Funk  1 , Katherine Truong, Tomoko Nagasaki, Shessy Torres, Nadia Floden, Ezequiel Balmori Melian, Judy Edmonds, Hongping Dong, Pei-Yong Shi, Alexander A Khromykh
Affiliations

RNA structures required for production of subgenomic flavivirus RNA

Anneke Funk et al. J Virol. 2010 Nov.

Abstract

Flaviviruses are a group of single-stranded, positive-sense RNA viruses causing ∼100 million infections per year. We have recently shown that flaviviruses produce a unique, small, noncoding RNA (∼0.5 kb) derived from the 3' untranslated region (UTR) of the genomic RNA (gRNA), which is required for flavivirus-induced cytopathicity and pathogenicity (G. P. Pijlman et al., Cell Host Microbe, 4: 579-591, 2008). This RNA (subgenomic flavivirus RNA [sfRNA]) is a product of incomplete degradation of gRNA presumably by the cellular 5'-3' exoribonuclease XRN1, which stalls on the rigid secondary structure stem-loop II (SL-II) located at the beginning of the 3' UTR. Mutations or deletions of various secondary structures in the 3' UTR resulted in the loss of full-length sfRNA (sfRNA1) and production of smaller and less abundant sfRNAs (sfRNA2 and sfRNA3). Here, we investigated in detail the importance of West Nile virus Kunjin (WNV(KUN)) 3' UTR secondary structures as well as tertiary interactions for sfRNA formation. We show that secondary structures SL-IV and dumbbell 1 (DB1) downstream of SL-II are able to prevent further degradation of gRNA when the SL-II structure is deleted, leading to production of sfRNA2 and sfRNA3, respectively. We also show that a number of pseudoknot (PK) interactions, in particular PK1 stabilizing SL-II and PK3 stabilizing DB1, are required for protection of gRNA from nuclease degradation and production of sfRNA. Our results show that PK interactions play a vital role in the production of nuclease-resistant sfRNA, which is essential for viral cytopathicity in cells and pathogenicity in mice.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
(A) Model of the WNVKUN 3′ UTR RNA structure. Highlighted in bold are the secondary structures investigated here. Dashed lines indicate putative PKs. The two sites of the putative PK interactions are shown in open boxes. sfRNA1, -2, -3, and -4 start sites are indicated by arrows. (R)CS, (repeated) conserved sequence; DB, dumbbell structure; PK, pseudoknot; SL, stem-loop. (B) Structural model of PK1 in SL-II with disruptive mutations. Nucleotide numbering is from the end of the 3′ UTR. The sfRNA1 start is indicated by an arrow. Nucleotides forming PK1 are on a gray background, and mutated nucleotides are white on a black background. (C) Sequences mutated in the different constructs. Nucleotides in the wt PK sequences used for mutations are bold and underlined. Introduced mutations are shown under the corresponding nucleotides in the wt sequence.
FIG. 2.
FIG. 2.
Defined 3′ UTR secondary structures are required for sfRNA formation. (A) Northern blot assay using a 3′-UTR-specific probe of RNA isolated from BHK-21 cells 72 h after electroporation with replicon RNAs containing indicated deletions. 18S rRNA is shown as a loading control. gRNA, genomic RNA; sfRNA, subgenomic flavivirus RNA; gRNA/sfRNA, ratio of gRNA to sfRNA. (B) Beta-galactosidase assay using lysates from electroporated BHK-21 cells 72 h after electroporation. The beta-galactosidase amount expressed from the wt construct was set at 100%. The standard deviations (SD) from 3 independent experiments are shown.
FIG. 3.
FIG. 3.
PK1 is required for sfRNA1 formation. (A) Northern blot assay using a 3′-UTR-specific probe of RNA isolated from BHK-21 cells 72 h after electroporation with replicon RNAs containing indicated mutations in PK1. 28S rRNA is shown as a loading control. gRNA, genomic RNA; sfRNA, subgenomic flavivirus RNA. (B) Beta-galactosidase assay using lysates from electroporated BHK-21 cells 72 h after electroporation. The beta-galactosidase amount expressed from the wt construct was set at 100%. The SDs from 3 independent experiments are shown. (C) Probing RNA structure by using RNase digestion. The RNA representing nucleotides −527 to −448 of the 3′ terminus of the WNVKUN genome (spanning SL-II, PK-1, and RCS3) was 5′ end labeled with 32P and digested with RNase I and RNase V1. The reaction mixtures were analyzed on a 6% (left panel) and a 20% (right panel) polyacrylamide denaturing gel. An OH ladder and G ladder were included to locate the positions of the digested nucleotides. The positions of G residues are labeled on the left sides of the gels. (D) Summary of RNase cleavages from panel C. Both weak and strong digestions are indicated. The nucleotides involved in the PK1 interactions (indicated by a dashed line) are shaded in gray.
FIG. 4.
FIG. 4.
Sequences in PK2 are required for sfRNA2 formation. (A) Northern blot assay using a 3′-UTR-specific probe of RNA isolated from BHK-21 cells 72 h after electroporation with replicon RNAs containing indicated mutations in PK2 and PK1. 18S rRNA is shown as a loading control. gRNA, genomic RNA; sfRNA, subgenomic flavivirus RNA. (B) Beta-galactosidase assay using lysates from electroporated BHK-21 cells 72 h after electroporation. The beta-galactosidase amount expressed from the wt construct was set at 100%. The SDs from 3 independent experiments are shown.
FIG. 5.
FIG. 5.
PK3 is required for sfRNA3 formation and efficient viral replication. (A) Northern blot assay of RNA isolated from electroporated BHK-21 cells 72 and 120 h after electroporation using a 3′-UTR-specific probe. The left panel shows 72 h after electroporation; the right panel shows 120 h after electroporation. Note that 4-fold (72 h) and 2-fold (120 h) more total RNA, respectively, is loaded for PK1′2′3′ than for the other samples. 18S rRNA is shown as a loading control. gRNA, genomic RNA; sfRNA, subgenomic flavivirus RNA. (B) Beta-galactosidase assay using lysates from electroporated BHK-21 cells 72 h after electroporation. The beta-galactosidase amount expressed from the wt construct was set at 100%. The SDs from 3 independent experiments are shown.
FIG. 6.
FIG. 6.
sfRNA1 is essential for efficient viral replication, cytopathicity, and pathogenicity. (A) Northern blot assay with RNA isolated from C6/36 and Vero cells infected with wild-type and mutant WNVKUN viruses. gRNA, genomic RNA; sfRNA, subgenomic flavivirus RNA. rRNA is shown as a loading control. (B) Growth kinetics of mutant viruses in mosquito (C6/36, left panel) and mammalian (Vero, right panel) cells infected with an MOI of 1. sfRNA species produced are indicated. Data are represented as averages ± SDs. (C) Cytopathicity assay. Vero cells were infected, fixed, and stained with crystal violet at the indicated time points. (D) Pathogenicity in mice. Three-week-old Swiss outbred mice (5 per group) were injected intraperitoneally with 104 PFU of indicated viruses. Mice were monitored daily and sacrificed when symptoms of encephalitis became evident. As a control, 5 mice were injected with medium only. These mice remained healthy over the observation period (data not shown). d pi, days postinfection.
FIG. 7.
FIG. 7.
sfRNA-deficient WNVKUN viruses protect against lethal challenge with highly pathogenic WNVNY99. (A) Antibody production in inoculated mice. Five-week-old Swiss outbred mice were intraperitoneally inoculated with 10,000 PFU of indicated viruses in groups of 5 mice. Two weeks after immunization, serum was collected and the production of WNVKUN-specific antibodies was investigated by ELISA. Values from the 1:320 serum dilution are shown. As a control, mice were mock inoculated with medium only, which did not lead to the development of WNVKUN-specific antibodies (data not shown). (B) Survival of inoculated mice after challenge with WNVNY99. Four weeks after immunization, mice were challenged with 1,000 PFU of WNVNY99, and survival was monitored for 14 days after infection.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alvarez, D. E., M. F. Lodeiro, S. J. Luduena, L. I. Pietrasanta, and A. V. Gamarnik. 2005. Long-range RNA-RNA interactions circularize the dengue virus genome. J. Virol. 79:6631-6643. - PMC - PubMed
    1. Bredenbeek, P. J., E. A. Kooi, B. Lindenbach, N. Huijkman, C. M. Rice, and W. J. Spaan. 2003. A stable full-length yellow fever virus cDNA clone and the role of conserved RNA elements in flavivirus replication. J. Gen. Virol. 84:1261-1268. - PubMed
    1. Brierley, I., S. Pennell, and R. J. Gilbert. 2007. Viral RNA pseudoknots: versatile motifs in gene expression and replication. Nat. Rev. Microbiol. 5:598-610. - PMC - PubMed
    1. Brinton, M. A., A. V. Fernandez, and J. H. Dispoto. 1986. The 3′-nucleotides of flavivirus genomic RNA form a conserved secondary structure. Virology 153:113-121. - PubMed
    1. Brion, P., and E. Westhof. 1997. Hierarchy and dynamics of RNA folding. Annu. Rev. Biophys. Biomol. Struct. 26:113-137. - PubMed

Publication types

LinkOut - more resources