Skip to main page content
U.S. flag
See More

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
. 2008 Dec 9;105(49):19294-9.
doi: 10.1073/pnas.0807211105. Epub 2008 Dec 1.

Storage of cellular 5' mRNA caps in P bodies for viral cap-snatching

M A Mir  1 , W A Duran, B L Hjelle, C Ye, A T Panganiban
Affiliations

Storage of cellular 5' mRNA caps in P bodies for viral cap-snatching

M A Mir et al. Proc Natl Acad Sci U S A. .

Abstract

The minus strand and ambisense segmented RNA viruses include multiple important human pathogens and are divided into three families, the Orthomyxoviridae, the Bunyaviridae, and the Arenaviridae. These viruses all initiate viral transcription through the process of "cap-snatching," which involves the acquisition of capped 5' oligonucleotides from cellular mRNA. Hantaviruses are emerging pathogenic viruses of the Bunyaviridae family that replicate in the cytoplasm of infected cells. Cellular mRNAs can be actively translated in polysomes or physically sequestered in cytoplasmic processing bodies (P bodies) where they are degraded or stored for subsequent translation. Here we show that the hantavirus nucleocapsid protein binds with high affinity to the 5' cap of cellular mRNAs, protecting the 5' cap from degradation. We also show that the hantavirus nucleocapsid protein accumulates in P bodies, where it sequesters protected 5' caps. P bodies then serve as a pool of primers during the initiation of viral mRNA synthesis by the viral polymerase. We propose that minus strand segmented viruses replicating in the cytoplasm have co-opted the normal degradation machinery of P bodies for storage of cellular caps. Our data also indicate that modification of the cap-snatching model is warranted to include a role for the nucleocapsid protein in cap acquisition and storage.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
N protects capped 5′ termini. (A) Binding of N to synthetic capped and uncapped TriEx RNA was examined by using radiolabeled RNA and filter binding with increasing concentrations of N as described in Materials and Methods. Dissociation constants (Kd) are indicated. Solid squares indicate capped RNA, open squares indicate uncapped RNA. (B) Parallel binding of N to a capped or uncapped synthetic decamer RNA corresponding to the 5′ terminus of TriEx RNA was examined using filter binding as in A. Solid squares indicate capped RNA, open squares indicate uncapped RNA. (C) Competition binding analysis using m7GTCTCTCCCA labeled with P32 CTP and unlabeled GTCTCTCCCA with either an m7 or 2′-O-methyl cap. This oligonucleotide was chosen to ensure quantitative capping as the sequence lacks internal G residues. N binds with this capped decamer RNA at an affinity (Kd = 130 nM) similar to that of the decamer in B. Reactions contained 0.01 nM of labeled decamer, 520 nM N, and increasing amounts of competitor RNA containing an m7 or 2′-O-methyl cap as indicated in the log scale. The amount of RNA binding in the absence of competitor (100% binding) is also depicted for clarity, although [0 nM] competitor cannot be plotted on a log scale. Closed and open squares indicate cold competitor decamer with an m7 cap or 2′-O-methyl cap, respectively. (D) Diagram of TriEx RNA expressed in transfected HeLa cells. Following cDNA synthesis by reverse transcriptase, real-time PCR was used to quantify the 5′ and 3′ ends using primers complementary to the indicated nucleotides. See Materials and Methods for a detailed description of quantitative real-time PCR and exact primer sequences. The RNA encodes a short, arbitrary ORF. (E) Effect of co-expression of N on the 5′ and 3′ ends of TriEx RNA. The quantified PCR products of the 5′ and 3′ termini in the absence of N were used for normalization.
Fig. 2.
Fig. 2.
N associates with P bodies. (A) Confocal detection of cytoplasmic P bodies and N. HeLa cells were transfected with plasmid expressing a GFP-N fusion protein. Intracytoplasmic DCP1 was detected with anti-DCP1 antibody. N was visualized by detection of GFP, and nuclei by DAPI. (B) Pull-down analysis to detect association of N with P bodies. N was recovered from the lysates of transfected cells by virtue of a C-terminal octahistidine tag using Ni-NTA columns. Recovered material was analyzed with Western blots with anti-N antibody to verify recovery of N and with anti-DCP1 to detect association of P body components with N. The indicated samples were treated with RNase A before recovery to verify that association of DCP1 with N was RNA-dependent. Dashes represent untransfected cells. Lysate (sample from pTriEX transfected cells before fractionation), pTriEx (an empty vector control), pT-GFP-N (a plasmid that expresses a GFP-N fusion peptide), and pT-GFP (a negative control plasmid that expresses the GFP portion of pT-GFP-N but that lacks N) are described in the text. (C) Co-immunoprecipitation analysis to further verify association of N with P bodies. DCP1 was recovered by immunoprecipitation with anti-DCP1 Ab and Sepharose-G beads. Recovered material was examined by Western analysis with anti-DCP1 Ab to verify recovery of DCP1, or with anti-N Ab to detect co-precipitation of N with DCP1. As in B, some samples were also treated with RNase A before recovery to verify that association between N and DCP1 is RNA-dependent. (n represents purified bacterially expressed N; dashes represent untransfected cells; pTriEx, pT-GFP-N, and pT-GFP are as described for B.)
Fig. 3.
Fig. 3.
N sequesters 5′ caps in P bodies. (A) We used an mRNA that expresses GFP, and a closely related nsRNA containing a premature termination codon to examine the effect of N on RNA stability. The GFP gene in the nsRNA contains a premature stop codon resulting from the insertion of two G residues (shown in bold). A primer pair corresponding to the first 180 nucleotides of both RNAs was used to quantify 5′ termini using real-time PCR following reverse transcription. A second primer pair was used to quantify a region near the 3′ termini of both RNAs. (B) The relative steady state levels of the 5′ and 3′ termini of the mRNA and nsRNA in the absence of N are shown. The quantified PCR products of the 5′ and 3′ termini in the GFP mRNA were used for normalization. (C) Comparison of the steady-state levels of 5′ and 3′ termini GFP mRNA in the presence and absence of N. (D) Comparison of the steady-state levels of 5′ and 3′ termini in GFP nsRNA in the presence and absence of N. (E) Effect of N on the relative abundance of 5′ and 3′ termini from GFP mRNA and nsRNA in P bodies. P body-associated material was recovered by immunoprecipitation with anti-DCP1 Ab as in Fig. 2. RNA was then prepared and the 5′ and 3′ termini quantified. 5′ termini in the absence of N were used for normalization. (n.d. = not detected.)
Fig. 4.
Fig. 4.
Use of mRNA and nsRNA caps in viral mRNA initiation. (A) Composite viral mRNAs containing caps from GFP mRNA or nsRNA were detected using a sense primer matching the 5′ end of the GFP RNA and primer complementary to SNV S segment mRNA as shown. The total length of S segment mRNA is 2,076 nucleotides, not including the cap. (B) Confocal detection of cytoplasmic P bodies and N in virus-infected cells. Twenty-four hours after infection, intracytoplasmic DCP1 was detected with anti-DCP1 antibody, N was visualized by detection with anti-N antibody, and nuclei by DAPI. (C) Quantification of virus-infected cells expressing GFP mRNA or nsRNA. “Virus” represents RNA from virus-infected cells; “mRNA + virus” represents RNA from virus-infected cells expressing GFP mRNA (used for normalization of the graph); and “nsRNA + virus” represents RNA from virus-infected cells expressing GFP nsRNA. (D) Sequence analysis of caps from GFP nsRNA on viral mRNA. RT-PCR products were cloned and 20 DNAs were randomly obtained and sequenced. Cap sequences are depicted in blue and viral UTR sequences in green. The triplet repeats present at the terminus of the viral UTR are underlined. The number of clones with each displayed sequence is indicated.
Fig. 5.
Fig. 5.
Cap-snatching and translation initiation by N. Turnover of cellular mRNA results in transport to P bodies, where viral N shelters the 5′ termini from decapping and degradation (A). The viral RdRp uses the capped 5′ termini during transcription initiation to generate nascent viral mRNA using the minus strand viral RNA template (B). N then recruits the 43S preinitiation complex during the process of translation initiation (C).
See this image and copyright information in PMC

References

    1. Li ML, Ramirez BC, Krug RM. RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same protein subunit constitute the two required RNA-binding sites. EMBO J. 1998;17:5844–5852. - PMC - PubMed
    1. Hagen M, Chung TD, Butcher JA, Krystal M. Recombinant influenza virus polymerase: requirement of both 5′ and 3′ viral ends for endonuclease activity. J Virol. 1994;68:1509–1515. - PMC - PubMed
    1. Duijsings D, Kormelink R, Goldbach R. In vivo analysis of the TSWV cap-snatching mechanism: single base complementarity and primer length requirements. EMBO J. 2001;20:2545–2552. - PMC - PubMed
    1. Estabrook EM, Tsai J, Falk BW. In vivo transfer of barley stripe mosaic hordeivirus ribonucleotides to the 5′ terminus of maize stripe tenuivirus RNAs. Proc Natl Acad Sci USA. 1998;95:8304–8309. - PMC - PubMed
    1. Ramirez BC, Garcin D, Calvert LA, Kolakofsky D, Haenni AL. Capped nonviral sequences at the 5′ end of the mRNAs of rice hoja blanca virus RNA4. J Virol. 1995;69:1951–1954. - PMC - PubMed

Publication types

MeSH terms