My
laboratory explores the molecular mechanisms of
replication of positive-strand RNA animal viruses.
Positive-strand RNA viruses are interesting because they
replicate exclusively via RNA intermediates.
Considering the "RNA World" view of the evolution of
life, RNA viruses represent modern day organisms with
evolutionarily ancient replication strategies.
Positive-strand viral RNA serves two important functions
within the cytoplasm of infected host cells:
(1)
as mRNA for the expression of the viral proteins and,
(2)
as the template for negative-strand RNA synthesis.
Viral RNA cannot simultaneously serve as a mRNA and as a
template for negative-strand RNA synthesis due to the 5'
to 3' movement of translating ribosomes and the 3' to 5'
movement of replicase during negative-strand RNA
synthesis. We study two viruses in great detail:
poliovirus and hepatitis C virus. We hypothesize that
direct interactions occur between the 5'- and
3'-terminal non-translated regions of viral RNA to
regulate the transformation of viral mRNA into a
template for viral negative-strand RNA synthesis.
Poliovirus RNA, as mRNA, first becomes part of a
messenger ribonucleoprotein (mRNP) complex with
communication between the 5'- and 3'-termini mediated by
the cellular translation machinery (eIF4G, poly(A)
binding protein, etc.). Following viral protein
synthesis, the viral mRNP complex must transform into a
preinitiation RNA replication complex to allow for the
initiation of viral negative-strand RNA synthesis. The
5'-terminal ribonucleoprotein complex of poliovirus
containing viral protein 3CD mediates, in part, the
initiation of viral negative-strand RNA synthesis at the
3'-terminus of poliovirus RNA. We propose that this
model of viral RNA replication, emphasizing
communication between the 5'- and 3'-termini of the
viral RNA, is broadly applicable to all positive-strand
RNA animal viruses.
In
another series of experiments, we discovered that
hepatitis C virus RNA is detected and destroyed by an
interferon-regulated antiviral pathway present in the
cytoplasm of cells; the 2'-5' oligoadenylate synthetase/ribonuclease
L pathway. Ribonuclease L cleaves viral RNA at
single-stranded UA and UU dinucleotides. Relatively
interferon-resistant genotype 1 hepatitis C viruses have
fewer ribonuclease L cleavage sites than more
interferon-sensitive genotype 2 and 3 viruses. These
discoveries may help to explain the clinical outcome of
interferon therapy in hepatitis C virus-infected
patients where patients infected with genotype 1 viruses
are cured less frequently by interferon therapy than
patients infected with genotype 2 or 3 viruses.
Selected Publications
Barton, D.J. and J.B. Flanegan. 1997. Synchronous replication of
poliovirus RNA: Initiation of negative-strand RNA
synthesis requires the guanidine-inhibited activity of
protein 2C. J. Virol. 71:8482-8489.
Barton, D.J.,
B.J. Morasco and J.B. Flanegan. 1999. Translating
ribosomes inhibit poliovirus negative-strand RNA
synthesis. J. Virol. 73:10104-10112.
Barton, D.J., B.J. O'Donnell and J.B. Flanegan.
2001. 5' Cloverleaf in poliovirus RNA is a cis-acting
replication element required for negative-strand
synthesis. EMBO 20:1-10.
Murray, K.E., A.W. Roberts and D.J. Barton.
2001. Poly(rC) binding proteins mediate poliovirus RNA
stability. RNA 7:1126-1141.
Lyons, T., K.E. Murray, A.W. Roberts and D.J. Barton.
2001. The 5' cloverleaf of poliovirus RNA is required
for VPg uridylylation and the initiation of
negative-strand RNA synthesis. J. Virol.
75:10696-10708.
Barton, D.J., B.J. Morasco, L.E. Smerage and J.B.
Flanegan. 2002. Poliovirus RNA replication and genetic
complementation in cell-free reactions. In Wimmer, E.,
and Semler, B. (eds.) "Molecular Biology of
Picornaviruses". ASM Press, pgs. 461-469.
Han, J-Q, and D.J. Barton. 2002. Activation and
evasion of the antiviral 2'-5' oligoadenylate synthetase/ribonuclease
L pathway by hepatitis C virus. RNA 8:512-525.
Pathak, H.B., S.K.B. Ghosh, A.W. Roberts, S.D. Sharma,
J.D. Yoder, J.J. Arnold, D.W. Gohara, D.J. Barton,
A.V. Paul, and C.E. Cameron. 2002. Structure-function
relationships of the RNA-dependent RNA polymerase from
poliovirus (3DPol): A surface of the primary
oligomerization domain functions in capsid precursor
processing and VPg uridylylation. J. Biol. Chem.
277:31551-31562.
Murray, K.E. and D.J. Barton. 2003. Poliovirus
CRE-dependent VPg uridylylation is required for
positive-strand RNA synthesis but not for
negative-strand RNA synthesis. J. Virol. In Press.
Murray KE, Steil BP, Roberts AW, Barton DJ. Replication of poliovirus RNA with complete internal ribosome
entry site deletions.
J Virol. 2004 Feb;78(3):1393-402.
Han JQ, Wroblewski G, Xu Z, Silverman RH, Barton DJ. Sensitivity of hepatitis C virus RNA to the
antiviral enzyme ribonuclease L is determined by a subset of efficient cleavage sites.
J Interferon Cytokine Res. 2004 Nov;24(11):664-76.
Jurgens CK, Barton DJ, Sharma N, Morasco BJ, Ogram SA, Flanegan JB. 2A(pro) is a
multifunctional protein that regulates the stability, translation and replication of poliovirus RNA.
Virology. 2005 Nov 16; [Epub ahead of print]
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