Inter-subunit interactions of the Autographa californica M
nucleopolyhedrovirus RNA polymerase
Erin A. Crouch, Leah T. Cox, Kristy G. Morales, A. Lorena Passarelli⁎
Molecular, Cellular, and Developmental Biology Program, Division of Biology, 116 Ackert Hall, Kansas State University, Manhattan, KS 66506-4901, USA
Received 29 January 2007; returned to author for revision 2 February 2007; accepted 18 May 2007
Available online 2 July 2007
Autographa californica M nucleopolyhedrovirus transcribes genes using two DNA-directed RNA polymerases; early genes are transcribed by
the host RNA polymerase II, and late and very late genes are transcribed by a viral-encoded multisubunit RNA polymerase. The viral RNA
polymerase is composed of four proteins: Late Expression Factor-4 (LEF-4), LEF-8, LEF-9, and P47. The predicted amino acid sequences of
lef-9 and lef-8 contain motifs that are similar to those that participate at the catalytic center of known RNA polymerases. The requirement for the
motif present in LEF-8 in late gene expression has been previously demonstrated. We have assessed the requirement of specific residues within
the motif in LEF-9 for late gene expression. The conserved aspartic acid residues within the LEF-9 motif, corresponding to those essential for
activity of the Escherichia coli RNA polymerase largest subunit, were required for late gene expression. Furthermore, we found that LEF-8 and
LEF-9 interacted in coimmunoprecipitation experiments. We determined possible interactions of all the RNA polymerase subunits in pairwise
combinations and found associations between LEF-9 and P47, LEF-4 and P47, and LEF-8 and P47. In contrast, LEF-4 and LEF-8 did not
coimmunoprecipitate but coimmunoprecipitated in the presence of P47, suggesting that they do not associate directly. A weak association was
observed between LEF-4 and LEF-9. Further analysis also suggested that LEF-8, LEF-9, and P47 have the ability to self-associate. Studies on
protein-protein interactions may provide insight into the structural design of the complex and mechanistic aspects affecting late and very late
© 2007 Elsevier Inc. All rights reserved.
Keywords: Baculovirus; RNA polymerase; Late gene expression
Transcription of Autographa californica M nucleopolyhe-
drovirus (AcMNPV) genes takes place in three temporal phases,
early, late, and very late, during the infection cycle. Transcrip-
tion of late and very late genes can be distinguished from that of
early genes by their requirement for protein and viral DNA
syntheses (Rice and Miller, 1986). Moreover, transcription of
early and late or very late genes is carried out by distinct trans-
criptosomes, where early genes are transcribed by the cellular
RNA polymerase II and the late and very late genes are trans-
cribed by a viral DNA-directed RNA polymerase (Beniya et al.,
1996; Grula et al., 1981; Grula and Weaver, 1981; Huh and
Weaver, 1990; Yang et al., 1991).
The subunits of the DNA-directed RNA polymerase were
initially identified by either characterizing viruses with tem-
perature sensitive mutations (Carstens et al., 1994; Knebel-
Mörsdorf et al., 2006; Partington et al., 1990) or by their
requirement in transient late gene expression assays (Lu and
Miller, 1994; Passarelli and Miller, 1993a; Passarelli et al.,
1994; Todd et al., 1995). Subsequently, purification of factors
necessary to transcribe a late promoter in vitro identified the
polypeptides in the viral RNA polymerase as four previously
identified late gene transcription factors: Late Expression
Factor-8 (LEF-8), LEF-9, LEF-4, and P47 (Guarino et al.,
1998b). This four-subunit complex was sufficient to recognize a
late gene promoter, bind DNA, and synthesize a late message in
vitro (Guarino et al., 1998b).
Two subunits, LEF-8 and LEF-9, encode motifs with homo-
logy to those present in other known RNA polymerases (Lu and
Miller, 1994; Passarelli et al., 1994). In other RNA polymerases
Virology 367 (2007) 265–274
⁎Corresponding author. Fax: +1 785 532 6653.
E-mail address: email@example.com (A.L. Passarelli).
0042-6822/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
these motifs participate at the active center including the
coordination of Mg+2during transcription (Lu and Miller, 1994;
Passarelli et al., 1994; Zaychikov et al., 1996). A 13-residue
sequence required for activity of LEF-8 in transient late gene
expression assays (Titterington et al., 2003) is also conserved in
the Escherichia coli RNA polymerase β subunit region H
(Passarelli et al., 1994). lef-9 encodes a motif similar to the
“invariable” RNA polymerase motif, NADFDGF, in the E. coli
RNA polymerase β′ subunit and other RNA polymerase largest
subunits (Lu and Miller, 1994). However, the role of this motif
in late gene transcription has not been explored.
The other two subunits, LEF-4 and P47, have been
characterized to different extents. LEF-4 has activities asso-
ciated with 5′ capping, guanylyltransferase and RNA tripho-
sphatase activities (Gross and Shuman, 1998; Guarino et al.,
1998a; Ho et al., 1998; Jin et al., 1998). The specific function of
p47 in late gene transcription is not known.
DNA-directed RNA polymerases are the engines responsible
for gene transcription and they play a major role in gene regu-
lation. RNA polymerase II is a multisubunit complex of 8 to
14 subunits that interacts with promoter recognition and pro-
a multisubunit and multifunctional four-subunit enzyme com-
plex, and baculoviruses are the only nuclear-replicating viruses
that encode a functional DNA-directed RNA polymerase. There
is no significant overall sequence homology between the
baculovirus RNA polymerase subunits and other RNA poly-
merases, except for the two motifs described above, implying
that active site sequence conservation is essential for function.
In this study we were interested in defining the AcMNPV
RNA polymerase subunit interaction map to gather information
on the overall architecture of this novel enzyme complex. In
addition, we performed experiments to define the region(s) of
LEF-8 that interfaced with two interactive partners. Finally, we
altered the putative catalytic domain of LEF-9 to evaluate its
contribution to late gene expression.
Results and discussion
Mutagenesis of the RNA polymerase motif within LEF-9
LEF-9 predicts a seven-amino acid region (NTDCDGD) that
is also conserved in the β′ subunit of the E. coli RNA poly-
merase and largest subunit of the eukaryotic RNA polymerase II
(NADFDGD) and the site coordinating the 3′-OH of the nascent
RNA and the α-phosphate of the NTP being incorporated
(Cramer et al., 2001). The aspartic residues bind Mg2+at the
catalytic center (Cramer et al., 2001). This region is also highly
conserved in the predicted lef-9 polypeptides of all sequenced
baculovirus genomes (Fig. 1). Although there are slight dif-
ferences in this sequence among different viruses, the three
aspartic acids are invariable. We introduced alanines in place of
aspartic acid residues at positions 282, 284, and 286 in the
AcMNPV LEF-9 NADFDGD motif to determine if the residues
were necessary for late gene expression (Fig. 2A). The results
indicated that mutation of the aspartic acid residues reduced
late promoter activity to background levels (Fig. 2B, compare
column 1 to columns 3 to 5). All of the LEF-9 mutants were
Fig. 1. Alignment of LEF-9 sequences. The region of AcMNPV LEF-9 that contains a seven amino acid region also conserved in other RNA polymerases,
NADFDGD, was aligned with selected baculovirus LEF-9 sequences from the baculoviruses indicated on the left. The position of the first amino acid in each region is
listed in parentheses.
266E.A. Crouch et al. / Virology 367 (2007) 265–274
Guarino, L.A., Jin, J., Dong, W., 1998a. Guanylyltransferase activity of the
LEF-4 subunit of baculovirus RNA polymerase. J. Virol. 72, 10003–10010.
Guarino, L.A., Xu, B., Jin, J., Dong, W., 1998b. A virus-encoded RNA poly-
merase purified from baculovirus-infected cells. J. Virol. 72, 7985–7991.
Ho, C.K., Pie, Y., Shuman, S., 1998. Yeast and viral RNA 5′ triphosphatases
comprise a new nucleoside triphosphatase family. J. Biol. Chem. 273,
Huh, N.E., Weaver, R.F., 1990. Identifying the RNA polymerases that syn-
thesize specific transcripts of the Autographa californica nuclear polyhe-
drosis virus. J. Gen. Virol. 71, 195–202.
Jin, J., Dong, W., Guarino, L.A., 1998. The LEF-4 subunit of baculovirus RNA
polymerase has RNA 5′-triphosphatase and ATPase activities. J. Virol. 72,
Knebel-Mörsdorf, D., Quadt, I., Li, Y., Montier, L., Guarino, L.A., 2006.
Expression of baculovirus late and very late genes depends on LEF-4, a
component of the viral RNA polymerase whose guanyltransferase function
is essential. J. Virol. 80, 4168–4173.
Lu, A., Miller, L.K., 1994. Identification of three late expression factor genes
within the 33.8- to 43.4-map-unit region of Autographa californica nuclear
polyhedrosis virus. J. Virol. 68, 6710–6718.
Markovtsov, V., Mustaev, A., Goldfarb, A., 1996. Protein-RNA interactions in
the active center of transcription elongation complex. Proc. Natl. Acad. Sci.
U.S.A. 93, 3221–3226.
Naryshkina, T., Rogulja, D., Golub, L., Severinov, K., 2000. Inter- and
intrasubunit interactions during the formation of RNA polymerase assembly
intermediate. J. Biol. Chem. 275, 31183–31190.
O'Reilly, D.R., Miller, L.K., Luckow, V.A., 1994. Baculovirus Expression
Vectors: A Laboratory Manual. W.H. Freeman and Company, New York.
Partington, S., Yu, H., Lu, A., Carstens, E.B., 1990. Isolation of temperature
sensitive mutants of Autographa californica nuclear polyhedrosis virus
phenotype characterization of baculovirus mutants defective in very late
gene expression. Virology 175 (1), 91–102.
Passarelli, A.L., Miller, L.K., 1993a. Identification of genes encoding late
expression factors located between 56.0 and 65.4 map units of the Autogra-
pha californica nuclear polyhedrosis virus genome. Virology 197, 704–714.
Passarelli, A.L., Miller, L.K., 1993b. Three baculovirus genes involved in
late and very late gene expression: ie-1, ie-n, and lef-2. J. Virol. 67,
Passarelli, A.L., Todd, J.W., Miller, L.K., 1994. A baculovirus gene involved in
late gene expression predicts a large polypeptide with a conserved motif of
RNA polymerases. J. Virol. 68, 4673–4678.
Potter, K.N., Miller, L.K., 1980. Transfection of two invertebrate cell lines with
DNA of Autographa californica nuclear polyhedrosis virus. J. Invertebr.
Pathol. 36, 431–432.
Prikhod'ko, E.A., Lu, A., Wilson, J.A., Miller, L.K., 1999. In vivo and in vitro
analysis of baculovirus ie-2 mutants. J. Virol. 73, 2460–2468.
Rapp, J.C., Wilson, J.A., Miller, L.K., 1998. Nineteen baculovirus open reading
frames, including LEF-12, support late gene expression. J. Virol. 72,
Rice, W.C., Miller, L.K., 1986. Baculovirus transcription in the presence of
inhibitors and in nonpermissive Drosophila cells. Virus Res. 6,
Sosunov, V., Sosunova, E., Mostaev, A., Bass, I., Nikiforov, V., Goldfarb, A.,
2003. Unified two-metal mechanism of RNA synthesis and degradation by
RNA polymerase. EMBO J. 22, 2234–2244.
Thiem, S.M., Miller, L.K., 1990. Differential gene expression mediated by late,
very late, and hybrid baculovirus promoters. Gene 91, 87–94.
Titterington, J.S., Nun, T.K., Passarelli, A.L., 2003. Functional dissection of the
baculovirus late expression factor-8: sequence requirements for late gene
promoter activation. J. Gen. Virol. 84, 1817–1826.
Todd, J.W., Passarelli, A.L., Miller, L.K., 1995. Eighteen baculovirus genes,
including lef-11, p35, 39K, and p47, support late gene expression. J. Virol.
Vaughn, J.L., Goodwin, R.H., Tompkins, G.J., McCawley, P., 1997. The
establishment of two cell lines from the insect Spodoptera frugiperda
(Lepidoptera: Noctuidae). In vitro 13, 213–217.
Wu, Y., Carstens, E.B., 1998. A baculovirus single-stranded DNA binding
protein, LEF-3, mediates the nuclear localization of the putative helicase
P143. Virology 247, 32–40.
Yang, C.L., Stetler, D.A., Weaver, R.F., 1991. Structural comparison of the
Autographa californica nuclear polyhedrosis virus-induced RNA polymer-
ase and the 3 nuclear RNA polymerases from the host, Spodoptera
frugiperda. Virus Res. 20 (3), 251–264.
Zaychikov, E., Martin, E., Denissova, L., Koslov, M., Markovtsov, V., Kashlev,
M., Heumann, H., 1996. Mapping of catalyltic residues in the RNA
polymerase active center. Science 273, 107–109.
274E.A. Crouch et al. / Virology 367 (2007) 265–274