Gene, 122 (1992) 345-348 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

345

0378-1119/92/$05.00

06807

Short

Communications

Sequence

and in vitro translational analysis of a 1629-nucleotide Autographa californica nuclear polyhedrosis virus strain E2 (AcMNPV-E2;

recombinant

DNA;

insect vector; proline-rich

protein;

anomalous

ORF, in

migration)

Daphne Q.-D. Pham and Natarajan Sivasubramanian Departmetzt of Entomology, Received

University of California, Riverside, CA 92521, USA

by J.A. Engler: 9 April 1992; Revised/Accepted:

25 June/8

July 1992; Received

at publishers:

20 August

1992

SUMMARY

The complete nucleotide (nt) sequence of an open reading frame (ORF) (map unit 5.1 to 3.8) from Autographa californica nuclear polyhedrosis virus strain E2 (AcMNPV-E2) has been determined. This 1629-nt ORF has a coding potential for a 61-kDa Pro-rich protein. However, in vitro translation of the 1629-nt ORF and sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) revealed a 7%kDa protein product. The discrepancy between the M, predicted by the nt sequence and that obtained from the in vitro translational analysis is due to the high Pro content of this protein. The high Pro content causes anomalous migration of this protein during SDS-PAGE.

INTRODUCTION

Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) particles are rod-shaped, enveloped virions with a 12%kb, circular, double-stranded DNA genome. A unique feature of the AcMNPV is that two forms of viral progeny are produced during viral infection. These two forms of AcMNPV have separate roles in the persistence of the virus in nature. In infected lepidopteran Sf21 tissue culture cells, both morphological forms are produced. However, whether all the tissues of the infected host

Correspondence to: Dr. D.Q.-D. versity of California, Riverside,

Pham, Department of Entomology, UniCA 92521, USA. Tel. (714) 787-3629;

Fax (714) 787-3086. Abbreviations: A., Autographa, aa, amino acids; AcMNPV, A. calijbmica NPV; AcMNPV-C6 or -E2, AcMNPV strain C6 or E2, respectively; BV, budded

virus;

nuclear

polyhedrosis

GCG,

Genetic

Computer

virus; nt, nucleotide(s);

PAGE, polyacrylamide virus; PRP, proline-rich scription start point(s).

Group,

Madison,

WI; NPV,

ORF, open reading

frame;

gel electrophoresis; PDV, polyhedra-derived protein; SDS, sodium dodecyl sulfate; tsp, tran-

larva can support the development of both viral forms is not known. For most NPVs, the occluded virus is rarely observed in the midgut but is seen extensively in other tissues of the infected host larva. These results suggest that the viral genes necessary for these two forms may be expressed differently in various tissues of the infected host larvae. To test this hypothesis, we studied the expression of several key genes of AcMNPV in an insect tissue culture system as well as in different tissues of host larvae. During our study, we found that a probe specific to a 1629-nt ORF detected RNA transcripts in uninfected tissues of Trichoplusia ni lepidopteran larvae even under highstringency conditions (Pham and Sivasubramanian, 1992). This 1629-nt ORF (map unit 5.1-3.8 on the AcMNPV-E2 genome) is located in opposite direction to and downstream from the pn gene in AcMNPV-E2. Expression of host transcripts homologous to a 1629-nt ORF appears to be tissuespecific, and the homology observed appears to be specific for the 1629-m ORF. The ‘1629-nt ORF’-specific probe hybridized to transcripts of 0.7 and 2.5 kb in uninfected midgut tissues as well as transcripts of 2.5, 8.8 and 11.0 kb in uninfected hemocytes. Our preliminary data suggest that

346 at least one of these host transcripts, the 0.7-kb transcript, is a poly(A)+mRNA in the uninfected midgut tissues (un-

of these sequences is used. The virus may also use a motif different from the two consensus sequences cited above to

published data). These results prompted us to study the complete nt sequence as well as the translatability of the 1629-nt ORF in an attempt to understand its role in viral development.

initiate transcription of this gene. The nt sequence (GGCATGA) surrounding the Met codon of this ORF conforms to the Kozak rules for a favorable eukaryotic translational initiation sequence (PuNNATGPu) (Kozak, 1983). The stop codon at position 1629 is followed by two polyadenylation recognition and addition sites. Four potential N-glycosylation sites are seen at aa 122,240,421 and 442. Although viral genomes from strains E2 and C6 of AcMNPV gave different restriction enzyme digestion patterns (Possee et al., 1991), the 1629-nt ORFs in AcMNPVE2 and -C6 show no sequence differences. The conservation of this ORF between the two strains: (1) suggests that this ORF probably plays an important role in the persistence and survival of the virus and (2) is consistent with the observation that a stop codon insertion in this ORF resulted in the production of unstable viruses (Possee et al., 1991).

EXPERIMENTAL

AND DISCUSSION

(a) Nucleotide sequence of the 1629-nt ORF The cloning, subcloning and sequencing strategies of the 1629-nt ORF are shown in Fig. 1; the entire nt sequence of the 1629-nt ORF is shown in Fig. 2. Consensus eukaryotic TATA and CAT boxes are found near the tsp and are located at positions -74 and - 136, respectively. Interestingly, a CAGT sequence often seen at the tsp of most AcMNPV early genes is found at position -5 1 of the 1629nt ORF, whereas an ATAAG sequence often seen at the tsp of most AcMNPV late genes is observed very far from the Met start codon at position -497. Yet, the 1629-nt ORF is not transcribed until late during the virus life cycle (Ooi and Miller, 1990; Pham and Sivasubramanian, 1992). Since the tsp has not been identified, it is not known which

(b) Computer analysis Extensive computer analyses of the 1629-nt ORF were carried out. TESTCODE analysis (GCG) indicated that this ORF has a very strong coding potential starting from

Fig. 1. Cloning and sequencing strategies. (A) Cloning and sucloning strategies. Clones AcuB and AcuB were made by inserting the BarnHI-F fragment (2 kb, nt 4254-6193, shown in figure as ‘F’) and the BarnHI-C fragment (8 kb, nt 6193 to approx. 15000, shown in figure as ‘C) into pUs13, respectively. Clone AcgHSl was created by inserting 1-kb HindIII-Sal1 fragment (nt 5264-6264 of the AcMNPV genome) into pGem1. Clone AcuB was further subcloned to give clone AcuHAO.5, which had the Asp718-Hind111 fragment (3’ end of 1629~nt ORF, nt 4714-5264) inserted into pUcl3. Clone AcuB was further subcloned to give clone AcuHpO.5, which had the HindIII-PvuII fragment (5’ end of 1629~nt ORF, nt 6306-6841) inserting in pUcl3. Clone AcgHSl

was further

RsaI (nt 5264-5719

subcloned

of the AcMNPV

to give the SspI (nt 5264 to 6063 of the AcMNPV genome) deletion clones. (II) Sequencing

strategies.

genome),

CZaI (nt 5264-5872

The original sequencing

of the AcMNPV

was done on AcgHSl,

genome)

and

AcuB2, AcuB

clones and their subclones. The entire nucleotide sequence was double checked by resequencing the opposite strand of the three parental clones using custom-designed primers. All sequencing was done using dideoxy method (Sanger et al., 1977) with Sequenase Version 2.0 (US Biochemical, Cleveland, OH). Sequencing was performed according to manufacturer’s recommendation using [ s(-35S]dATP (1000 Ci/mmol, Amersham) as the radioactive label. All templates were double-stranded DNA. Primers used in sequencing of clones inserted in pGem1 were the SP6 or T7 promoter sequencing primers (Promega, Madison, WI). The primers used in the sequencing of clones inserted in pUcl3 were the universal pUc or reverse pUc primers (US Biochemical). The numbers

next to the restriction

sites denote their nt. The nt positions

are based on Possee et al. (1991).

TCTGGTGC~CTCCTTTA~~~~~~~e~~~~~~e~~e~~~~~~~~~cc~~~~e~~e*~~~~~~~e~~~~ec

Fig. 2. The nt sequence tial glycosylation

of the 1629-m ORF and the deduced

sites, underlined:

2301

aa sequence.

and the aa Pro, zigzag-underlined.

Eukaryotic

Motives

consensus

CAT and TATA boxes are doubly underlined;

for early and late AcMNPV

genes are italicized

poten-

and zigzag-underlined.

Lower-case letters represent noncoding regions; capital letters represent coding regions. Plain numbers denote nt numbers; italicized numbers denote aa numbers. OCH stands for ochre stop codon. Each base pair was sequenced on the average five times. All of the plus strand and 90% (the complete ORF) of the minus strand

were sequenced.

The sequence

was deposited

in EMBL database

the ‘ATG’ start codon (nt 6486 or 5.1 map unit of the AcMNPV genome) and stopping around the stop codon (nt 4857 or 3.8 map unit of the AcMNPV genome). Indirect prediction of aa sequence (StriderTM) showed that the 1629-nt ORF has the potential to code for a 61-kDa protein. This putative protein is very Pro-rich (15% overall), especially between aa 143 and 320 (Fig. 2). TFASTA search (GCG) of the 1629-nt ORF against GenBank and EMBL databases reveals that many Pro-rich proteins are either transcriptional factors or cell ‘envelope’ proteins (i.e., cellular proteins that are part of an enclosing structure; Eckert and Green, 1986; Mehrel et al., 1990; Hong et al., 1990; Selkirk et al., 1991). Since the 1629-nt ORF is expressed late in infection (Ooi and Miller, 1990; Pham and Sivasubramanian, 1992), it is unlikely that this ORF would code for a regulatory transcriptional activator. AcMNPV polymerase and transcriptional factor genes are known to express very early in the virus life cycle (Tolmaski et al., 1988; Carson et al., 1988; 1991a,b; Guarino and Summers, 1986; 1987). Furthermore, the putative protein coded by the 1629-nt ORF has the Pro-rich domain situated in the central region of the protein. Conversely, DNA-binding proteins often have a Pro-rich domain located within either or both of the termini (Santoro et al., 1988; Mermod et al., 1989; Mitchell and Tjian, 1989). Thus, this putative protein coded by the 1629~nt ORF is probably not a DNA-binding protein. It nonetheless may still have a regulatory role in RNA-protein or protein-protein interactions during viral development. This putative protein may also be a structural

under accession

No. Z 11662.

component in the ‘envelope’ of the polyhedron or the envelope of the PDV or BV. Although Kyte-Doolittle hydrophobicity analysis of this protein indicate that its N and C termini are hydrophilic, the two Pro-rich hydrophobic domains in the center of the protein potentially can span the membrane twice. In doing so, it would leave the N and C termini on one side of the lipid bilayer and therefore could fuction as a membrane protein in the envelope of PDV or BV, both of which are true bilipid membranes. Alternatively, if it is a component of the polyhedral ‘envelope’, it need not be a membrane glycoprotein because this ‘envelope’ is not a bilipid membrane, but rather an enclosing structure consisting primarily of carbohydrate and protein. Whether this protein is a structural or nonstructural protein remains to be determined. (c) In vitro translation of the 1629-nt ORF In vitro translation of the 1629-nt ORF gene revealed a protein product that migrated at 78 kDa during SDSPAGE (Fig. 3). However, computer-assisted translation of the 1629-nt ORF predicted only 61 kDa. This discrepancy is due probably to the anomalous mobility of the protein during SDS-PAGE rather than a true difference between the two data. This 1629-nt ORF protein is very Pro-rich [especially between aa residues 143 and 322) and PRPs are known to have abnormally low mobility during SDSPAGE (Robinson et al., 1989; Ziemer et al., 1982). Moreover, other ATGs upstream from the coding region do not give ORF more than 100 nt long.

348

kDa 97-

Carson,

kDa

D.D., Summers,

a baculovirus

-78

Guarino,

L.A.

and

Guarino,

L.A. and Summers,

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and the number

The numbers

on the left represent

on the right of the gel denotes

size of the in vitro translated

protein.

the es-

The symbols ( + ) and ( - )

stand for the in vitro translation of plus-strand and minus-strand RNAs, respectively. Both plus- and minus-strand RNAs were made from PvuIIdigested AcPBA2.1 clone using the Promega Riboprobe Gemini11 Core System. T7 RNA polymerase was used in the making of the plus-strand RNA; SP6 RNA polymerase

was used in the making of the minus-strand

RNA. The in vitro translation reticulocyte

lysate

was performed (Promega),

and [a5S]methionine

with the nuclease-treated

minus-methionine

ucts was performed resolving

with a 0.1%

gel (Laemmli,

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mixture

(1000 Ci/mmol, Trans 35S-labelTM meta-

bolic labeling from ICN). The gel electrophoresis SDS/lo:;

of the translation

PAGE

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phoresis, the gel was stained for 30 min in 0.125% Coomassie blue/lo?; acetic acid/50:/; methanol solution. destained for 1 h in 10”; acetic acid/ 50”; methanol

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regulatory

of

of a proline-rich

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PAGE

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of a baculovirus

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29-

(Promega)

Summers,

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43 -

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+

68-

timated

M.D. and Guarino,

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solution

and treated

with a 16”; Na.salicylate

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was performed subsequently at X-OMATTM AR film. The AcP-

by inserting

2.1-kb PvuII-Asp718

(nt -358 to 1771 of the 1629~nt ORF) of the EcoRI-I NPV genome into %a1

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The authors thank Dr. B.A. Federici and Mr. CA. Post for their comments on this manuscript. We also want thank Mr. R.H. Hice for his invaluable inputs throughout this work. This work was supported by a grant from the Sigma Xi Scientific Foundation and U.C. Biotechnology Research and Education Program.

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