Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Perera, O. Articles by Becnel, J. J. Search for Related Content PubMed PubMed Citation Articles by Perera, O. Articles by Becnel, J. J.

 Previous Article  |  Next Article 

Journal of Virology, May 2007, p. 4585-4590, Vol. 81, No. 9
0022-538X/07/$08.00+0     doi:10.1128/JVI.02391-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Proteins Associated with Culex nigripalpus Nucleopolyhedrovirus Occluded Virions

Omaththage Perera,^1* Terry B. Green,² Stanley M. Stevens Jr.,³ Susan White,² and James J. Becnel²

Southern Insect Management Research Unit, USDA-ARS, 141 Experiment Station Road, P.O. Box 346, Stoneville, Mississippi 38776,¹ Center for Medical, Agricultural and Veterinary Entomology, USDA/ARS, 1600-1700 SW 23rd Avenue, Gainesville, Florida 32608,² Proteomics Core, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, Florida 32610³

Received 31 October 2006/ Accepted 2 February 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Occlusion-derived virions (ODVs) of the nucleopolyhedrovirus of Culex nigripalpus (CuniNPV) were purified by Ludox density gradient ultracentrifugation, and the proteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were identified by using Edman sequencing, matrix-assisted laser desorption ionization-time of flight mass spectrometry, nanoelectrospray quadrupole time-of-flight mass spectrometry, or a combination of these methods. Half of the 44 polypeptide sequences identified in this analysis were unique open reading frames (ORFs) encoded by the CuniNPV genome and did not show similarity to any other sequences present in protein databases. Of the 22 polypeptides that showed similarities to other baculovirus-encoded proteins, only 17 sequences have previously been identified as structural proteins. The newly identified CuniNPV structural proteins cun058, cun059, cun087, cun106, and cun109 are homologues of Autographa californica nucleopolyhedrovirus (AcMNPV) ORFs 68, 62, 98, 81, and 2, respectively. The products of four genes, namely, lef-1 (cun045), alkaline exonuclease (cun054), helicase (cun089), and DNA polymerase (cun091), were not detected in the CuniNPV ODV preparations. These four genes are conserved among all annotated baculovirus genomes, and their homologues have been detected in the ODV of AcMNPV.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Baculoviruses that infect mosquitoes are of growing interest since they are currently believed to represent a separate branch within the Baculoviridae that existed prior to the split of lepidopteran nucleopolyhedroviruses and granuloviruses (8, 15, 19). These baculoviruses may also be basal to the baculoviruses from hymenoptera, which form a branch distinct from the lepidopteran baculoviruses (11).

Culex nigripalpus nucleopolyhedrovirus (CuniNPV) is highly pathogenic to Culex sp. mosquitoes, which are important vectors of the West Nile, St. Louis, and Eastern encephalitis viruses. This virus is responsible for epizootics in field populations of C. nigripalpus larvae (6). CuniNPV development is restricted to the nuclei of midgut epithelial cells in the gastric ceca and posterior stomach (6, 22). It has two virion phenotypes: the occlusion-derived virion (ODV) that initiates infection in the midgut epithelial cells and a budded form that spreads the infection within the midgut (22). The genome of CuniNPV contains 109 open reading frames (ORFs), and only 36 of these show similarities to the ORFs of other nucleopolyhedroviruses (2).

Transmission of CuniNPV ODVs to mosquito larvae is facilitated by divalent cations: magnesium is essential for horizontal transmission, whereas the presence of calcium inhibits virus transmission (6). Cascading events are required for infection of a host with a baculovirus. A typical sequence consists of the following: (i) the virions are released from occlusion bodies (OBs) in the insect gut, (ii) the virions attach to and cross the peritrophic matrix, and (iii) the virions attach and enter midgut epithelial cells and nuclei where replication occurs. Envelope proteins of the ODV are of particular importance in this process, since they interact with the mosquito peritrophic matrix, as well as with the plasma membrane of the midgut epithelial cells. Identifying the structural proteins of the CuniNPV ODV with particular emphasis on the envelope proteins will provide insight into the biology of CuniNPV and will allow investigation of host specificity and virulence (9).

One widely used method for resolving proteins from complex mixtures for subsequent identification by mass spectrometry (MS) is two-dimensional polyacrylamide gel electrophoresis (PAGE), wherein proteins are separated by their molecular weights and isoelectric points (29). However, this technique has proven to be challenging and time-consuming in terms of method development and is limited due to the under-representation of certain protein components such as membrane and extremely acidic or basic proteins (24). An alternate method for rapid identification of proteins from complex mixtures is the use of liquid chromatography (LC) separation techniques in conjunction with tandem MS (MS/MS); this approach is commonly known as multidimensional protein identification technology. This method involves processing a sample by enzymatic digestion of the protein mixture, separation of the corresponding peptides via two-dimensional LC (typically, strong cation-exchange and reversed-phase high-pressure LC [HPLC]), and subsequent MS/MS analysis. Protein identity is ultimately determined by searching the MS data against protein sequence databases using various database search algorithms (20, 29). Another common approach for the identification of proteins from complex mixtures uses one-dimensional gel electrophoresis, followed by nanoelectrospray MS/MS analysis of trypsin-digested regions derived from the one-dimensional-gel-separated mixtures (GeLC-MS/MS). This particular method was utilized to identify the composition of proteins involved with the ODVs from CuniNPV. The present study begins the systematic analysis of the structural proteins of CuniNPV ODVs with emphasis on those localized to the envelope.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Virus preparations and purification. Viral OB preparations were carried out as described in Moser et al. (22). Briefly, late-second-instar Culex quinquifasciatus larvae were exposed to infected larvae or purified OBs. Larvae were collected 48 h after inoculation by straining them through a nylon mesh and rinsing them with deionized water to remove food particles. Collected larvae were homogenized in a glass jar using a blender. The homogenate was filtered through a 5-µm low-protein binding Durapore disk (Millipore) to eliminate cuticular debris. The filtrate was layered on top of a 30% HS-40 Ludox (Dupont) gradient and centrifuged at 15,000 x g for 30 min in a Sorvall high-speed centrifuge (model RC-5B) using a swinging-bucket rotor. The OB band formed at a density of approximately 1.14 to 1.18 g/ml and was carefully removed by using a Pasteur pipette and washed twice with 0.1 mM NaOH solution, followed by pelleting with centrifugation at 10,000 x g. Alkaline release of ODVs was achieved by adding 0.5 M NaOH to an OB suspension to achieve 10 to 15 mM final concentration. Transformation of the turbid OB suspension into a translucent viscous solution indicated the breakdown of OB and the release of ODVs. Gradient centrifugation of the samples immediately after the release of ODVs led to banding of the ODVs at a density of 1.06 ± 0.01 (n = 11) in continuous HS-40 Ludox gradients. Negatively stained ODV preparations were examined by transmission electron microscopy to determine the success of ODV purification, and the viability of ODVs was tested by larval bioassays.

PAGE and protein blots. Purified ODVs were denatured by resuspending them in 10 mM Tris-HCl containing ß-mercaptoethanol and sodium dodecyl sulfate (SDS), followed by heating at 95°C for 10 min. Denatured ODVs were separated by SDS-PAGE on an 8 to 16% gel (18) at 25 V for 15 min, followed by 100 V for 1 h by using the Protean Mini-gel system (Bio-Rad, Hercules, CA). Gels were washed for 20 min in distilled water and stained with Coomassie blue R250 (Bio-Rad) according to the manufacturer's instructions. Gels were destained for 45 min with two changes of destaining solution (40% [vol/vol] methanol, 7% [vol/vol] acetic acid).

Blots for N-terminal sequencing were made by equilibrating freshly run SDS-PAGE protein gels in transfer buffer (10 mM NaHCO₃, 3 mM Na₂CO₃, 20% methanol, 0.1% SDS; pH 9.9) for 15 min and electroblotting them onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P^sq; Millipore, Bedford, MA) by using a Transblot mini apparatus (Bio-Rad). Electroblotting was carried out at constant amperage of 350 mA for 2 h. PVDF membranes were stained with Coomassie blue R250 (Bio-Rad) by using standard protocols.

Protein identification. All protein analyses were carried out at the Proteomics Core Facility at the Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, Florida. The proteins blotted on the PVDF membranes were excised and subjected to sequence analysis by the Edman degradation method. Protein bands that were analyzed by matrix-assisted laser desorption/ionization time-of-flight MS or the GeLC-MS/MS technique using nanoelectrospray quadrupole time-of-flight MS were excised from Coomassie blue-stained polyacrylamide gels and digested in-gel with trypsin (20, 29). Capillary reversed-phase HPLC separation of protein digests was performed on a 15-cm-by-75-µm (inner diameter) TARGA C18 column (Higgins Analytical, Mountain View, CA) in combination with an Ultimate Capillary HPLC system (LC Packings, Sunnyvale, CA) operated at a flow rate of 200 nl/min. MS/MS analysis was accomplished by a hybrid quadrupole time-of-flight MS instrument (QSTAR; Applied Biosystems, Foster City, CA) equipped with a nanoelectrospray source. MS/MS data generated by the QSTAR were searched against the National Center for Biotechnology Information nr sequence database using the Mascot (Matrix Science, Boston, MA) database search algorithm. Proteins identified by Mascot were considered significant if individual ion scores exceeded the threshold value calculated for identity or extensive homology (P 0.05). The percent coverage of each protein by peptide fragments identified by one or more techniques was calculated using only the unique peptide fragments with 100% amino acid identity (i.e., amino acid sequences from recurring peptide fragments or overlapping amino acid sequences were counted only once).

Informatics. Protein sequence analyses were performed by using the MacVector 7.5 software package (Accelrys, San Diego, CA). Database searches (GenBank, EMBL, SWISS-PROT, and PIR) for protein comparisons were carried out by using BLAST and PSI-BLAST (3, 4, 27). Online software packages on the ExPASy (12; http://www.expasy.org/), SignalP 3.0 (7; http://www.cbs.dtu.dk/services/SignalP/), TMPred (16; http://www.ch.embnet.org/software/TMPRED_form.html), and Swiss EMBNet Node (www.ch.embnet.org) servers were used for the prediction of secondary structures, to search for domains and transmembrane regions, and for subcellular localization (28).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
To characterize the proteins associated with CuniNPV ODVs, OBs obtained from infected mosquito larvae were purified, and the virions were released and isolated by Ludox density centrifugation. Transmission electron microscopy, as well as larval bioassays, of ODVs confirmed their purity and viability. SDS-PAGE of the virion band and the protein-containing top layer (mostly OB proteins) recovered from the Ludox gradient indicated that some of the protein bands were present in both fractions, although in different proportions (data not shown). Therefore, we assumed that either contamination of ODV bands with proteins from denatured OBs or proteins of the virion structure were released from the virions during processing. However, we made no attempt to determine the specific location of the novel proteins identified in the present study. The lack of mosquito cell lines that could propagate CuniNPV has restricted the production of ODVs to density gradient centrifugation of alkaline-denatured OBs produced in mosquito larvae.

An electron micrograph of negatively stained ODVs from a typical purification is shown in Fig. 1A. The arrows indicate two of the ODVs. The denaturing polyacrylamide gel used to separate the protein bands for GeLC-MS/MS analysis is shown in Fig. 1B. The 13 gel regions used in the analysis and the approximate molecular masses (in kilodaltons) are indicated on each side of Fig. 1B. The CuniNPV structural proteins associated with the ODV, the methods of identification, the percent coverage of each protein identified by various techniques, and the proteins identified in each gel band (shown in Fig. 1B) by GeLC-MS/MS technique are presented in Table 1. It should be noted that many proteins were detected in more than one gel band, often in bands of molecular mass range different from those calculated using the translated CuniNPV ORFs. We postulated that this was a result of incomplete denaturation of ODV, the breakdown of protein complexes, or protein processing and not because the actual proteins differed in size from those predicted by the ORFs. A summary of CuniNPV structural proteins and their conserved counterparts, when applicable, from a few selected NPVs is presented in Table 2. A total of 45 proteins, including the previously characterized major OB protein (cun085 [26]), were identified by one or more of the techniques used here. The total number of structural proteins identified is comparable to that of Autographa californica nucleopolyhedrovirus (AcMNPV) (9). Of the 44 proteins identified, 22 were derived from unique ORFs encoded by the CuniNPV genome, whereas the remainder were conserved among all sequenced baculovirus genomes available to date. Of these 22 conserved proteins, 5 had not been previously been found to be associated with ODV. These five proteins—cun058, cun059, cun087, cun106, and cun109—and, by homology, their counterparts in other baculoviruses can be considered structural components of the OB or ODV. These ORFs show amino acid sequence similarity to ORFs 68, 62, 98, 81, and 2 of AcMNPV, respectively (Table 2).

View larger version (71K): [in this window] [in a new window]

FIG. 1. (A) Electron micrograph of negatively stained ODVs with arrows pointing to two of the ODVs. (B) SDS-PAGE of purified ODV proteins used for GeLC-MS/MS analysis. This gel contained more ODV proteins and was run for a shorter duration than the gel in Fig. 1A. Molecular masses of Bio-Rad broad-range marker (in kilodaltons) are indicated on the left side of the gel. The gel bands used in the analysis are numbered and indicated by the arrows.

View this table: [in this window] [in a new window]

TABLE 1. Structural proteins identified from CuniNPVa

View this table: [in this window] [in a new window]

TABLE 2. ORF comparison of CuniNPV proteins identified in this study to other baculovirusesa

The majority of CuniNPV structural genes appeared to be organized into two gene clusters. Thirteen structural genes were clustered between cun008 and cun024 (with the exception of cun009, cun016, cun017, and cun020), and a second cluster of 10 genes was found between cun029 to cun038. The two clusters contained 76 and 100% structural genes, respectively, of the total number of genes located within the clusters and include 56% of all of the CuniNPV structural genes identified to date. Of the 13 genes in the first cluster of structural genes, 9 were unique to CuniNPV. The second cluster contains genes encoding for proteins such as pif-1, pif-2, odv-ec27, gp41, and vp91 that have been localized to ODV of AcMNPV (9). If cun046 (homolog of AcMNPV pif-3 (23), cun048, and cun049 (structural genes of unknown function) were combined with the above two gene clusters, the 26 structural genes identified from this region would account for 62% of all of the genes present in this stretch of the CuniNPV genome and for 59% of all of the structural genes identified from CuniNPV. Although other baculovirus genomes contain some structural genes in close proximity, clustering of structural gene at the densities found in CuniNPV has not been found in any other published baculovirus genome sequence.

Bioinformatic analyses predicted that 19 of the 44 CuniNPV structural proteins contained one or more transmembrane regions. In addition, 12 sequences were predicted to contain transmembrane domains and a signal or signal anchor sequences with significant probabilities (P > 0.8), while 4 others contained strong nuclear localization signals (Table 2). SignalP and TMPred domain predictions of AcMNPV proteins homologous to CuniNPV structural proteins containing functional domains (transmembrane and/or signal/anchor) revealed that all but AcMNPV ORF 54 (vp1054) contained corresponding functional domains in their amino acid sequences. However, vp1054 homologs of the nucleopolyhedroviruses of Helicoverpa zea, Lymantria dispar, Neodiprion lecontei, and Neodiprion sertifer contained predicted transmembrane domains, whereas other NPVs listed in Table 2 did not.

Four of the CuniNPV genes—cun045, cun054, cun089, and cun091—that showed amino acid sequence similarity to AcMNPV ORFs 14 (lef-1), 133 (alk exo), 95 (helicase), and 65 (DNA polymerase), respectively, were not detected in the present study. All four of these sequences are conserved among all baculovirus genomes sequenced to date and have been identified as ODV components of AcMNPV (9). The sizes of CuniNPV homologs of these polypeptides ranged from 235 to 1,332 amino acids in length. In the present study, the GeLC-MS/MS method allowed for detection of proteins that were present in relatively low quantities and with sizes as small as 65 amino acids. Therefore, we conclude that either these four proteins are not components of the ODVs of CuniNPV or were present in quantities below the detection capabilities of the techniques used in the present study. In addition, seven conserved proteins (cun046, cun058, cun059, cun087, cun090, cun106, and cun109) that were not identified as structural components by Braunagel et al. (9) were identified in the present study. Homologs of two of the seven proteins (cun046 and cun090) have recently been confirmed as components of the ODV (23, 30).

Eight of the CuniNPV proteins identified in the present study had less than 10% amino acid coverage as determined by GeLC-MS/MS analysis (Table 1). Although these proteins had only one to three unique peptide fragments, these peptide fragments were identified in more than one gel band and were considered components of bona fide structural proteins. It should be noted that all of these peptide fragments were identified from gel bands that contained proteins of lower molecular weight than that calculated from the CuniNPV ORFs. Therefore, we postulated that these peptide fragments were products of proteins degraded during the release of ODVs by alkaline treatment.

The identification of structural proteins in CuniNPV provided the opportunity to compare and contrast various baculovirus proteomes. Although the total number of structural proteins in CuniNPV is comparable to that of AcMNPV, 50% of the CuniNPV structural proteins are unique. The remaining 50% of the CuniNPV structural proteins accounted for 61% of the total number of conserved baculovirus proteins (n = 36) identified in the CuniNPV genome (2). The existence of similar domains between CuniNPV and AcMNPV proteins with low similarities may indicate functional conservation among structural proteins of highly divergent baculovirus species. In the present study, five conserved baculovirus proteins were also identified for the first time as proteins associated with the OB or ODV. These results will provide the groundwork for understanding the function of critical structural proteins of CuniNPV and their potential role in key biological features such as infectivity, host range, and virulence. In addition, the identification of the major structural proteins of CuniNPV will facilitate comparative studies with other baculoviruses adapted to aquatic hosts and environments when they become available.

 
We acknowledge the technical support of Heather Furlong (USDA/ARS Gainesville, FL) and Linda Ballard (USDA/ARS, Stoneville, MS). We also extend our sincere gratitude to George Rohrmann (Oregon State University, Department of Microbiology), Johannes Jehle (Agricultural Service Center Palatinate, Phytopathology and Plant Protection, Neustadt, Germany), and Gordon Snodgrass (Southern Insect Management Research Unit, USDA/ARS, Stoneville, MS) for critical reviews of the manuscript.

The mention of trade names or commercial products in this report is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

 
* Corresponding author. Mailing address: Southern Insect Management, Research Unit, 141 Experiment Station Road, P.O. Box 346, Stoneville, MS 38776. Phone: (662) 686-5248. Fax: (662) 686-5421. E-mail: operera{at}msa-stoneville.ars.usda.gov

Published ahead of print on 14 February 2007.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

1
1. Ahrens, C. H., R. L. Q. Russell, C. J. Funk, S. H. Harwood, and G. F. Rohrmann. 1997. The sequence of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus genome. Virology 229:381-399.[CrossRef][Medline]
2
2. Afonso, C. L., E. R. Tulman, Z. Lu, C. A. Balinsky, B. A. Moser, J. J. Becnel, D. L. Rock, and G. F. Kutish. 2001. Genome sequence of a baculovirus pathogenic for Culex nigripalpus. J. Virol. 75:11157-11165.[Abstract/Free Full Text]
3
3. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.[CrossRef][Medline]
4
4. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.[Abstract/Free Full Text]
5
5. Ayres, M. D., S. C. Howard, J. Kuzio, M. Lopez-Ferber, and R. D. Possee. 1994. The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology 202:586-605.[CrossRef][Medline]
6
6. Becnel, J. J., S. White, B. A. Moser, T. Fukuda, M. Rotstein, A. Undeen, and A. F. Cockburn. 2001. Epizootiology and transmission of a newly discovered baculovirus from the mosquitoes Culex nigripalpus and C. quinquefasciatus. J. Gen. Virol. 82:275-282.[Abstract/Free Full Text]
7
7. Bendtsen, J. D., H. Nielsen, G. von Heijne, and S. Brunak. 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340:783-795.[CrossRef][Medline]
8
8. Blissard, G., B. Black, N. Crook, B. A. Keddie, R. Possee, G. Rohrmann, D. Theilmann, and L. Volkman. 2000. Family Baculoviridae, p. 195-202. In M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle, and R. B. Wickner (ed.), Virus taxonomy: classification and nomenclature of viruses. Seventh report of the International Committee on Taxonomy of Viruses. Academic Press, Inc., San Diego, CA.
9
9. Braunagel, S. C., W. K. Russell, G. Rosas-Acosta, D. H. Russell, and M. D. Summers. 2003. Determination of the protein composition of the occlusion-derived virus of Autographa californica nucleopolyhedrovirus. Proc. Natl. Acad. Sci. USA 100:9797-9802.[Abstract/Free Full Text]
10
10. Chen, X., W. J. Zhang, J. Wong, G. Chun, A. Lu, B. F. McCutchen, J. K. Presnail, R. Herrmann, M. Dolan, S. Tingey, Z. H. Hu, and J. M. Vlak. 2002. Comparative analysis of the complete genome sequences of Helicoverpa zea and Helicoverpa armigera single-nucleocapsid nucleopolyhedroviruses. J. Gen. Virol. 83:673-684.[Abstract/Free Full Text]
11
11. Garcia-Maruniak, A., J. E. Maruniak, P. M. A. Zanotto, A. E. Doumbouya, J. C. Liu, T. M. Merritt, and J. S. Lanoie. 2004. Sequence analysis of the genome of the Neodiprion sertifer nucleopolyhedrovirus. J. Virol. 78:7036-7051.[Abstract/Free Full Text]
12
12. Gasteiger, E., A. Gattiker, C. Hoogland, I. Ivanyi, R. D. Appel, and A. Bairoch. 2003. ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 31:3784-3788.[Abstract/Free Full Text]
13
13. Gomi, S., K. Majima, and S. Maeda. 1999. Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus. J. Gen. Virol. 80:1323-1337.[Abstract]
14
14. Hayakawa, T., R. Ko, K. Okano, S. I. Seong, C. Goto, and S. Maeda. 1999. Sequence analysis of the Xestia c-nigrum granulovirus genome. Virology 262:277-297.[CrossRef][Medline]
15
15. Herniou, E. A., J. Olszewski, J. S. Cory, and D. R. O'Reilly. 2003. The genome sequence and evolution of baculoviruses. Annu. Rev. Entomol. 48:211-234.[CrossRef][Medline]
16
16. Hofmann, K., and W. Stoffel. 1993. TMbase: a database of membrane spanning proteins segments. Biol. Chem. Hoppe-Seyler's 374:166.
17
17. Kuzio, J., M. N. Pearson, S. H. Harwood, C. J. Funk, J. T. Evans, J. M. Slavicek, and G. F. Rohrmann. 1999. Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria dispar. Virology 253:17-34.[CrossRef][Medline]
18
18. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:681-685.
19
19. Lauzon, H. A., C. J. Lucarotti, P. J. Krell, Q. Feng, A. Retnakaran, and B. Arif. 2004. Sequence and organization of the Neodiprion lecontei nucleopolyhedrovirus genome. J. Virol. 78:7023-7035.[Abstract/Free Full Text]
19
20. Li, Q., C. Donly, L. Li, L. G. Willis, D. A. Theilmann, and M. Erlandson. 2002. Sequence and organization of the Mamestra configurata nucleopolyhedrovirus genome. Virology 294:106-121.[CrossRef][Medline]
20
21. Link, A. J., J. Eng, D. M. Schieltz, E. Carmack, G. J. Mize, D. R. Morris, B. M. Garvik, and J. R. Yates. 1999. Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17:676-682.[CrossRef][Medline]
21
22. Luque, T., R. Finch, N. Crook, D. R. O'Reilly, and D. Winstanley. 2001. The complete sequence of the Cydia pomonella granulovirus genome. J. Gen. Virol. 82:2531-2547.[Abstract/Free Full Text]
22
23. Moser, B. A., J. J. Becnel, S. E. White, C. Afonso, G. Kutish, S. Shanker, and E. Almira. 2001. Morphological and molecular evidence that Culex nigripalpus baculovirus is an unusual member of the family Baculoviridae: J. Gen. Virol. 82:283-297.[Abstract/Free Full Text]
23
24. Ohkawa, T., J. O. Washburn, R. Sitapara, E. Sid, and L. E. Volkman. 2005. Specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to midgut cells of Heliothis virescens larvae is mediated by products of pif genes Ac119 and Ac022 but not by Ac115. J. Virol. 79:15258-15264.[Abstract/Free Full Text]
24
25. Pandey, A., and M. Mann. 2000. Proteomics to study genes and genomes. Nature 405:837-846.[CrossRef][Medline]
25
26. Pang, Y., J. Yu, L. Wang, X. Hu, W. Bao, G. Li, C. Chen, H. Han, S. Hu, and H. Yang. 2001. Sequence analysis of the Spodoptera litura multicapsid nucleopolyhedrovirus genome. Virology 287:391-404.[CrossRef][Medline]
26
27. Perera, O. P., S. M. Valles, T. B. Green, S. E. White, C. Strong, and J. J. Becnel. 2006. Molecular analysis of an occlusion body protein from Culex nigripalpus nucleopolyhedrovirus (CuniNPV). J. Invertebr. Pathol. 91:35-42.[CrossRef][Medline]
27
28. Pearson, W. R. 1990. Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol. 183:63-98.[Medline]
28
29. Reinhardt, A., and T. Hubbard. 1998. Using neural networks for prediction of the subcellular location of proteins. Nucleic Acids Res. 26:2230-2236.[Abstract/Free Full Text]
29
30. Washburn, M. P., D. Wolters, and J. R. Yates III. 2001. Large-scale analysis of the yeast proteome via multidimensional protein identification technology. Nat. Biotechnol. 19:242-247.[CrossRef][Medline]
30
31. Xu, H. J., Z. N. Yang, F. Wang, and C. X. Zhang. 2006. Bombyx mori nucleopolyhedrovirus ORF79 encodes a 28-kDa structural protein of the ODV envelope Arch. Virol. 151:681-695.[CrossRef]

---

Journal of Virology, May 2007, p. 4585-4590, Vol. 81, No. 9
0022-538X/07/$08.00+0     doi:10.1128/JVI.02391-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Wu, W., Liang, H., Kan, J., Liu, C., Yuan, M., Liang, C., Yang, K., Pang, Y. (2008). Autographa californica Multiple Nucleopolyhedrovirus 38K Is a Novel Nucleocapsid Protein That Interacts with VP1054, VP39, VP80, and Itself. J. Virol. 82: 12356-12364 [Abstract] [Full Text]  
• Xu, H.-J., Yang, Z.-N., Zhao, J.-F., Tian, C.-H., Ge, J.-Q., Tang, X.-D., Bao, Y.-Y., Zhang, C.-X. (2008). Bombyx mori nucleopolyhedrovirus ORF56 encodes an occlusion-derived virus protein and is not essential for budded virus production. J. Gen. Virol. 89: 1212-1219 [Abstract] [Full Text]  
• Ge, J.-Q., Yang, Z.-N., Tang, X.-D., Xu, H.-J., Hong, J., Chen, J.-G., Zhang, C.-X. (2008). Characterization of a nucleopolyhedrovirus with a deletion of the baculovirus core gene Bm67. J. Gen. Virol. 89: 766-774 [Abstract] [Full Text]  
• Deng, F., Wang, R., Fang, M., Jiang, Y., Xu, X., Wang, H., Chen, X., Arif, B. M., Guo, L., Wang, H., Hu, Z. (2007). Proteomics Analysis of Helicoverpa armigera Single Nucleocapsid Nucleopolyhedrovirus Identified Two New Occlusion-Derived Virus-Associated Proteins, HA44 and HA100. J. Virol. 81: 9377-9385 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Perera, O. Articles by Becnel, J. J. Search for Related Content PubMed PubMed Citation Articles by Perera, O. Articles by Becnel, J. J.