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Short Communication |
1 Department of Biological Sciences, University of Lethbridge, Lethbridge, AB T1K 3M4, Canada
2 Faculty of Dentistry, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC
Correspondence
Oliver Lung
oliver.lung{at}inspection.gc.ca
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ABSTRACT |
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Present address: Canadian Centre for Behavioural Neuroscience, University of Lethbridge, AB T1K 3M4, Canada. 
Present address: Lethbridge Laboratory, Canadian Food Inspection Agency, PO Box 640, Lethbridge, AB T1J 3Z4, Canada.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Theilmann & Blissard, 2008). Occlusion-derived viruses (ODVs) contain one or more nucleocapsids, and are encapsulated within the nucleus by polyhedrin proteins to form environmentally stable occlusion bodies (OBs; Slack & Arif, 2006). Upon ingestion of OBs by susceptible hosts, OB disassembly and ODV release occurs within the host's midgut after which a second virion phenotype, budded viruses (BVs), spread the infection systemically (Granados & Lawler, 1981; Keddie & Volkman, 1985; Keddie et al., 1989; Engelhard et al., 1994).
Of the two different envelope fusion protein genes identified in baculoviruses, f is more widely distributed than gp64, being found in both viruses with and without gp64 genes (Pearson & Rohrmann, 2002). F proteins from several gp64-minus baculoviruses have membrane fusion activity and contain conserved features of viral fusion proteins (IJkel et al., 2000; Pearson et al., 2000, 2002b; Westenberg et al., 2002; Yin et al., 2008; Long et al., 2008). In contrast, F proteins from baculoviruses with gp64 genes such as AcMNPV's Ac23 and Orgyia pseudotsugata MNPV's Op21 have no detectable membrane fusion activity and lack common features of functional viral fusion proteins such as proteolytic cleavage site, fusion peptide and heptad repeat regions (Pearson et al., 2000; Lung et al., 2003; Long et al., 2008); GP64 serves as the viral fusion protein in these viruses (Blissard & Wenz, 1992; Monsma et al., 1996).
Characterization of an Ac23null AcMNPV showed that Ac23 is a pathogenicity factor that is not essential for viral replication and infectivity (Lung et al., 2003). Animals infected with mutant viruses survived 
24 h longer than animals infected with control viruses (Lung et al., 2003). Ac23 is associated with both BVs (Lung et al., 2003; Braunagel et al., 2003) and ODVs (Braunagel et al., 2003). Existing evidence suggests Ac23's role in BV infectivity and production can increase the rate of kill (Lung et al., 2003; Wang et al., 2008). However, the impact of Ac23 on OBs and ODVs has not been characterized. This study was undertaken to determine if deletion of Ac23 has an effect on OB size and composition.
Genetic differences between the Ac23null mutant, control and Ac23–green fluorescent protein (gfp)–expressing viruses used in this study are summarized in Fig. 1. Generation of mutant and control viruses and OB preparation were described previously (Lung et al., 2003). Since OBs are polyhedral crystals, the average of the two largest perpendicular length measurements was used as a measure of OB size. Scanning electron microscopy (SEM) analysis showed significant size differences between wild-type (AcMNPV E2) OBs and OBs from all three bacmid-derived viruses (ANOVA and Tukey test, P<0.0001). Wild-type OBs (n=160, mean±SD, 2.48±0.60 µm) are generally larger than OBs of Ac23null-repair (n=162, 1.62±0.44 µm), Acbacmid (n=154, 1.74±0.44 µm) and Ac23null mutant (n=113, 1.75±0.54 µm) viruses. However, there was no significant difference between the sizes of OBs from the two bacmid-derived controls (Ac23null-repair and Acbacmid) and between the bacmid-derived controls and the Ac23null mutant. The sizes of OBs were also determined by measuring the widest OB cross section found in transmission electron microscopy (TEM) serial sections of purified agarose-embedded OBs. TEM was performed as described previously by Hong et al. (1994). Again, statistically significant size differences (P<0.0001) were observed only between wild-type OBs and OBs of the two bacmid-derived viruses (Ac23null and Acbacmid) analysed in this study. The average size of the widest section of wild-type OBs (n=74, 2.39±0.65 µm) is greater than that of Acbacmid (n=64, 1.87±0.41 µm) and the Ac23null mutant (n=49, 1.71±0.46 µm). Thus, both SEM and TEM analysis indicate that bacmid-derived OBs were on average significantly smaller than those from wild-type virus. However, the sizes of Ac23null OBs are not significantly different from the size of OBs from bacmid-derived controls. Since the bacmid is derived from the AcMNPV E2 variant (Luckow et al., 1993), the smaller size of bacmid-derived OBs is likely the consequence of changes in expression of polyhedrin and/or flanking ORF603 and ORF1629 genes. These changes in gene expression could be the result of insertion of non-baculovirus sequences such as a kanamycin resistance marker, mini-F replicon and attTn7 into the polyhedrin locus during construction of the bacmid.
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Examination of a large number of ODV cross sections shows that nucleocapsids in Ac23null and control ODVs are arranged in a similar pattern. The majority of ODVs with fewer than 11 nucleocapsids are arranged in one or two distinct patterns, examples of prevalent patterns are shown in Fig. 2(b)
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The hallmark of multiple nucleopolyhedrovirus (MNPV) is the presence of multiple nucleocapsids within each ODV. For AcMNPV, when multiple ODV nucleocapsids are delivered to host midgut cells, an alternate pathway of infection that accelerates the progression of infection by many hours may occur (Adams et al., 1977; Granados & Lawler, 1981). Thus, the results presented here suggest that Ac23null mutant's slower rate of kill may also be due to Ac23null ODVs having fewer nucleocapsids for the faster alternative pathway of infection. Packaging of multiple nucleocapsids has been suggested to offer a selective advantage over baculovirus that package a single nucleocapsid per virion (SNPV) (Washburn et al., 1999, 2003). The biological basis for multiple nucleocapsid envelopment has not been determined; however, Ac142 was recently shown to be essential for ODV nucleocapsid envelopment (McCarthy et al., 2008). The observations that Ac23null ODVs with high nucleocapsid counts are present and that the percentage of Ac23null ODVs with two to four nucleocapsids are comparable to the controls (data not shown) suggest that Ac23 somehow facilitates, but is not essential for multiple nucleocapsid envelopment. Interestingly, deletion of Sf29 from Spodoptera frugiperda MNPV reduces ODV numbers in OBs, but had no apparent affect on ODV nucleocapsid content (Simón et al., 2008).
Viruses expressing Ac23 with a C-terminal gfp fusion were used to examine Ac23 localization in infected Sf9 cells by confocal microscopy (Fig. 3). At later times (48 h post-infection, h p.i.) Ac23–gfp fluorescence frequently overlapped with the peripheral ring of DAPI staining, suggesting that Ac23–gfp may localize to the inner nuclear membrane (INM). This localization was not observed at earlier time points (e.g. 12 and 18 h p.i.). Ac23–gfp fluorescence was also detected in the cytoplasm and the plasma membrane, but not in the nucleoplasm. The spatial changes in Ac23–gfp fluorescence over time suggest that Ac23 produced early is primarily targeted to BVs via the secretory pathway, while some Ac23 produced later are diverted to ODVs via the INM. Ac23 contains both early and late promoter sequences (Pearson et al., 2002a). Ac23–gfp's perinuclear localization is consistent with Ac23 being involved in ODV envelopment as ODV envelopes are suggested to be derived from either the nuclear membrane or by de novo synthesis within the nucleus (Hong et al., 1994, 1997).
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). NLS has been identified in the majority of INM proteins (Lusk et al., 2007), and deletion of the NLS from the INM protein Heh2 results in its exclusion from the INM (King et al., 2006). Lusk et al. (2007) proposed that steady-state distribution of INM proteins to the INM and outer nuclear membrane/endoplasmic reticulum (ER) depend on factors such as the size of the extralumenal domain, the affinity of the NLS for karyopherin-
, the presence of INM retention factors, interaction with nuclear architecture, as well as the protein's biophysical properties. Thus Ac23's broad distribution to the outer nuclear membrane/ER and INM could be due to a low-affinity NLS insufficient to confer exclusive INM localization. Putative NLSs are also present in F homologues from other baculoviruses. For example, the F protein of Bombyx mori MNPV has the same NLS as Ac23, while a different NLS is present in Op21 (PRRRFNY) and SeF (PKYKRGK). In contrast to Ac23, Pearson et al. (2001) showed by Western blot analysis that Op21 is not associated with ODVs, and by immunocytochemistry that Op21 localizes to the plasma membrane, but not to the nuclear membrane. These discrepancies could be due to differences in NLSs, differences in host or other viral factors, and an amino acid identity of only 47.9 % between mature Ac23 and Op21. Alternatively, differences in experimental methodologies, such as the lack of HgCl2 for protease inhibition during Orgyia pseudotsugata MNPV ODV purification, may have prevented the detection of Op21 in ODVs. The use of non-cell permeabilizing protocols for immunostaining of Op21 may also have prevented detection of Op21 at the nuclear membrane. Since Ac23 is also associated with BVs (Lung et al., 2003), and since most BVs contain single nucleocapsids, factors in the cytosol/plasma membrane or BV nucleocapsid may inhibit Ac23 promotion of multiple capsid packaging in BV. Alternatively, unidentified factors found exclusively in the nucleus or ODV nucleocapsids could be enabling Ac23 to promote envelopment of multiple nucleocapsids into the ODV. In SNPVs, the F homologues and/or their interaction partners may have enough sequence divergence to prevent the envelopment of multiple capsids into a single ODV. The long cytoplasmic tail domain of Op21 and other F proteins relative to GP64 has been suggested to mediate the interaction with the nucleocapsid (Pearson et al., 2001). Thus the identification of host and viral interacting partners of F protein's cytoplasmic domain will help elucidate the similarities and differences in the function of these proteins. Analysis performed in cultured cells indicate Ac23null mutant BVs have retarded growth and infection kinetics, when compared with control viruses (Lung et al., 2003). This phenotype, however, may be caused by deficiencies in viral attachment or entry. Recently, Wang et al. (2008) showed the production of a SeF-pseudotyped Ac23 and gp64 double mutant was three orders of magnitude lower than SeF-pseudotyped gp64null viruses, suggesting that Ac23 has a significant effect on BV production. Together, these results suggest Ac23's affects on both ODVs and BVs impact the rate of kill.
Ac23's distinct roles in ODVs and in BVs could be due to different post-translational modifications resulting from Ac23 being targeted to the INM and to the plasma membrane via the secretory pathway, respectively. Consistent with this hypothesis, Ac23 from purified BVs had a higher Mr than Ac23 from purified ODVs on Western blots (Braunagel et al., 2003). Generation of ODVs with higher nucleocapsid numbers could facilitate rapid establishment of primary infection, while facilitating BV infection and production promotes systemic infection and OB formation. In contrast, GP64, the BV envelope fusion protein, is not associated with ODVs. These intriguing observations further support the contention that the F protein plays a different role from that of GP64 in viruses that contain both proteins. f is likely to have been acquired from a host, and has been evolving in baculovirus genomes over a longer period of time than the more recently acquired gp64 (Pearson & Rohrmann, 2002; Lung & Blissard, 2005). Thus it is not surprising to discover that F proteins may have evolved pleotrophic functions beyond envelope fusion. Existing data suggest that, although Ac23 is not essential, the multiple auxiliary functions that it performs offer selective advantages to AcMNPV.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Blissard, G. W. & Wenz, J. R. (1992). Baculovirus gp64 envelope glycoprotein is sufficient to mediate pH-dependent membrane fusion. J Virol 66, 6829–6835.
Braunagel, S. C., Russell, W. K., Rosas-Acosta, G., Russell, D. H. & Summers, M. D. (2003). Determination of the protein composition of the occlusion-derived virus of Autographa californica nucleopolyhedrovirus. Proc Natl Acad Sci U S A 100, 9797–9802.
Dai, X., Stewart, T. M., Pathakamura, J. A., Li, Q. & Theilmann, D. A. (2004). Autographa californica multiple nucleopolyhedrovirus exon0 (orf141), which encodes a RING finger protein, is required for efficient production of budded virus. J Virol 78, 9633–9644.
Engelhard, E. K., Kam-Morgan, L. N. W., Washburn, J. O. & Volkman, L. E. (1994). The insect tracheal system: a conduit for the systemic spread of Autographa californica M nuclear polyhedrosis virus. Proc Natl Acad Sci U S A 91, 3224–3227.
Granados, R. R. & Lawler, K. A. (1981). In vivo pathway of Autographa californica baculovirus invasion and infection. Virology 108, 297–308.[CrossRef][Medline]
Hong, T. S., Braunagel, S. C. & Summers, M. D. (1994). Transcription, translation, and cellular localization of PDV-E66: a structural protein of the PDV envelope of Autographa californica nuclear polyhedrosis virus. Virology 204, 210–222.[CrossRef][Medline]
Hong, T. S., Summers, M. D. & Braunagel, S. C. (1997). N-terminal sequences from Autographa californica nuclear polyhedrosis virus envelope proteins ODV-E66 and ODV-E25 are sufficient to direct reporter proteins to the nuclear envelope, intranuclear microvesicles and the envelope of occlusion derived virus. Proc Natl Acad Sci U S A 94, 4050–4055.
IJkel, W. F. J., Westenberg, M., Goldbach, R. W., Blissard, G. W., Vlak, J. M. & Zuidema, D. (2000). A novel baculovirus envelope fusion protein with a proprotein convertase cleavage site. Virology 275, 30–41.[CrossRef][Medline]
Keddie, B. A. & Volkman, L. E. (1985). Infectivity difference between the two phenotypes of Autographa californica nuclear polyhedrosis virus: importance of the 64K envelope glycoprotein. J Gen Virol 66, 1195–1200.
Keddie, B. A., Aponte, G. W. & Volkman, L. E. (1989). The pathway of infection of Autographa californica nuclear polyhedrosis virus in an insect host. Science 243, 1728–1730.
King, M. C., Lusk, C. P. & Blobel, G. (2006). Karyopherin-mediated import of integral inner nuclear membrane proteins. Nature 442, 1003–1007.[CrossRef][Medline]
Long, G., Pan, X. & Vlak, J. M. (2008). Conserved leucines in N-terminal heptad repeat HR1 of envelope fusion protein F of group II nucleopolyhedroviruses are important for correct processing and essential for fusogenicity. J Virol 82, 2437–2447.
Luckow, V. A., Lee, S. C., Barry, G. F. & Olins, P. O. (1993). Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol 67, 4566–4579.
Lung, O. & Blissard, G. W. (2005). A cellular Drosophila melanogaster protein with similarity to baculovirus F envelope fusion proteins. J Virol 79, 7979–7989.
Lung, O. Y., Cruz-Alvarez, M. & Blissard, G. W. (2003). Ac23, an envelope fusion protein homolog in the baculovirus Autographa californica multicapsid nucleopolyhedrovirus, is a viral pathogenicity factor. J Virol 77, 328–339.[CrossRef][Medline]
Lusk, C. P., Blobel, G. & King, M. C. (2007). Highway to the inner nuclear membrane: rules for the road. Nat Rev Mol Cell Biol 8, 414–420.[CrossRef][Medline]
McCarthy, C. B., Dai, X., Donly, C. & Theilmann, D. A. (2008). Autographa californica multiple nucleopolyhedrovirus ac142, a core gene that is essential for BV production and ODV envelopment. Virology 372, 325–339.[CrossRef][Medline]
Miller, L. K. (1997). Baculovirus interaction with host apoptotic pathways. J Cell Physiol 173, 178–182.[CrossRef][Medline]
Monsma, S. A., Oomens, A. G. & Blissard, G. W. (1996). The GP64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection. J Virol 70, 4607–4616.[Abstract]
Pearson, M. N. & Rohrmann, G. F. (2002). Transfer, incorporation, and substitution of envelope fusion proteins among members of the Baculoviridae, Orthomyxoviridae, and Metaviridae (insect retrovirus) families. J Virol 76, 5301–5304.
Pearson, M. N., Groten, C. & Rohrmann, G. F. (2000). Identification of the Lymantria dispar nucleopolyhedrovirus envelope fusion protein provides evidence for a phylogenetic division of the Baculoviridae. J Virol 74, 6126–6131.
Pearson, M. N., Russell, R. L. & Rohrmann, G. F. (2001). Characterization of a baculovirus-encoded protein that is associated with infected-cell membranes and budded virions. Virology 291, 22–31.[CrossRef][Medline]
Pearson, M. N., Groten, C. & Rohrmann, G. F. (2002a). Transcriptional mapping of two genes encoding baculovirus envelope-associated proteins. J Gen Virol 83, 937–943.
Pearson, M. N., Russell, R. L. & Rohrmann, G. F. (2002b). Functional analysis of a conserved region of the baculovirus envelope fusion protein, LD130. Virology 304, 81–88.[CrossRef][Medline]
Simón, O., Williams, T., Cabrera-Asensio, A., Ros, S., Gaya, A., Caballero, P. & Possee, R. D. (2008). Sf29 gene of Spodoptera frugiperda multiple nucleopolyhedrovirus is a viral factor that determines the number of virions in occlusion bodies. J Virol 82, 7897–7904.
Slack, J. & Arif, B. M. (2006). The baculoviruses occlusion-derived virus: virion structure and function. In Advances in Virus Research, vol. 68, pp. 99–165. Edited by K. Maramorosch, A. Shatkin & B. C. Bonning. New York: Academic Press.
Theilmann, D. A. & Blissard, G. W. (2008). Baculoviruses: molecular biology of nucleopolyhedroviruses. In Encyclopedia of Virology, 3rd edn, pp. 254–265. Edited by B. W. J. Mahy & M. H. V. van Regenmortel. New York: Academic Press.
Wang, M., Tan, Y., Yin, F., Deng, F., Vlak, J. M., Hu, Z. & Wang, H. (2008). The F-like protein Ac23 enhances the infectivity of the budded virus of gp64-null Autographa californica multinucleocapsid nucleopolyhedrovirus pseudotyped with baculovirus envelope fusion protein F. J Virol 82, 9800–9804.
Washburn, J. O., Lyons, E. H., Haas-Stapleton, E. J. & Volkman, L. E. (1999). Multiple nucleocapsid packaging of Autographa californica nucleopolyhedrovirus accelerates the onset of systemic infection in Trichoplusia ni. J Virol 73, 411–416.
Washburn, J. O., Chan, E. Y., Volkman, L. E., Aumiller, J. J. & Jarvis, D. L. (2003). Early synthesis of budded virus envelope fusion protein GP64 enhances Autographa californica multicapsid nucleopolyhedrovirus virulence in orally infected Heliothis virescens. J Virol 77, 280–290.[CrossRef][Medline]
Westenberg, M., Wang, H., IJkel, W. F. J., Goldbach, R. W., Vlak, J. M. & Zuidema, D. (2002). Furin is involved in baculovirus envelope fusion protein activation. J Virol 76, 178–184.
Yin, F., Wang, M., Tan, Y., Deng, F., Vlak, J. M., Hu, Z. & Wang, H. (2008). A functional F analog of Autographa californica nucleopolyhedrovirus GP64 from Agrotis segetum granulovirus. J Virol 82, 8922–8926.
Zuleger, N., Korfali, N. & Schirmer, E. C. (2008). Inner nuclear membrane protein transport is mediated by multiple mechanisms. Biochem Soc Trans 36, 1373–1377.[CrossRef][Medline]
Received 24 November 2008;
accepted 12 February 2009.
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