Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102, USA
Correspondence
Loy E. Volkman
lvolkman{at}nature.berkeley.edu
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ABSTRACT |
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Present address: State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.
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INTRODUCTION |
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During the early stages of AcMNPV pathogenesis in penultimate larvae of the permissive hosts, Trichoplusia ni, Spodoptera exigua and Heliothis virescens, secondary infection by BV of even a single tracheolar cell leads to overwhelming infection and death (Engelhard & Volkman, 1995; Washburn et al., 1995
; Zhang et al., 2004
). Host larvae, however, can clear primary infection by sloughing ODV-infected midgut cells, a defensive response that varies qualitatively among host species and temporally within instars of a single species (Inoue & Miyagawa, 1978
; Briese, 1986
; Keddie et al., 1989
; Engelhard & Volkman, 1995
; Washburn et al., 1995
, 1998
, 1999
, 2003
). If a host can eliminate ODV-infected midgut cells prior to BV transmission to secondary targets, systemic infection fails and the insect survives. The ability to slough infected cells increases as larvae age and this response is an important component of developmental resistance (Engelhard & Volkman, 1995
; Washburn et al., 1998
). It is not surprising, therefore, that selection has favoured an AcMNPV infection strategy that incorporates both the timely onset of primary midgut infection and the rapid transmission of BV to nearby tracheolar cells. Two classes of viral factors impact these events and contribute to virulence of per os infection without affecting virulence of BV.
The so-called pif (per os infectivity factor) genes of AcMNPV and their homologues are representative of the first class of factors. The pif genes are essential for establishing midgut infection and are highly conserved among all sequenced baculoviruses. Moreover, their absence is inconsequential to BV infectivity. AcMNPV p74, the founding member of this class, was described over a decade ago (Kuzio et al., 1989). Two more genes were identified subsequently in Spodoptera littoralis NPV and S. exigua NPV; these were SlNPV ORF 7 (pif) and SeNPV ORF 35 (pif-2), homologues of Ac119 and Ac022, respectively (Kikhno et al., 2002
; Pijlman et al., 2003
). AcMNPV p74 and pif encode ODV structural proteins and AcMNPV P74 is involved in the specific binding of ODV to midgut cells (Haas-Stapleton et al., 2004
). The functions of PIF and PIF-2 are still unknown.
Members of the second class of factors promote rapid transmission of BV to tracheolar cells and thereby enhance virulence of infection initiated per os. This class of factors is diverse and includes PE38 (Milks et al., 2003) and GP64 expressed early, prior to virus replication (Washburn et al., 2003
; Zhang et al., 2004
). Such factors are of interest because, whilst not essential for in vivo infection, they fine-tune virulence in host insects and their effects may vary among susceptible species.
Recently, Lapointe et al. (2004) reported that two members of the 11K gene family, Ac145 and Ac150, enhance virulence of AcMNPV occlusions without affecting BV infectivity. The 11K genes' are predicted to encode small proteins of 90110 aa that contain hydrophobic N termini and single copies of the so-called C6 motif or peritrophin-A domain, thought to bind chitin (Dall et al., 2001
; Tellam et al., 1999
). The C6 motif also occurs within proteins encoded by diverse species within the ecdysozoan clade. Such proteins include various chitinases, mucins, peritrophins and other proteins incorporated within peritrophic membranes lining the guts of caterpillars and basal laminae of insect tracheae (Dall et al., 2001
). Between the hydrophobic N terminus and the peritrophin-A domain, Ac150 also encodes a short stretch of basic and then acidic amino acids, with an RGD sequence separating the two. This is of note because RGD is an integrin-binding domain, and integrins make transmembrane connections to the cytoskeleton and may activate cellular signalling pathways (Hynes, 2002
).
All baculovirus species infecting lepidopteran or hymenopteran larvae that have been sequenced to date contain one or more of the 11K homologues, and the apparent affinity of the proteins for chitin suggests a role during primary infection, possibly at the peritrophic-membrane interface. Lapointe et al. (2004), however, were unable to demonstrate chitin-binding activity for either Ac150 or Ac145, nor were they able to show that the absence of Ac150 alone had any adverse effect on virulence. The latter was a surprising result because deletion of Ac145 alone, or together with Ac150, reduced virulence in orally infected H. virescens larvae by 6- and 39-fold, respectively. Moreover, Ohkawa (1997)
found that deletion of the Bombyx mori NPV homologue of Ac150, BmNPV ORF 126, reduced virulence in orally infected B. mori larvae. Our long-term interest in baculovirus pathogenesis in vivo led us to revisit the question of a possible role for Ac150 in oral infection. We generated an Ac150 deletion mutant, Ac
150, in which the hsp70/lacZ reporter cassette was inserted into the Ac150 ORF. In comparative bioassays with wild-type occlusions, we found that virulence of Ac
150 occlusions was decreased significantly in larvae of all three species tested (H. virescens, T. ni and S. exigua). Comparison of pathogenesis revealed that the only discernible role of Ac150 was to enhance establishment of primary midgut-cell infection, rather than to facilitate rapid transmission of BV. In this regard, Ac150 is in the same class as the pif genes.
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METHODS |
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Virus preparation.
Four viruses were used in the experiments described in this report: Ac150 and Ac
150R (described above), AcMNPV-hsp70/lacZ (Engelhard et al., 1994
) and AcMNPV E2, the parental wild-type virus (Smith & Summers, 1978
). AcMNPV-hsp70/lacZ BV and ODV both have wild-type virulence levels in vivo (Engelhard et al., 1994
; Washburn et al., 1995
). Occlusion populations of each virus were generated from infected Sf-9 cells, harvested at 5 days post-infection and partially purified by sucrose-gradient centrifugation (Summers & Smith, 1987
). Occlusions were suspended in a neutrally buoyant solution of glycerine and water (3 : 2, v/v) and quantified by using a haemocytometer (Washburn et al., 1995
). ODV used in bioassays was liberated from occlusions by exposure to dilute alkaline saline and neutralized with 1 M Tris buffer. Undissolved occlusions and empty calyxes were removed by pelleting at 2000 g for 10 min; subsequently, ODV in the supernatant was banded by density-equilibrium centrifugation on continuous 2559 % sucrose gradients for 1 h at 90 000 g. The resulting ODV bands were harvested and pooled, diluted 1 : 3 in PBS and pelleted at 90 000 g for 30 min. ODV pellets were collected in a minimal volume of PBS and aliquots of the two ODVs were quantified by using a BSA protein assay (Pierce) (Haas-Stapleton et al., 2004
). To stabilize ODV, we added BSA to a final concentration of 10 µg ml1 and dispensed small aliquots, which were stored at 20 °C until use. For bioassays, ODV inocula were thawed and diluted to the appropriate concentration in PBS immediately before use. BV was harvested at 72 h post-infection from the supernatant of Sf-9 cells infected with each of the viruses and titrated by immunoplaque assay on Sf-9 cells (Volkman & Goldsmith, 1982
). For bioassays, BV stocks were diluted to the appropriate concentration with PBS and BSA (10 µg ml1, final concentration). Stocks of all viruses were stored at 4 °C in the dark until use.
ODV content and nucleocapsid-packaging characteristics.
To compare ODV content of occlusions and nucleocapsid-packaging characteristics of Ac150, Ac150
R and AcMNPV wild-type, ODV was harvested from 1·8x109 occlusions of each virus and equal volumes were subjected to density-equilibrium centrifugation as described above. The banding patterns of each virus were compared by visual inspection and photographed prior to fractionation with an ISCO density-gradient fractionator (model 640); A254 was measured and the relative areas beneath the peaks were calculated.
Insects and virus inoculation.
For all experiments, we used fourth-instar larvae of H. virescens, T. ni or S. exigua reared from eggs provided by the USDA Western Cotton Research Laboratory, Phoenix, AZ, USA. All larvae were reared in groups at 28±2 °C on a modified wheatgerm diet (Stoneville) until the onset of quiescence at the end of the third instar, indicative that larvae are preparing to moult to the fourth instar. For some experiments, large numbers of quiescent third instars were held between 4 and 15 °C until sufficient insects of the same developmental stage were available for testing (Washburn et al., 1995). Each larva was inoculated individually with occlusions, ODV or BV in 0·51·5 µl aliquots, using a microapplicator (Burkhard) fitted with a blunt- or sharp-tipped 32-gauge needle (for oral and intrahaemocoelic inoculations, respectively) mounted on a 1 ml tuberculin syringe [for details, see Washburn et al. (1995)
]. For one experiment, suspensions of Ac
150 and AcMNPV wild-type occlusions additionally contained 1 % M2R dissolved in DMSO or just DMSO for control inocula (see Washburn et al., 1998
).
Occlusions and ODV were administered orally by inserting the blunt-tipped needle through the mouth until the tip was well within the midgut lumen. BV was injected into the haemocoel by inserting the sharp-tipped needle through the planta of one of the prolegs, as described previously (Washburn et al., 1995). Larvae were inoculated orally within 15 min after moulting to the fourth instar (i.e. newly moulted larvae or 40) or 16 h after the moult (416). For all BV inoculations, we used fourth-instar larvae 24±6 h post-moult. After inoculation, test larvae were maintained individually in 25 ml plastic cups containing diet ad libitum in a growth chamber at 28±2 °C.
Bioassays and time-course experiments.
Bioassays were performed to compare the virulence of Ac150 and Ac
150R occlusions, ODV and BV relative to those of AcMNPV wild-type in H. virescens, T. ni and S. exigua. For these and all additional assays described below, individual larvae were inoculated with varying dosages of inoculum (n=2232 larvae per dosage) administered orally or intrahaemocoelically as described above. All larvae were maintained until pupation or death from polyhedrosis disease, and baculovirus-induced mortality was confirmed by microscopic examination (400x) of cadaver tissues for occlusions. For each of the three species, we established the oral dosemortality relationships for Ac
150 by inoculating 40 larvae with various occlusion numbers. The dosemortality relationship for each species was quantified by the method of least squares and regression equations were used to calculate the LD50 for each species. These values were compared with the LD50 of 40 larvae inoculated with wild-type AcMNPV occlusions. A minimum of five assays was used to calculate the wild-type LD50 for each species.
M2R is a stilbene-derived optical brightener known to bind chitin and damage the peritrophic membrane (Wang & Granados, 2000). To determine whether M2R affected the virulence of Ac
150, 40 and 416 T. ni were inoculated orally with 50 and 10 occlusions of Ac
150 or AcMNPV wild-type virus, respectively, in the presence or absence of 1 % M2R. These dosages were predicted to generate final mortalities of between 30 and 50 %, levels sufficiently low to quantify M2R mortality enhancement, if present, for both developmental cohorts. Additional bioassays were conducted to compare the virulence of Ac
150 and AcMNPV wild-type ODV in H. virescens and T. ni. In these experiments, identical dosages of between 0·1 and 100 pg of either Ac
150 or wild-type ODV were administered orally to larval cohorts of each species.
To evaluate the effects of deleting Ac150 on pathogenesis in vivo, we conducted a time-course experiment using 40 S. exigua inoculated with occlusions of either Ac150 or AcMNPV-hsp70/lacZ. In this experiment, we used a dosage for each virus (determined from bioassays described above) that yielded final mortalities of
85 %. At 4 h intervals during the first 24 h post-inoculation (p.i.), cohorts of between 26 and 32 larvae from each viral treatment were dissected and their midguts and associated tissues were removed. These tissues were processed to elucidate the blue
-galactosidase reporter signal and examined using stereo (1050x) and/or compound microscopy (100480x) in order to quantify infection foci and identify infected cell types (Engelhard et al., 1994
; Washburn et al., 1995
, 2003
). For each host species, an additional cohort of 32 insects was inoculated orally with Ac
150 or AcMNPV-hsp70/lacZ to confirm that the dosages used yielded the same final mortality.
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RESULTS |
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DISCUSSION |
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Interestingly, whilst the occlusions of Ac150 were less infectious orally than wild-type occlusions, the isolated ODVs had the same infectivity. These results suggested that exposure to dilute alkaline saline inactivated Ac150 or that it was lost during ODV purification, or both. The lack of Ac150 activity associated with purified ODV is consistent with the findings of Braunagel et al. (2003)
, who found no evidence of Ac150 in AcMNPV ODV by using multiple analytical approaches.
To test whether Ac150 facilitated passage of ODV across the peritrophic membrane (which could explain the reduced efficiency of primary infection), we inoculated Ac150 occlusions in the presence of the stilbene-derived optical brightener, M2R, which is known to release proteins from the peritrophic membrane and cause holes to form (Wang & Granados, 2000
). In our experiments, addition of 1 % M2R failed to enhance mortality levels generated by the deletion mutant to expected levels if Ac150 worked by a similar mechanism to M2R. This result was consistent with the lack of chitin-binding activity reported by Lapointe et al. (2004)
.
It is possible that Ac150 has a role in signalling. Integrins are known to propagate signalling when bound by a ligand, and Ac150 has an RGD integrin-binding motif in the middle of a cluster of charged amino acids. Alternatively, microvilli of midgut cells are coated heavily with glycosylated proteins and the peritrophin-A domain of Ac150 may bind one of these. A number of membrane-bound receptors for growth factors and cytokines are glycosylated, and evidence has indicated that oligosaccharide moieties are crucial for the functions of some of those receptors (Takahashi et al., 2004). Whether or not Ac150 binds to midgut cells at all, however, remains to be determined.
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ACKNOWLEDGEMENTS |
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Received 28 January 2005;
accepted 14 March 2005.