1084
Current Drug Targets, 2007, 8, 1084-1095
Molecular Biology of the Baculovirus Occlusion-Derived Virus Envelope
Sharon C. Braunagel* and Max D. Summers
Department of Entomology, Texas A&M University, Texas Agricultural Experiment Station, College Station, TX 77843, USA
Abstract: Study of the biology of the occlusion-derived virus (ODV) of the baculovirus Autographa californica nucleopolyhedrovirus
provides opportunities to reveal new discoveries into the mechanism of several cellular pathways. The synchronous pulse of multiple
ODV envelope proteins that integrate into the endoplasmic reticulum (ER) and traffic to the nuclear membranes on their way to the ODV
envelope provide a unique tool to study the mechanisms of integral membrane protein trafficking from the ER to the outer and inner nuclear membrane. Studies of the formation of virus-induced, intranuclear membrane microvesicles provide insight on mechanisms that alter fluidity and regulate budding of the inner nuclear membrane. Since ODV is specially adapted for primary infection of the insect gut,
studies of the structure and function of ODV envelope proteins reveals insights on the mechanism of viral invasion of the gut and this
knowledge is fundamental for the development of new strategies for insect control.
This review focuses on recent advances in understanding the source of the ODV envelope and the molecular events that sort and traffic
integral membrane proteins from the ER to the ODV envelope. The composition of ODV is reviewed, however it is worth noting that the
function of many ODV proteins are currently unknown.
I. OVERVIEW OF BACULOVIRUS BIOLOGY
Autographa californica nucleopolyhedrovirus (AcMNPV) undergoes a biphasic life cycle in its lepidopteran host. Progeny nucleocapsids that assemble in the nucleus have two fates; during the
early phase of infection, approximately 16% of the intracellular
copies of viral DNA are targeted to nucleocapsids that mature at the
cell surface to produce budded virus (BV) [1], while the remaining
nucleocapsids acquire their envelope from membranes within the
nucleus. After envelopment in the nucleus, virions are incorporated
within a protein crystal called the viral occlusion and these virions
are termed the occlusion-derived virus (ODV). The occluded virus
is highly stable, which allows it to persist in the environment and
promote seasonal infections of susceptible insects.
II. SOURCE OF INTRANUCLEAR MICROVESICLES
Small membrane vesicles appear within the infected cell nucleus in the same temporal sequence as the appearance of progeny
ODV. In 1973, Stoltz et al. [2] proposed that these microvesicles
were formed as a result of de novo intranuclear membrane morphogenesis. Considering the knowledge of membrane morphogenesis at
the time, this was an appropriate proposal. Today however, we know
that while all major classes of membrane phospholipids [phosphatidylcholine (PC), phospatidylethanolamine (PE), phosphatidylserine
(PS), and phosphatidylinositol (PI)] are present within the nucleus,
they are associated with the nuclear matrix and are distinct from the
lipids of the nuclear envelope. Nuclear synthesis of PC has been
documented, however it is not related to synthesis of the nuclear
envelope [3-5]. To date, de novo membrane morphogenesis of the
nuclear envelope has not been demonstrated.
The current hypothesis is that the formation of the intranuclear
membranes is the result of budding of discrete regions of the inner
nuclear membrane (INM) into the nucleoplasm. While the overall
morphology of the infected cell nuclear envelope remains intact,
regions appearing more fluid, and in close association with foci of
membrane microvesicles were reported as early as 1969 [6]. Electron microscopy shows microvesicles appearing near perturbed
regions of the nuclear envelope (Fig. (1); closed arrow), and accumulating in foci within the nucleoplasm (Fig. (1); double arrow).
Consistent with the EM observation, confocal analyses of infected
*Address correspondence to this author at the Department of Entomology,
Texas A&M University, Texas Agricultural Experiment Station, College
Station, TX 77843, USA; Tel: (979) 847-9037; Fax: (979) 845-6305;
[email protected]
1389-4501/07 $50.00+.00
Fig. (1). Electron microscopy of a 36 h p.i. AcMNPV infected cell. At
discrete regions, the nuclear envelope is perturbed. At these regions, microvesicles appear (solid arrow). The newly formed microvesicles appear to
migrate more interior into the nucleus (double arrow). Proteins destined for
the ODV envelope accumulate in these intranuclear microvesicles.
cells show that microvesicles initially appear at the nuclear periphery and as infection progress, these foci of membrane vesicles migrate toward the interior of the nucleus [7]. If the INM is indeed the
intermediary in the production of the virus-induced intranuclear
microvesicles, then it must proliferate during infection. A ready
source of membranes that would allow such proliferation are the
contiguous membranes of the outer nuclear membrane (ONM) and
ER, however analysis suggests that the lipid composition of these
© 2007 Bentham Science Publishers Ltd.
Molecular Biology of the Baculovirus Occlusion-Derived Virus Envelope
membranes must be significantly remodeled before being utilized
as a precursor of the ODV envelope [8].
AcMNPV infection arrests cells in late G2/early M phase of the
cell cycle [9]. This observation is significant because during this
phase of the cell cycle in normal cells, the INM has been shown to
migrate into the nucleus to produce various types of intranuclear
membrane structures. The nuclear envelope of oocytes derived from
the beetle Zenos peckii Kirby, becomes highly convoluted during
late prophase/early metaphase and the INM invaginates and buds
into the nucleus to produce an abundant quantity of intranuclear
membrane vesicles [10]. In mitotically active Drosophila melanogaster brain, stem and follicle cells, the nuclear envelope fenestrates and the INM serves as a source of the envelope that forms
around the mitotic spindles [11-13]. Thus, these observations are
consistent with the idea the during viral induced cell cycle arrest,
the INM becomes more fluid and serves as the source of the intranuclear membrane microvesicles.
Fluxes in nuclear calcium are known to regulate nuclear envelope breakdown. The production of many types of membrane vesicles includes mobilization and binding of inositol 1,4,5-trisphosphate (InsP3) with its receptor, with a subsequent release of sequestered calcium (Ca2+). The InsP3 receptor is present on the nuclear
envelope [14] and nuclear PI turnover is regulated by cell cycle
phase with InsP3 release at it highest levels during G2/M phase [15].
If the inositol pathway and subsequent mobilization of Ca2+ is utilized to promote INM budding and production of intranuclear microvesicles in infected cells, then one would expect intranuclear
calcium levels to increase during infection. A preliminary study
using Indo I dye to label Ca2+ and laser cytometry to quantitate
intracellular Ca2+ shows that the nuclei of AcMNPV infected cells
contain approximately 15% more intranuclear Ca2+ than wild type
nuclei (Fig. (2A) and (2B)). Within the infected cell nucleus, the
increase in intranuclear Ca2+ is present in regions consistent with
foci of virus-induced microvesicles (Fig. (2C)). Thus, not only is
the INM the most likely source of the intranuclear membrane vesicles, but also Ca2+ fluxes may regulate budding of the INM and
formation of these membranes.
Fig. (2). Sf9 and infected cells labeled with Indo 1 dye. A and B. Laser
cytometry localization and quantitation of intranuclear Ca2+. The scale on
the right shows an increase in Ca2+ from bottom to top (red images show
high levels of Ca2+). C. Z-stack series of confocal images through the nucleus of an infected cell showing location of Indo-1 labeled Ca2+. Strong
labeling is detected within the cytoplasmic region, consistent with ER
stores, and foci of labeling is detected within the nucleus, which is consistent with foci of virus induced intranuclear microvesicles.
Current Drug Targets, 2007, Vol. 8, No. 10
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III. MICROVESICLES AND VIRAL ENVELOPMENT
No recombinant virus has been described that has a phenotype
of decreased intranuclear microvesicles. However, several mutant
viruses exhibit phenotypes that include altered morphology of these
membranes. In ‘few polyhedra” (FP) mutants, intranuclear membranes assume convoluted shapes that are extreme examples of
normal structures [16-18]. The causes of these structural alterations
are not known, and since deletion of FP25K results in changes in
the abundance and temporal synthesis of several viral proteins, they
are not easy to discern [19]. We note that in FP-mutant infected
cells, ODV envelopment is significantly decreased. This suggests
that the basis for altered morphology of the intranuclear membranes
also affects the efficiency of ODV maturation.
Formation of Microvesicles and Viral Envelopment are Independent Events
During studies of the ODV envelope protein ODV-E56 (E56),
an unanticipated observation was made. When a second copy of
E56 was expressed under the control of the polyhedrin promoter
(increased levels and delayed expression of E56 compared to wild
type), there was a major affect on ODV maturation [20]. When
analyzed by confocal microscopy, the pattern of E56 localization
appeared normal; E56 localized to microvesicles and co-localized
with other envelope protein markers. Electron microscopy showed
that microvesicles were produced in apparently normal quantities
and ODV envelope proteins were incorporated within them. In
addition, nucleocapsids associated with the microvesicles in a normal fashion (Fig. (3A-D)). However, electron microscopy observation of many nuclei also revealed that the presence of mature, enveloped ODV was rare in the recombinant virus infected cells. Instead, nucleocapsids accumulated in abundant quantity within the
virogenic stroma and were present in stacked arrays within the nucleoplasm (Fig. (3B) and (3D)). This result was also obtained with
two other recombinant viruses, E56-GFPpolh (C-terminal in-frame
fusion with green fluorescent protein) and E56+polh (polyhedrin gene
is unaffected, the recombinant virus has an occlusion positive phenotype). Thus, it appears that altering the stoichiometry of E56
relative to other viral proteins is sufficient to diminish ODV maturation. These results prompt two conclusions: i) the formation of
microvesicles and viral envelopment are two distinct events, with
the formation of microvesicles preceding envelopment; and ii) E56
plays a vital role in the maturation and envelopment of ODV. Since
ODV envelopment is blocked in the presence of increased quantities of E56, these recombinant viruses provide a unique tool to
study the biochemical events of integral membrane protein trafficking prior to viral envelopment.
IV. TARGETING OF INTEGRAL MEMBRANE PROTEINS
TO THE ODV ENVELOPE
Identification of an INM-Sorting Motif
A sequence consisting of the N-terminal amino acids of the
ODV envelope protein ODV-E66 (E66) is sufficient to traffic fusion proteins to intranuclear membranes and the ODV envelope.
This sequence functions as an N-terminal signal anchor and has
been termed an INM-sorting motif (INM-SM), [7,21]. It consists of
two distinct features:
1. A hydrophobic domain of approximately 18 amino acids.
2. Within 4-8 amino acids from the end of the hydrophobic domain is a positively charged amino acid that is positioned in the
cyto/nucleoplasm.
The relevant sequences of ODV-E66 and fusion marker proteins are shown in Fig. (4).
When E66 and other ODV envelope proteins are expressed
under their native promoters, they locate quickly to the intranuclear
microvesicles and ODV envelope, thus it is difficult to detect intermediates in the trafficking pathway. However, the presence of
1086 Current Drug Targets, 2007, Vol. 8, No. 10
Braunagel and Summers
Fig. (3). ODV-E56polh virus, 48 h p.i. probed with antibody to E56 and gold-labeled secondary antibody. B and D are enlargements of the regions outlined in A and C. Note the abundance of intranuclear microvesicles and their association with nucleocapsids. The appearance of normal enveloped ODV is
very rare. m = microvesicles; v = virogenic stroma; solid arrow = nucleocapsid; open arrow = gold label of E56 in microvesicles.
ODV-E66
23-βgal
SM-GFP
+
+
MSIVLIIVIVVIFLICFLYLSNSNNKNDANKNN.....E66....*
+
MSIVLIIVIVVIFLICFLYLSNSGTKDPVVL-βgalactosidase...*
+
+
MSIVLIIVIVVIFLICFLYLSNSNNKNDANKPVEL.....GFP....*
79 kDa
110 kDa
27 kDa
Fig. (4). ODV-E66 derived INM-SM and INM-SM fusion constructs. Recombinant viruses were developed expressing these constructs under the control of
the polyhedrin promoter. Each of these proteins accumulated in the ER, ONM, INM, intranuclear microvesicles and ODV envelope. If the N-terminal 33
amino acids of E66 were deleted, E66 no longer associated with membranes [7].
small quantities of ODV envelope proteins at the ER membrane,
outer nuclear membrane (ONM) and INM suggest that these continuous membranes function in the trafficking pathway of these
proteins to the viral-induced intranuclear membranes. When recombinant viruses are produced with an additional copy of E66polh,
E25polh or a variety of fusion proteins (like those shown in Fig. 4),
the marker proteins accumulate in the membranes of the ER, ONM,
pore membrane and INM. Moreover, the formation of microvesicles, progeny ODV, and other observable viral phenotypes in these
recombinant virus infected cells are similar to wild type. This suggests that large quantities of E66 or SM-fusions are not visibly altering events of infection but rather “backing up” in their normal
pathway [7].
The structural features of the E66-derived INM-SM are shared
among resident INM proteins from a variety of species and other
ODV envelope proteins [22]. Studies of INM-SM-fusion marker
proteins have been useful in trapping and identifying transient intermediate states during protein movement from the ER membrane
to the nuclear envelope. These studies have revealed insights into
similar events occurring in uninfected cells during integral membrane trafficking to the nuclear envelope.
Similarity of the N-Terminal Sequences of ODV Proteins
A perusal of N-terminal sequences of baculovirus proteins reveals a number of proteins that contain single transmembrane sequences located at the N-terminus of the protein. Except for E25,
the AcMNPV orf’s that encode such a N-terminal hydrophobic
sequence also contain a positively charged amino acid closely associated with it (Figs. (4 and 5)). Thus, all of these proteins contain an
INM-SM-like sequence. Based upon the known function of the
Molecular Biology of the Baculovirus Occlusion-Derived Virus Envelope
ODV-E25
Current Drug Targets, 2007, Vol. 8, No. 10
1087
MWFIVLLIVLLILFYLYWTNALNFNSLTESSPSLGQS...
(orf 94)
ODV-E66
MSIVLIIVIVVIFLICFLYLSNSNNK
KNDANK
KNNAFI...
(orf 46)
pif 2
HIGH
HAQQDYN...
MYRVLIVFFLFVFLYIVYQPFYQAYLH
(orf 22)
p91
KRL...
MMSGVMLLMLAIFLIIAFTLMYLAIYFEFDETTFTK
(orf 83)
ORF 91
KILDDIQELYPNP...
MWYIIVIIIIIILNLFIIILLTLK
KNH
HHPFLH
HRIETLIQ...
ODV-E28 MLSIMLAIVFVLFVLIYLIISIK
(orf 96)
pif 3
KFVQK
KFILQDAYMH
H...
MLNFWQILILLVIILIVYMTFK
(orf 115)
pif 1
MHFAIILLFLLVIIAIVYTYVDLIDVH
HHEEVR
RYPIT...
(orf 119)
ORF 150
KSK
KRGDDDESDD...
MLKPNMLIIIFIIVCLFSIIIFTVLK
Fig. (5). N-terminal sequences encoded by AcMNPV orf’s. The region shown is yellow is the single N-terminal transmembrane sequence. Positively
charged amino acids flanking the transmembrane sequence are in highlighted in red.
INM-SM sequence, one would predict that all of these orf’s encode
proteins that locate to the ODV envelope. With the exception of orf
91 (which remains uncharacterized), this has proved to be the case.
Edman degradation and N-terminal sequencing of E66 and E25
show that these N-terminal sequences function as non-cleaved signal anchors. By extrapolation, we would predict that the N-terminal
sequences of the other ODV envelope proteins also remain uncleaved in the mature protein and function as non-cleaved Nterminal signal anchors.
Regulation of Trafficking of ODV-E66 or INM-Directed Integral Membrane Proteins: Sorting Begins During Co-Translational Membrane Integration
At first glance it is difficult to discern what property makes the
INM-SM functionally unique: the calculated free energies for
membrane integration of transmembrane sequences from viral and
cellular INM-SM like sequences vary widely; and the presence of
charged amino acids flanking a transmembrane sequence are not
unique to INM-directed proteins. To address this, Saksena et al.
[21] analyzed the initial stages of ER membrane targeting and integration. By showing that membrane integration of E66 is dependent
upon interaction of the N-terminal transmembrane sequence and the
signal recognition particle, Saksena determined that E66 co-translationally inserted into the ER membrane. Using photocrosslinking,
the transmembrane sequence within the INM-SM was shown to
occupy a different binding site within the translocon, and interact
differently with translocon proteins than transmembrane sequences
of non-INM directed proteins. Since the transmembrane sequences
of E25 and E66 interact in the same manner with proteins of the
translocon, Saksena concluded that the first cotranslational sorting
event is mediated by interactions between the INM-SM transmembrane sequence and proteins of the translocon. Braunagel et al. [22]
demonstrated that the spacing between the charged amino acids and
the end of the transmembrane sequence is an important feature of
the INM-SM sequence and if altered, the efficiency of protein targeting to the INM decreases.
Once the transmembrane sequence is properly positioned within
the translocon, chemical crosslinking experiments show that the
appropriately spaced positively charged amino acid is positioned
proximal to two cellular proteins. One of these has a molecular
mass of approximately 25 kDa, but its identity is unknown. The
second protein has a molecular mass of 16 kDa and is an isoform of
cellular importin- that comprises the C-terminal armadillo helicalrepeat domains 7–10 [23]. This protein has been named importin-16. Importin--16 associates with the translocon and is spatially
positioned adjacent to the translocon protein Sec61. This position
presumably allows it to survey, discriminate and bind with INMSM containing protein nascent chains as they enter the translocon.
Thus the recognition of the INM-SM by importin--16 occurs before membrane integration. Importin--16 remains proximal to the
INM-SM sequence even after it is released from the translocon and
integrated into the ER membrane. This part of the sorting pathway
also appears to be utilized by cellular INM proteins and mammalian
cells encode a protein similar to Sf9 importin--16 [24].
Potential Regulation of Trafficking of ODV-E66 or SM-fusion
Marker Proteins: Sorting Continues After Membrane Integration
Chemical crosslinking studies show that during infection, the
positively charged amino acids in the INM-SM are proximal to two
viral proteins: FP25K and BV/ODV-E26 (E26). Unlike the cellular
protein importin--16, FP25K or E26 do not recognize the INMSM sequence until it has been released from the translocon and
integrated into the ER membrane. The identification of an interaction of the E66-derived INM-SM with FP25K is consistent with
previous results showing that FP25K plays some role in E66 trafficking to intranuclear microvesicles [19,25]. The observation that
deletion of FP25K affects the trafficking of E66 to the ODV envelope more severely than that observed for E25 may suggest that the
lack of positively charged amino acids in the E25 N-terminal sequence allows E25 to utilize a somewhat different molecular pathway during its trafficking to the ODV envelope.
Chemical crosslinking shows that the positively charged lysine
within the INM-SM is proximal to E26 and immunoprecipitation
studies demonstrate that E26 and the cellular protein importin--16
share epitopes [23]. It remains to be determined if the N-terminal
1088 Current Drug Targets, 2007, Vol. 8, No. 10
INM-SM-like sequence of the other ODV-envelope proteins
crosslink to FP25K and E26, or the cellular protein importin--16.
Such knowledge would reveal if these proteins transit from the ER
membrane to the intranuclear microvesicles through a common
pathway, or if other regulatory features function during ER to INM
trafficking.
While the role in trafficking of FP25K and E26 are unknown, it
has been proposed that these proteins facilitate movement from the
ER to the nuclear envelope by interacting with cellular motors. This
interaction with motor proteins would provide directionality and
force to move the INM-SM complex within the lipid matrix toward
the nucleus [23]. Clearly future studies designed to trap and identify
other proteins participating in the trafficking of ODV envelope
proteins will reveal a great deal about the mechanism of integral
membrane protein trafficking from the ER toward the INM and in
infected cells, to viral induced intranuclear membranes.
V. COMPOSITION OF THE OCCLUSION-DERIVED VIRUS
ENVELOPE: LIPIDS
A lipid analyses of the ODV envelope showed that it is densely
packed with protein [8]. This was demonstrated by a phosphate/protein ratio of 0.11 (mol:g) and a cholesterol/protein ratio
of 0.02 (g/g). The lipid analysis showed that the ODV envelope
is also densely packed with cholesterol (cholesterol/phosphate ratio
of 0.58 mol/mol). If intranuclear microvesicles are derived from the
INM and serve as direct precursors of the ODV envelope, then the
lipid analysis is surprising. One would expect these ratios to more
closely reflect the composition of intracellular membranes. Yet
these ratios are very different from that obtained from an analysis of
intact nuclei of Sf9 cells (predominantly ER and nuclear envelope).
Sf9 nuclei have a cholesterol/phosphate ratio of 0.13 (mol/mol) and
this ratio is very close that observed with ER membranes from other
cell types [26]. These findings prompt the hypothesis that baculovirus infection induces a remodeling of the lipid composition of those
domains of the nuclear envelope that serve as the source of the
virus induced intranuclear microvesicles and ODV envelope.
If baculovirus is remodeling the nuclear envelope by increasing
its concentration of cholesterol, then this should be detected using a
semi-permeabilization assay based upon the detergent digitonin,
which selectively extracts cholesterol. In this assay, the high concentration of cholesterol in the plasma membrane of a cell allows
the plasma membrane to be selectively permeabilized by low concentrations of digitonin, leaving the ER/nuclear envelope remain
intact [27]. When this assay was tested on Sf9 cells, digitonin concentrations ranging from 75-150 g/ml permeabilized the plasma
membrane and left the ER/nuclear envelope intact. A similar result
was obtained for AcMNPV infected cells at early time points post
infection. However at approximately 18 h p.i., dramatic differences
were noted. At this time and continuing throughout infection, digitonin concentrations as low as 50 g/ml fully permeabilized the
infected cell nuclei, allowing dextrans as large as 250 kDa free
access to the nucleoplasm (unpublished data). This is strong evidence that the increase in cholesterol in the ODV envelope reflects
virus regulated lipid remodeling of the nuclear envelope. However,
there are other possibilities that would explain a permeabilized
nuclear membrane at late times during infection. It is possible that
virus-induced morphological changes in the nuclear envelope result
in regions that demonstrate no practical exclusion barrier to the
nucleoplasm; or that fundamental characteristics of nuclear pores
are being altered during infection. We think these explanations are
unlikely because Jarvis et al. [28] showed that when the nuclear
localization signal of the polyhedrin protein was altered, polyhedrin
protein did not freely migrate into the nucleus, rather occlusions
assembled in the cytoplasm. This suggests that a diffusion barrier at
the nuclear envelope is maintained throughout infection.
The potential ability of baculovirus to remodel nuclear membranes may have significant impact on regulation and transport of
Braunagel and Summers
ODV envelope proteins from the ER to the nucleus. The entire
question of the role and mechanism(s) utilized by baculovirus to
control lipid composition of the ODV is fertile ground with abundant opportunities to reveal significant insights into protein trafficking and viral-host cell interactions.
VI. COMPOSITION OF THE OCCLUSION-DERIVED VIRUS: PROTEINS
ODV is the viral form specially adapted for primary infection of
the host midgut epithelium. As such, the virion must contain the
proteins essential for host range determination and initiation of
infection. As important as ODV is to the control of lateral infection,
surprising little is known about the functions of the proteins residing within ODV. However, their importance is clearly demonstrated
when one notes that most of the structural proteins of ODV envelope are highly conserved and are members of the 30 core baculovirus genes [29,30].
This section summarizes what is known about ODV envelope
proteins, however it will be limited to information that has become
available since the comprehensive review of baculovirus structural
proteins authored by Funk et al. [31]. Since AcMNPV is the baculovirus type species, the orf encoding each protein in the AcMNPV
genome is referenced.
ODV-E25 (AcMNPV orf 94)
ODV-E25 (E25) was the first ODV envelope protein identified
for any baculovirus [32] and it is conserved among all lepidopteran
baculoviruses [29]. Several studies of E25 from various viral strains
confirm the original observation that it is an ODV-envelope specific
protein [33]. Kawasaki et al. [34] showed that E25 localized at the
periphery of IE-1 associated structures or virogenic stroma. While
immunogold localization was not performed, this observation
would be consistent with the accumulation of E25 within intranuclear microvesicles. Li et al. [33] show that E25 of SpltMNPV is
clearly an envelope protein of ODV, however confocal microscopy
detects E25 predominantly within the cytoplasm of infected cells. It
is difficult to rationalize the cytoplasmic localization of E25 unless
it has additional functions yet to be described. Repeated attempts to
produce a recombinant OpMNPV lacking E25 have been unsuccessful, suggesting that E25 is essential [31].
ODV-E66 (AcMNPV orf46)
Identification and study of ODV-E66 (E66) provided the first
evidence that ODV envelope proteins were present in the viralinduced intranuclear microvesicle membranes [35]. Like E25, E66
is also conserved among lepidopteran baculoviruses [29] and is
found in the newly proposed virus genus, Nudivirus [36,37]. While
the location of E66 within the ODV envelope is firmly established,
E66 function has only recently been demonstrated. Data suggests
that E66 is a hyaluronan lyase and this activity may be important
for penetration of extracellular barriers during primary infection
[38].
An early study suggested that E66 was not essential and that
mutants could be generated that replaced the wild type protein in its
native locus [7]. However, a more thorough analysis of these mutant viruses show that a truncated E66 protein was generated in
cells infected with these viruses and repeated attempts to delete the
entire E66 gene have not been successful. These observations suggest that like E25, E66 is an essential gene (Braunagel and Summers; unpublished data).
Studies of E66 have predominantly focused upon the Nterminal 33 amino acids and their role in sorting proteins from the
ER to the nuclear envelope. These studies have been very informative and revealed much about the possible mechanisms of regulated
trafficking of ODV envelope proteins to the nuclear envelope. E66
trafficking to viral-induced microvesicles within the nucleus is
decreased in the absence of FP25K [19,25]. When this observation
Molecular Biology of the Baculovirus Occlusion-Derived Virus Envelope
was first made, it was unclear whether FP25K itself, or another
protein affected by the presence of FP25K, was responsible for
altered trafficking of E66. However, the identification that FP25K
covalently crosslinks to the E66-derived INM-SM sequence strongly
suggests that FP25K directly interacts, and may actively participate
in directed trafficking of E66 to the nuclear envelope [22].
P74 (AcMNPV orf 138)
P74 was the first ODV protein shown to be important for primary infection of the insect gut [39,40]. There are now several such
proteins identified and collectively these have been named per os
infectivity factors (pif’s). P74 is a highly conserved envelope protein of ODV and it is one of the 30 core baculovirus proteins
[29,41,42]. A transmembrane sequence residing within the carboxyl
terminus of the protein is required for ODV envelope localization
[40,43]. In 1993, Horton and Burand [44] postulated that ODV
binds to specific receptors on the surface of gut epithelia and this
hypothesis was supported when Haas-Stapleton et al. [45] demonstrated that p74 binds ODV to the primary target cells within the
insect gut. This is supported by subsequent studies that show p74
binding with brush border membrane vesicles displays saturation
kinetics and that p74 binds to a midgut membrane protein of approximately 30 kDa [46,47]. However, attachment alone cannot
explain the functional significance of p74. When p74 deletion mutants are tested, a 100,000-fold difference in infectivity is observed
however; only a threefold decrease in ODV binding to midgut cells
can be detected [45]. These data suggest that binding of ODV with
the gut cells does not ensure productive infection. It has been proposed that the alkaline conditions of the midgut prime p74 for Nterminal cleavage by a brush border membrane-specific factor [48].
While there is precedence for proteolytic activation of baculovirus
virion proteins, it is currently unknown if p74 cleavage is relevant
for its function during primary infection.
Per os Infectivity Factor 1 (pif1; AcMNPV orf 119)
Pif1 is an ODV envelope protein that was identified from infectivity studies performed using SpltNPV [49]. Like p74, pif1 is one
of the 30 core baculovirus genes [29]. Pif1 is essential for midgutmediated infection and was originally proposed to function during
the period after the binding of ODV to midgut cells and before viral
DNA replication [49]. However, pif1 has since been shown to function during the binding and fusion of ODV to midgut target cells
[50] and when deleted, oral infectivity is substantially reduced [51].
Like p74, pif1 is expressed in low quantities in infected cells. The
low level of expression of pif1 appears to be regulated at the transcriptional level [52]. The low quantity of protein of both p74 and
pif1 explains why neither protein was identified during a proteomic
screen using mass spectrometry of ODV proteins [53]. Indeed, it
seems logical that other ODV envelope proteins were missed in this
proteomic screen for a similar reason.
Per os Infectivity Factor 2 (pif2; AcMNPV orf 22)
Direct evidence is not yet available that pif2 is an ODV envelope protein, however functional evidence clearly suggests it resides
within the ODV envelope. SeMNPV or HearNPV mutant viruses
lacking pif2 demonstrate a significant loss of oral infectivity
[54,55]. Ohkawa et al. [50] provided evidence that pif2 mediates
binding to primary target cells within the insect midgut. Like pif1,
pif2 is one of the 30 core baculovirus genes [29].
Per os Infectivity Factor 3 (pif3; AcMNPV orf 115)
The fourth pif gene was identified in a comprehensive screen of
viral mutants of BmNPV’s [50]. Like the other pif genes, pif3 is one
of the 30 core genes of baculoviruses [29]. Unlike the other pif
proteins, pif3 does not appear to affect binding or fusion of ODV
with target gut cells. Ohkawa et al. [50] concluded that pif3 mediates an unidentified, but critical early event during primary infec-
Current Drug Targets, 2007, Vol. 8, No. 10
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tion. This laboratory has generated antisera to pif3 and western
blotting shows that it locates to ODV but not BV (Fig. (6)).
Fig. (6). Western Blot using orf115 antibody. 30g of cell extract from
uninfected or 30 h p.i. infected Sf9 cells or purified ODV or BV were used
per lane. Star denotes the placement of orf115 protein.
ODV-E56 (E56; AcMNPV orf 148)
E56 was simultaneously identified in two laboratories and the
protein was assigned two names: ODV-E56 and odvp-6e. One of
the most distinguishing features of E56 is that it is detected at a
variety of molecular weights when various baculoviruses are analyzed, and even within the same baculovirus, immunoreactive proteins with various masses are detected [56,57]. An initial study
showed that it was possible to generate a recombinant virus containing a disrupted E56 gene, yet repeated attempts to generate a
deletion mutant have been unsuccessful [20]. E56 is highly conserved; even among distantly related members of the baculoviridae
family [58-61] and E56 is a core baculovirus gene [29]. Considering its level of conservation and the observation that if stoichiometric amounts of E56 are altered, ODV envelopment decreases to
almost undetectable levels (Fig. (3)), it seems reasonable to conclude that E56 plays a vital role in ODV morphogenesis.
Orf 150 (AcMNPV orf 150)
Like E26, Ac150 is also detected in the envelope of both ODV
and BV [62]. Orf 150 is not essential; deletion mutant viruses can
be generated and are infectious both in vivo and in vitro [62,63].
When a comparative study was performed to identify differences in
virulence using oral and intra-haemocoelic routes of infection between wild-type and an Ac150-deletion mutant virus, the mutant
was significantly less virulent in the larvae of three tested host species (Heliothis virescens, Spodoptera exigua and Trichoplusia ni).
Ac150 apparently affects virulence by increasing the efficiency of
establishing primary focus formation. Thus, Ac150 may also be
classified as a pif factor however, unlike the other pif’s, it mediates,
but is not essential for oral infection.
F-Protein (AcMNPV orf 23)
F-protein is another member of the 30 core baculovirus proteins
[29] and it has been suggested that F-protein is the primordial baculovirus envelope fusion protein [64]. F-protein is present in both
BV and ODV. This protein is required for fusion of the BV envelope with host endosomal membranes to release nucleocapsids for
initiation of infection [64,65]. Indeed, F-protein can rescue virion
production and infectivity in a gp64-null AcMNPV mutant, suggesting these two proteins have related, yet apparently different
activities during viral infection [66]. While the presence of F-
1090 Current Drug Targets, 2007, Vol. 8, No. 10
protein in BV is firmly established, it was only recently identified
in ODV and is only detected in ODV when comprehensive measures are used to inhibit proteases present during ODV purification
[53]. Functional assays show that F-protein accelerates the mortality of infected insect hosts; and as such, represents a new member
of baculovirus pathogenicity factors [67]. Studies of the F-protein
are the focus of several laboratories and this protein is deserving of
a comprehensive review devoted to its structure and function. The
following references will aid the interested reader in a more detailed exploration of baculovirus F-protein [68-71].
ODV-E18 (E18; AcMNPV orf 143)
E18 is present in all lepidopteran baculoviruses [29], but little is
known about its function. Repeated attempts to generate deletion
mutant viruses have been unsuccessful, and this is not due to the
location of the gene within the IE0 intron. If a second copy of E18
is available in the polyhedrin locus, the E18 gene in its native locus
can be deleted. However further attempts to delete the E18 gene
residing within the polyhedrin locus are not successful (unpublished
observations). It is possible that E18 is related to another envelope
protein, ODV-E35. The origin of E35 is unclear; it is apparently
encoded by a transcript that encompasses both BV/ODV-EC27
(EC27) and E18, however translation would require ribosomal
frame shifting [72]. The only evidence for the existence of E35 is
cross-reactivity to a protein with a Mr of 35 kDa using antibodies
generated to E18 or EC27. E18 is one of nine genes of Group 1
NPV’s that undergo positive selection, and it has been proposed
that such genes provide species-specific virulence factors or hostrange determinants [73].
ODV-E28 (AcMNPV orf 96)
The fact that the N-terminal region of orf 96 demonstrates such
similarities with other ODV-E proteins and that this orf belongs to
the 30 cores baculovirus genes, has made many predict that this
protein resides within the envelope of ODV. However, evidence
demonstrating this has only recently been provided. Xu et al. [74]
showed that BmMNPV orf 79 located to the ODV envelope and
was detected using SDS-PAGE as a 28 kDa protein.
Gp41 (AcMNPV orf 80)
Gp41 is a major protein within the ODV [75] and is included
here because it routinely fractionates with the viral envelope
[53,76], and resides in the tegument region between the envelope
membrane and the capsid [77]. Gp41 is a member of the 30 core
genes of baculovirus. About 20% of gp41 is glycosylated and it
contains single residues of GlcNac O-glycosidically linked to the
polypeptide chain [77-79]. As such, it is unique among baculovirus
proteins that associate with the ODV envelope; no other glycosylated ODV-envelope proteins have been identified. Gp41 is highly
conserved in the central region while the C- and N-terminal regions
are divergent [79,80]. Mutational analyses suggest that gp41 is
required for the egress of nucleocapsids from the nucleus to the
plasma membrane for the production of BV [81]. Since the only
gp41 deletion mutant that has been generated is a temperaturesensitive mutant, it is believed that gp41 is essential [81].
BV/ODV-E26 (E26; AcMNPV orf 16)
E26 is one of the least conserved of all the envelope proteins of
ODV. E26 is only present in about half of the sequenced baculovirus genomes. The information available on AcMNPV E26 and its
encoded protein currently has some contradictory observations.
There is agreement that E26 is an early gene and transcripts accumulate rapidly after infection (1-2 hrs p.i.), [82]. E26 was proposed
to be involved in the activation of baculovirus late gene expression,
however a later report studying a virus with a disrupted E26 gene
showed that there was no delay in late gene expression [83]. Rather,
based upon results of assays of virus growth in cell culture and
Braunagel and Summers
insect larvae, O’Reilly et al. [83] suggested that E26 is nonessential. Beniya et al. [84] reported that E26 encoded a structural
protein of baculovirus that was incorporated into the envelope of
both BV and ODV, and named the gene product BV/ODV-E26
(E26). However a subsequent report by Imai et al. [85] and Kang et
al. [86] could not confirm this observation. Rather, they showed
that the Bombyx mori homologue, orf 8 encoded a protein that accumulated in the nucleus, co-localized with IE1 and bound to nucleic acids. They were unable to isolate a null mutant and proposed
that Bm8 was essential and this was supported by subsequent observations by J. Burks [87]. Indeed, data has only recently become
available that may explain many of the apparent discrepancies reported for the structure and function of E26. Multiple isoforms of
E26 are present in the infected cell and the different isoforms present uniquely different epitopes that are discriminated by antiserum
produced to bacterially or virally produced antigen. One form of
E26 associates with viral DNA or DNA-binding proteins while a
second form associates with intracellular membranes. E26 membrane association is likely mediated via palmitoylation [88].
Regardless of the controversy surrounding E26, mass spectrometry has shown that E26 associates with microsomal membranes (composed predominantly of ER), and that it is in close
proximity with the E66-derived INM-SM [22]. Antibodies generated to E26 (expressed in bacteria) precipitate the cellular protein
importin--16 and while these two proteins clearly share epitopes
that are recognized by antibody preparations, they only share limited amino acid similarity [23]. E26 association with the E66derived INM-SM sequence occurs after it has been released from
the translocon and integrated into the ER membrane. The function
of this interaction has yet to be defined.
VII. PROTEINS RESIDING WITHIN BOTH THE ENVELOPE AND NUCLEOCAPSID OF ODV
ODV-EC27 (EC27; AcMNPV orf 144)
EC27 is a member of the 30 core baculovirus genes [29]. It
encodes the first structural protein of baculovirus shown to reside
within both the envelope and capsid of ODV [72]. Repeated attempts at generating a deletion mutant of EC27 have been unsuccessful. Like E18, the inability to delete EC27 is not due to its location within the intron of IE0 (unpublished observations). EC27 has
homology with cellular cyclins within the cyclin box region and it
associates with the cellular cyclin kinases cdc2 and cdc6 [89]. The
EC27cdk6 complex also associates with proliferating cell nuclear
antigen. It is not clear what the functional role of EC27-cyclin-like
activity is during infection, however several observations offer
clues. AcMNPV targets at least two different checkpoints to prevent normal cell-cycle progression of Sf9 cells, and neither viral
DNA replication nor expression of viral late genes is necessary for
AcMNPV-induced cell cycle arrest [90]. The midgut cells that are a
target of ODV invasion with subsequent viral DNA replication are
in a G0 state. As such, cellular factors required for DNA replication
are either not present or in a repressed state. It is possible that EC27
(EC27cdc6) interact with the gut cell and facilitate progression of
the cellular machinery to an S-like phase, thus allowing optimal
conditions for viral DNA replication. If EC27 only functioned during primary infection however, then a deletion mutant should easily
be generated using Sf9 cells and in vitro conditions. However, the
data strongly indicate that EC27 is essential. It is possible that the
cyclin-like activity of EC27 also functions to phase the infected
host cell at G2/M (EC27cdc2) [9], which in insect cells allows
increased fluidity of the INM. In this manner, EC27 may play a
pivotal role in the production of the intranuclear microvesicles and
ODV envelope.
P91 (AcMNPV orf 83)
p91 is also a member of the 30 core baculovirus genes [29]. It
was identified during a screen of an OpMNPV expression library
Molecular Biology of the Baculovirus Occlusion-Derived Virus Envelope
Current Drug Targets, 2007, Vol. 8, No. 10
1091
pif2, pif3, EC43, F-protein), and 3 others are retained in all lepidopteran baculoviruses (E18, E25, E66). The high degree of gene conservation speaks to the functional significance of these proteins in
the life cycle of baculoviruses. If ODV envelope proteins only functioned during lateral transmission of the virus in nature, then one
would expect these genes to be readily deleted in the laboratory,
where the only requirement is the maintenance of viability of the
budded virus progeny. However, current experience shows that
many of these genes cannot be deleted from the genome. It is not
clear why these genes are essential for BV-mediated infection.
While determining functions of structural proteins is a daunting
task, much can be learned through identification of protein interactions. However, little is currently known about protein interactions
of ODV envelope proteins. Chemical crosslinking shows that the
E66-derived INM-SM is proximal to E26 and FP25K [22] and both
proteins, as well as E25, co-precipitates with E66 [19]. A yeast 2hybrid direct cross assay predicts that FP25K and E26 interact, and
this is supported by the observation that FP25K co-precipitates with
E26. Actin also co-precipitates with E26 [84]. Blue native gels
predict that EC27 is present in the infected cell in a complex that
also contains p78/83 and BV/ODV-C42 (C42) [94].
Yeast 2-hybrid direct cross assays predict a number of proteinprotein interactions (Fig. (7)). While the goal of this experiment was to explore proteinprotein interactions of ODV envelope
proteins, other viral structural proteins already implicated as potential partners were included. When polyhedrin and IE1 were cloned
into the binding domain vector, a positive reaction was detected
when crossed with every protein. This suggests they contain intrinsic transcriptional activation activity of the GAL4-responsive promoters, which is not informative in these assays. Gp41 appeared to
be lethal to yeast except in the presence of E26, in which case the
yeast not only grew, but a positive interaction was detected between
these two proteins. When FP25K was placed in the activation domain, it interacted with E26, however this interaction was not detected when the cross is reversed. IE1 interacted with E26 and p39.
using antisera generated to ODV and its presence within ODV is
clearly established. Like E26, p91 is also detected in BV [91]. Confocal microscopy detects p91 in infected cell nuclei and by immunoelectron microscopy p91 is detected in both the capsid and envelope of ODV [91]. A p91-GFP fusion protein shows p91 accumulates in the peristromal region of the nucleus in a manner similar to
other ODV-E proteins [34]. The functional role of p91 in either BV
or ODV is unknown.
ODV-EC43 (EC43; AcMNPV orf 109)
EC43 was simultaneously identified as a protein residing within
ODV in two laboratories. Fang et al. [92] identified that the
HaSNPV homologous gene (Ha98) encoded an ODV protein that
was located in both envelope and nucleocapsid, and they named the
protein EC43. Braunagel et al. [53] identified that orf 109 encoded
an ODV protein in a proteomic screen using both MADLI-TOF and
MS/MS spectrometry. The function of EC43 is unknown, however
the fact that it is one of the 30 core baculovirus core genes suggests
that it plays a vital role during infection [29].
Others
Although this review has focused on the protein composition of
AcMNPV, other baculoviruses have unique proteins comprising the
structural composition of ODV. One such example is Orf 122 of
Helicoverpa armigera SNPV. Ha 122 has no immediate homologues in other biological systems and encodes a protein with a Mr
of 21 kDa that is present in the ODV nucleocapsid [93]. Studies of
such unique proteins may be the key to revealing strategies for determination of host-range and productive infection in various insect
hosts.
VIII. PROTEIN-PROTEIN INTERACTIONS OF ODV
STRUCTURAL PROTEINS
Of the list of 30 core baculovirus genes, 10 of these encode
known ODV envelope proteins (gp41, EC27, E56, p74, vp91, pif1,
Activation Domain
X
BV/ODV-C42
X
X
X
NG
X
FP25K
ODV-EC27
X
IE1
X
X
ODV-E66
X
X
ODV-E25
X
ODV-E56
X
BV/ODVE26
X
X
X
X
NG NG
NG
X
X
X
X
NG
NG
X
X
gp41
X
orf 91
X
X
pif3
X
X
gp41
p78/83
BV/ODV-E26
X
ODV-E56
X
ODV-E25
p39
IE1
X
ODV-E66
ODV-EC27
X
FP25K
p39
polyhedrin
p78/83
polyhedrin
BV/ODV-C42
Binding Domain
NG
NG
X
NG
X
NG
X
NG
X
NG
X
NG
NG NG NG NG NG NG NG NG
X
NG
pif3
X
X
X
NG
orf 91
X
X
X
NG
NG NG
Fig. (7). Yeast-2-Hybrid Direct Cross Assay. Crosses were performed of the entire matrix, if the square is blank, then no interaction was detected. A blue
(X) square shows that a positive interaction was detected, while the yellow (NG) square indicates that the gene was lethal and the transformed yeast did not
grow.
1092 Current Drug Targets, 2007, Vol. 8, No. 10
The major capsid protein p39 had the highest number of interactions with other proteins. When tested in the activation domain, p39
interacted with itself, EC27 and E56; and in the binding domain,
p39 interacted with itself, polyhedrin, p78/83, C42, IE1, E66, E56,
pif 3 and orf 91.
Yeast 2-hybrid libraries were constructed from genomic
AcMNPV DNA and cDNA’s prepared from AcMNPV infected Sf9
cells at 4, 6, 18 and 24 h p.i. These libraries were screened with
appropriate constructs containing several structural proteins of
ODV and the positive interactions are listed in Fig. (8). In addition
to E66, a deletion construct missing the N-terminal INM-SM sequence was tested, and as expected, deleting the transmembrane
sequence resulted in the detection of more interactions than did the
membrane associated E66. Three p39 interactions detected in the
direct cross analysis were also detected with the library screen: p39
interacted with C42, 23-E66 and E56 (Fig. 8). IE1 identified a
ODV-E66
Braunagel and Summers
potential interaction with p78/83, E66 and E26. The interaction of
E66 and FP25K that was originally predicted using chemical
crosslinking reagents was detected in the screen of the 18 h p.i.
library. E25 has been shown to co-precipitate with E66 [19], and
this interaction was detected in the library screen (18 and 24 h p.i.).
EC27, p78 and C42 have been predicted to exist in complex [94]
and the library screen detected an interaction between C42 and
EC27 and p78/83 (Fig. 8). A number of potential interactions were
detected with 23-E66 and of these, C42, lef 5 (orf 199), and alkaline exonuclease (orf 133) were identified multiple times (Fig. (8)).
The advantage of using a library to detect interacting proteins is
the ability of such a screen to also identify cellular proteins that
may interact with bait ODV proteins. Indeed, several cellular proteins were identified in this screen. When EC27 was used as bait, a
cellular protein with a high homology to the pocket protein p130
was identified. Considering the possible role of EC27 in the regula-
ODV-E18
4 h p.i.
BV/ODV-C42 (orf 101)
4 h p.i.
Sec6
18 h p.i.
ORF 60
none
18 h p.i.
ORF 142
ODV-E66 (orf 46)
24 h p.i.
none
24 h p.i.
none
AcMNPV
none
AcMNPV
none
ODV-EC27
23-E66
4 h p.i.
alkaline exonuclease (orf 133)
α-adaptin
ubiquitin/ribosomal protein L40
18 h p.i.
BV/ODV-C42 (orf 101)
ODV-E25 (orf 94)
FP25K (orf 61)
gp67 (orf 128)
helicase (orf 95)
lef 7 (orf 125)
lef 5 (orf 199 - 2 clones)
p39 (orf 89)
gp37 (orf 64)
ORF 69
24 h.p.i.
alkaline exonuclease (orf 133)
ODV-E25 (orf 94)
IE1 (orf 147)
AcMNPV
BV/ODV-C42 (orf 101)
BV/ODV-E26
6 h p.i.
4 h p.i.
p130-like protein
18 h p.i.
BV/ODV-C42 (orf 101 - 4 clones)
24 h p.i.
BV/ODV-C42 (orf 101 - 2 clones)
AcMNPV
none
p78/83
IE1 (orf 147 - 9 clones)
cytoplasmic dynein-like protein
4 h p.i.
18 h p.i.
none
24 h p.i.
AcMNPV
none
AcMNPV
ODV-E56
none
18 h p.i.18 none
BV/ODV-C42 (orf 101 - 7 clones)
IE1 (orf 147 )
none
FP25K
4 h.p.i.
none
4 h p.i.
none
18 h p.i.
none
18 h p.i.
none
24 h p.i.
AcMNPV
none
p39 (orf 100)
24 h p.i.
AcMNPV
none
lef 11 (orf 37)
p39
4 h p.i.
none
18 h p.i.
none
24 h p.i.
AcMNPV
BV/ODV-C42 (orf 101)
none
Fig. (8). Yeast-2-hybrid library Screen. Yeast 2-hybrid cDNA libraries of AcMNPV infected Sf9 cells were prepared at 4, 6, 18, and 24 h .p.i. An AcMNPV
genomic library was also prepared. The libraries were then screened with viral-bait proteins and the results are shown.
Molecular Biology of the Baculovirus Occlusion-Derived Virus Envelope
tion of cell cycle this interaction may have functional significance.
The identification of a cytoplasmic dynein-like protein interacting
with E26 and the observation that actin co-precipitates with E26
and FP25K, was used to predict a mechanism by which FP25K or
E26 might be regulating the transport of E66 or other INM-directed
integral membrane proteins from the endoplasmic reticulum to the
nuclear envelope [23]. 23-E66 interacted with the cellular protein
-adaptin. If this interaction is functionally important, it may indicate that E66 may function as a recognition factor for ODV and
midgut cells during primary infection: -adaptin is a marker for
endocytosis that is located in the plasma membrane of Drosophila
melanogaster midgut [95]. It plays a critical function during the
attachment of clathrin to the membrane, selection of vesicle cargo
and recruitment of accessory proteins that regulate vesicle formation during endocytosis [96]. The future identification of proteinprotein interactions of viral proteins with other viral and cellular proteins will reveal much about the mechanism of the recognition, invasion and maturation of baculovirus.
CONCLUSION
While the study of the biology of viruses is interesting in its
own right, history has shown us that knowledge of virus invasion
and interactions with their host cells reveals significant insights into
normal biochemical mechanisms of the host cells. Baculoviruses
have had millennia to interact with their invertebrate hosts, and as
such, these viruses have evolved with an intimate and extensive
genetic knowledge of their host cell systems. Thus, the study of
baculoviruses is rich with potential to reveal insights into their host
cells and potential intervention strategies.
Insight revealing the molecular mechanisms of the secretory
pathway were rapidly advanced using mammalian enveloped virus
models. Indeed, during infection, cells infected with these viruses
are turned into professional secretory cells abundantly expressing
envelope proteins that are transported to specific cellular membranes. The ability to provide a synchronous, amplified pulse of a
single type of membrane protein allowed detailed kinetic descriptions of transit through secretory organelles [97]. Baculovirus provides a unique tool to study integral membrane protein trafficking
from the ER to the nuclear envelope. Infection results in a synchronous, amplified pulse of viral integral membrane proteins that integrate into the ER membrane and are then directed toward the nuclear envelope and utilize the INM in their passage to the virusinduced intranuclear membranes. Studies of the mechanism of
ODV envelope protein trafficking have revealed surprising observations. They have identified a formerly unknown isoform of importin
, importin--16. Importin--16 associates with the ER membrane
and positions itself in close proximity to the translocon protein
Sec61. As a newly discovered translocon-associated protein, importin--16 has functional properties clearly different from other
translocon-associated proteins. Importin--16 appears to discriminate between INM and non INM-directed sequences and is proximal to those integral membrane proteins directed to the INM. Importin--16 remains associated with the INM-directed protein after
it is released from the translocon and integrated into the ER membrane. While it is unknown if ODV envelope proteins are recognized by cellular importin -16, we know that after they are integrated into the ER membrane, the E66-derived INM-SM is proximal to E26 and FP25K. Considering that E26 shares epitopes and
sequence homology to cellular importin -16, it is possible that it
assumes the function of importin -16 during infection [23]. Considering that integral membrane proteins of the INM are implicated
in multiple diseases [98], knowledge of the trafficking mechanism(s) of integral membrane proteins to the INM has important
implications for human health.
Much of the molecular, biochemical and physical interactions
of pathogens with the insect midgut remains unexplored, yet this
knowledge is fundamental to the development of new genetic ap-
Current Drug Targets, 2007, Vol. 8, No. 10
1093
proaches for insect control. Since ODV is responsible for primary
recognition and invasion in the insect gut, study of ODV envelope
proteins should provide insights that are fundamental for new approaches to insect control strategies. In addition, most insect vectored pathogens invade the insect through the gut, to then propagate
and be transmitted to their vertebrate hosts. As such, knowledge
gained of the invasion strategy of ODV, with subsequent optimization of cell components for viral DNA replication, can reveal much
about potential intervention strategies for the control of insectvectored diseases that affect more than one billion people worldwide.
ACKNOWLEDGEMENTS
A number of people have contributed to work included here: M.
Belyavskyi, R. Barhoumi-Mouneimne and R. Burkardt performed
the Indo I labeling and Ca2+ determination (Image Analysis Laboratory, College of Veterinary Medicine, Texas A&M University,
College Station, TX); H. Ma generated the E56polh recombinant
virus and prepared samples for electron microscopy and G. RosasAcosta and S. Williamson performed the digitonin permeabilization
assays. A number of laboratory members participated in the yeast2-hybrid screens, but special thanks go to G. Marcano and S. Williamson for making the libraries and B. Sweeney and T. Adams for
execution of the library screen and direct cross assays. This work
was supported by the Texas Agriculture Experiment Station Project
Grant TEXO-08078 (MDS).
ABBREVIATIONS
AcMNPV
=
BmMNPV
=
OpMNPV
=
LdMNPV
=
SeMNPV
=
HaSNPV
=
HearNPV
SpltMNPV
SlNPV
=
=
=
Autographa californica nucleopolyhedrovirus
Bombyx mori nucleopolyhedrovirus
Orgyia pseudotsugata nucleopolyhedrovirus
Lymantria dispar nucleopolyhedrovirus
Spodoptera exigua nucleopolyhedrovirus
Heliocoverpa armigera single nucleopolyhedrovirus
Heliocoverpa armigera nucleopolyhedrovirus
Spodoptera litura nucleopolyhedrovirus
Spodoptera littoralis nucleopolyhedrovirus
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Received: August 3, 2007
Accepted: August 7, 2007
Current Drug Targets, 2007, Vol. 8, No. 10
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