Establishment of systemic infection by AcM
NPV requires a coordinated series of activation steps that are reminiscent of findings during palatogenesis and aorta myogenesis described above; namely, cross talking between signaling pathways, involving FGFs, MMPs and caspases, and culminating in basal lamina remodeling. One can hypothesize that baculoviruses captured the cellular fgf
and use it to stimulate similar cellular pathways that ultimately lead to the productive invasion of larval tissues. If this is the case, since the basic factors involved in cellular developmental processes have been conserved in invertebrates, studies on baculovirus pathogenesis can help define protein functions in poorly understood human developmental programs and pathologies. The biochemical pathway downstream of and dependant on AcFGF signaling that allows for basal lamina remodeling and efficient midgut escape was recently described (Means and Passarelli, 2010
First, since baculoviruses may escape the midgut by crossing the midgut basal lamina, it was determined whether the basal laminae lining the midgut showed any ultrastructural changes in orally infected T. ni
larvae, and if AcFGF affected any of the observed changes. The integrity of midgut cell basal laminae infected with AcM
NPV encoding vFGF did not appear to have defects in the distribution or overall amounts of laminin, a major protein component of basal laminae (Means and Passarelli, 2010
). It is possible that subtle fissures or rearrangements in the midgut basal lamina may allow budded virions to access the hemocoel, but these were not obvious by laser confocal microscopy.
Second, baculoviruses may exit the midgut using tracheal elements; thus, basal lamina surrounding midgut-associated tracheae in orally infected T. ni
with viruses expressing AcFGF was examined by transmission electron microscopy. In the presence of AcFGF, the basal lamina of tracheal cells associated with the midgut was disorganized; instead of a thin uniform sheath surrounding the cells, it appeared fragmented (Means and Passarelli, 2010
) and reminiscent of that observed when larvae were infected with viruses expressing basal laminae degrading enzymes (Tang et al., 2007
). Analysis with a virus lacking Acfgf
showed some rearrangement of tracheal cell basal lamina but not as drastically as with the virus carrying Acfgf
. This may explain why vfgf
is not required for secondary infections but in its absence it takes longer to infect beyond the primary site. Also, a reduction in laminin was also observed following immunostaining (). Finally, immunoblots of laminin or collagen type IV using lysates from infected midguts and associated trachea indicated that these proteins were being proteolytically cleaved (Means and Passarelli, 2010
). Cleavage was drastically reduced in T. ni
infected with an Acfgf
mutant, suggesting that laminin cleavage and basal lamina degradation was stimulated by AcFGF.
Fig. 3 Delamination of trachea infected with AcMNPV. T. ni larvae were mock-infected or orally infected with a virus carrying vfgf (AcBAC) or lacking vfgf (AcBAC-vfgfKO). Midguts and associated trachea were dissected at 6 h p.i., incubated with anti-laminin (more ...)
It has been reported that tracheal elements reach into the midgut cell basal lamina of insects (Romoser et al., 2005
). It is not clear if the terminal tracheoblast actually protrudes past the midgut cell basal lamina where it would secrete its own basal lamina and come in closer contact with midgut epithelial cell membranes. Cells are known to be able to break through basement membranes or apposed basal laminae (Sherwood et al., 2005
). Alternatively, the tracheoblast may not cross the midgut basal lamina and instead may terminate within the basal lamina of the midgut epithelium, oxygenating the midgut epithelium by diffusion. Tracheoblasts protected by their basal lamina or buried within the midgut cell basal lamina may be targets for vFGF-mediated cell motility. Upon stimulation of cell motility, the tracheoblast would shed its basal lamina and become more accessible to the virus as it extends toward the signal. In this scenario, infection would need to take place prior to the secretion of new basal lamina post-cell motility.
Further investigation revealed that at least one caspase was directly responsible for cleaving both laminin and collagen IV in tracheal cell basal lamina (; Means and Passarelli, 2010
). Caspases are cysteine proteases that are best known for their roles in apoptosis or cell death. This is consistent with the implication of cell death events during organogenesis and disease mentioned above. However, caspases also play roles in other cellular processes and cell death would not necessarily occur upon caspase activation. The caspase(s) involved appear to be effector caspases or the final protease executioners based on their substrate preference. Inclusion of caspase inhibitors during virus feeding blocks both basal lamina degradation and caspase activation in vivo and in vitro (Means and Passarelli, 2010
). The purified Drosophila
effector caspase Drice was able to cleave mammalian laminin in vitro, and this activity was specifically inhibited by the effector caspase inhibitor zVAD-fmk but not by two initiator caspase inhibitors (Means and Passarelli, 2010
). This implies that the common caspase activation pathway, where an initiator caspase activates an effector caspase, is being circumvented during this process.
Fig. 4 Caspase activation in AcMNPV-infected tracheal cells. T. ni larvae were orally infected with a virus carrying (AcBAC) or lacking (AcBAC-vfgfKO) vfgf. Midguts and associated trachea were dissected at 12 h p.i., incubated with an antibody against the active (more ...)
Caspases are normally thought of as intracellular enzymes in charge of dismantling the cell. Given that this process is occurring extracellularly, the effector caspase may be secreted and then activated by other proteases and this extracellular compartmentalization could result in cleavage of different substrates, e.g.
, basal lamina protein components. The peptide sequences of laminin and collagen type IV contain predicted caspase cleavage sites, supporting the possibility of their degradation during virus infection. In addition, effector caspase activity was detected in the hemolymph of infected insects (Means and Passarelli, 2010
). It is not clear yet whether the caspases are shuttled extracellularly or if apoptotic cells release them, or whether the caspase(s) responsible for cleavage of basal laminae are not normally involved in apoptosis. Nevertheless, the extent of apoptosis in caspase activated tissues did not correlate with infection (Means and Passarelli, 2010
The presence of caspase activity in the extracellular matrix prompts the question of how effector caspases are activated extracellularly. Candidate proteins on the cell surface that have a role in basal lamina turnover are matrix metalloproteases, which have been associated with cell death and/or basal lamina remodeling in other systems (Cuervo and Covarrubias, 2003
; Kim et al., 2007
; You et al., 2003
). In vitro, purified human MMP-9, a basal lamina remodeling protein, was able to activate human pro-caspase-3. Caspase activation was enhanced in infected midgut lysates in the presence of MMP activity and vFGF expression, and blocking MMP activity also blocked caspase activation. Mock-infected midgut lysates could trigger caspase activity only if purified MMP-9 was added exogenously to an in vitro reaction. Together, this suggests that MMPs were activated during virus infection and active MMPs stimulated the activation of an effector caspase. Also, caspase activation and vFGF expression coincided with tyrosine phosphorylation (), indicative of vFGF receptor engagement (Means and Passarelli, 2010
). Finally, including either MMP or caspase inhibitors in the virus inoculums blocked both tracheal cell basal lamina remodeling and virus escape from the midgut, tying together the roles of vFGF, MMPs and caspases in basal lamina remodeling and virus dissemination (Means and Passarelli, 2010
). Although these enzymatic events leading to basal lamina remodeling have been identified, it remains possible that there are additional intermediary factors in this pathway that have not yet been identified.
Fig. 5 Colocalization of phosphotyrosines and active effector caspases in infected larvae trachea. Laser scanning confocal micrograph of midgut-associated trachea from insect larvae infected with AcMNPV showing anti-phosphotyrosine (red) and anti-active effector (more ...)