Suppose that f=(u,v) is a homeomorphism in the plane of the Sobolev class Wloc1,1 such that its inverse is of the same Sobolev class. We prove that u and v have the same set of critical points. As an ...application we show that u and v are distributional solutions to the same non-trivial degenerate elliptic equation in divergence form. We study similar properties also in higher dimensions.
Baculoviruses encode fibroblast growth factor (vfgf) homologs whose function during virus infection is unknown. We constructed a recombinant bacmid of Autographa californica M nucleopolyhedrovirus ...(AcMNPV) lacking a functional vfgf and characterized it in two insect cell lines. The kinetics of budded virus production were similar in the parental and vfgf-deficient viruses in both cell lines at both high and low multiplicities of infection. In addition, no obvious differences were observed between the mutant and parental viruses in protein or DNA synthesis. Finally, coinfection of vfgf-containing and -deficient viruses and passage for several generations did not reveal a consistent growth advantage for either virus.
Aedes aegypti
is the principal vector of dengue virus (DENV) throughout the tropical world. This anthropophilic mosquito species needs to be persistently infected with DENV before it can transmit the ...virus through its saliva to a new vertebrate host. In the mosquito, DENV is confronted with several innate immune pathways, among which RNA interference is considered the most important. The
Ae. aegypti
genome project opened the doors for advanced molecular studies on pathogen–vector interactions, including genetic manipulation of the vector for basic research and vector control purposes. Thus,
Ae. aegypti
has become the primary model for studying vector competence for arboviruses at the molecular level. Here, we present recent findings regarding DENV–mosquito interactions, emphasizing how innate immune responses modulate DENV infections in
Ae. aegypti
. We also describe the latest advancements in genetic manipulation of
Ae. aegypti
and discuss how this technology can be used to investigate vector transmission of DENV at the molecular level and to control transmission of the virus in the field.
Virus stability and infectivity during stressful conditions was assessed to establish guidelines for future virus filtration experiments and to contribute to the body of knowledge on a widely used ...virus. A recombinant baculovirus of
Autographa californica M nucleopolyhedrovirus (Ac
MNPV), vHSGFP, was incubated at 15–65
°C. A 2-log decrease in virus infectivity occurred after virus incubation above 45
°C. The activation energy of virus deactivation was circa 108
kJ/mol. Dynamic light scattering revealed an increase in apparent virus particle size from 150
±
19 to 249
±
13
nm at 55
°C. Protein and DNA concentrations in solution correlated well with virus aggregation as temperature was increased. Infectivity of vHSGFP stored for 5 months at 4
°C or exposed to shear stress from stirring (100
rpm, 1.02
×
10
−5
psi) and pumping (50–250
ml/min, 1.45
×
10
−5 to 7.25
×
10
−5
psi) did not change with time. Unlike temperature variations, cold storage and shear stress appeared to have little impact on infectivity.
Abstract Autographa californica M nucleopolyhedrovirus transcribes genes using two DNA-directed RNA polymerases; early genes are transcribed by the host RNA polymerase II, and late and very late ...genes are transcribed by a viral-encoded multisubunit RNA polymerase. The viral RNA polymerase is composed of four proteins: Late Expression Factor-4 (LEF-4), LEF-8, LEF-9, and P47. The predicted amino acid sequences of lef-9 and lef-8 contain motifs that are similar to those that participate at the catalytic center of known RNA polymerases. The requirement for the motif present in LEF-8 in late gene expression has been previously demonstrated. We have assessed the requirement of specific residues within the motif in LEF-9 for late gene expression. The conserved aspartic acid residues within the LEF-9 motif, corresponding to those essential for activity of the Escherichia coli RNA polymerase largest subunit, were required for late gene expression. Furthermore, we found that LEF-8 and LEF-9 interacted in coimmunoprecipitation experiments. We determined possible interactions of all the RNA polymerase subunits in pairwise combinations and found associations between LEF-9 and P47, LEF-4 and P47, and LEF-8 and P47. In contrast, LEF-4 and LEF-8 did not coimmunoprecipitate but coimmunoprecipitated in the presence of P47, suggesting that they do not associate directly. A weak association was observed between LEF-4 and LEF-9. Further analysis also suggested that LEF-8, LEF-9, and P47 have the ability to self-associate. Studies on protein-protein interactions may provide insight into the structural design of the complex and mechanistic aspects affecting late and very late gene expression.
We used a well established transient expression assay to test the ability of the baculovirus
Spodoptera exigua M nucleopolyhedrovirus (Se
MNPV) homologs of
Autographa californica MNPV (Ac
MNPV) late ...expression factors (
lefs) to activate a late promoter–reporter gene cassette in SF-21 cells. This insect-derived cell line is fully permissive for Ac
MNPV infection but not for Se
MNPV. In the assay, 19 Ac
MNPV
lefs stimulate optimal levels of late gene promoter activity. Se
MNPV
lef-5 successfully replaced the corresponding Ac
MNPV gene in the context of the remaining set of Ac
MNPV
lefs, whereas Se
MNPV
dnapol and
39k exhibited partial activity. When all the Se
MNPV
lefs were assayed together or in the presence of four
lefs encoded only in Ac
MNPV, it resulted in background levels of late promoter-driven reporter gene activity. However, Se
MNPV genomic DNA and the four Ac
MNPV-specific
lefs stimulated low levels of reporter gene activity. Moreover, Se
MNPV IE-1, but not Ac
MNPV IE-1, further stimulated late gene expression in the presence of Se
MNPV DNA. Ac
MNPV IE-1 was able to mediate early gene expression
cis-linked to homologous regions (
hrs) derived from Ac
MNPV and Se
MNPV. In contrast, Se
MNPV IE-1 was more specific for Se
MNPV-derived
hr elements.
Baculovirus infection of permissive cells proceeds in a cascade fashion with the transcription of early, late and very late genes. The structure of a number of baculovirus early gene promoters has ...been dissected in detail and the viral factors necessary to stimulate their expression have been identified. Early baculovirus gene promoters in general have a resemblance to host promoters while late and very late gene promoters are different from early baculovirus promoters and are more defined. In this study we investigated whether two key Autographa californica M nucleopolyhedrovirus (AcMNPV) transactivators have the ability to regulate the commonly used cellular promoter from the Drosophila heat shock 70 protein gene, during transient gene expression assays in two insect cell lines permissive for AcMNPV infection, SF-21 and TN-368, or during viral infection. The AcMNPV ie-1 transactivator gene stimulated gene expression of this cellular promoter in both cell lines when the promoter was cis-linked to an enhancer element, but stimulation in the absence of enhancer elements was either undetected or lower than in the presence of enhancer elements in SF-21 and TN-368 cells, respectively. The transactivator ie-2 stimulated gene expression in the presence of cis-linked enhancer elements and ie-1 in SF-21 cells. During viral infection, the heat shock 70 promoter was maximally activated at 12 hours post infection. We discuss how these results affect the interpretation of transient gene expression assays performed in the presence of viral transcription factors.
We compared the abilities of late gene transcription and DNA replication machineries of the baculoviruses
Autographa californica nucleopolyhedrovirus (Ac
MNPV) and
Bombyx mori NPV (BmNPV) in SF-21 ...cells, an insect-derived cell line permissive for Ac
MNPV infection. It has been well established that 19 Ac
MNPV late expression factors (
lefs) stimulate substantial levels of late gene promoter activity in SF-21 cells. Thus, we constructed a set of clones containing the BmNPV homologs of the Ac
MNPV
lefs under control of the constitutive
Drosophila heat shock 70 protein promoter and tested their ability to activate an Ac
MNPV late promoter–reporter gene cassette in SF-21 cells. We tested the potential of individual or predicted functional groups of BmNPV
lefs to successfully replace the corresponding Ac
MNPV gene(s) in transient late gene expression assays. We found that most, but not all, BmNPV
lefs were able to either fully or partially substitute for the corresponding Ac
MNPV homolog in the context of the remaining Ac
MNPV
lefs with the exception of BmNPV
p143,
ie-2, and
p35. BmNPV
p143 was unable to support late gene expression or be imported into the nucleus of cells in the presence of the Ac
MNPV or the BmNPV LEF-3, a P143 nuclear shuttling factor. Our results suggest that host-specific factors may affect the function of homologous proteins.
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