Insect virus proteins (FALPE and p10) self-associate to form filaments in infected cells

The main symptom of Morgellons Disease is the appearance of fibers emerging from intact skin or in other cases emerging from open sores.

All, namely 100%, Morgellons Sufferers report this condition.

According to scientists investigating this disease it has not been proven to this day what causes the formation of these fibers.

The exact chemical structure of the fibers is not fully understood nor investigated and still remains unknown.

This article will show how the Entomopoxvirus, and the Baculovirus, both used in combination as a bio-insecticide, is kept suspiciously responsible for the cause of this life threatening disease.

Here are a few excerpts from this article which I’ve found quite interesting:

Entomopoxviruses and baculoviruses are pathogens of insects which replicate in the cytoplasm and nuclei of their host cells.

During the late stages of infection, both groups of viruses produce occlusion bodies which serve to protect virions from the external environment.

* We remember that occlusion bodies are also part of the Baculovirus.

Description Occlusion Bodies: http://www.uniprot.org/keywords/842

occlusion body    image

Baculoviruses and Entomopoxviruses belong to the family of Cypoviruses.

continuing excerpt:

Immunofluorescence and electron microscopy studies have shown that large bundles of filaments are associated with these occlusion bodies.

Entomopoxviruses produce cytoplasmic fibrils which appear to be composed of the filament-associated late protein of entomopoxviruses (FALPE).

Cytoplasmic fibrils image image

Evidence that FALPE and p10 could produce filaments in the absence of other viral proteins is presented.

When FALPE was expressed in insect cells from a recombinant baculovirus, filaments similar to those produced by the wild-type Amsacta moorei entomopoxvirus were observed.

In addition, when expression plasmids containing FALPE or p10 genes were transfected into Vero monkey kidney cells, filament structures similar to those found in infected insect cells were produced.

The manner in which FALPE and p10 subunits interact to form polymers was investigated through deletion and site-specific mutagenesis in conjunction with immunofluorescence microscopy, yeast two-hybrid protein interaction analysis, and chemical cross-linking of adjacent molecules.

* As we know, scientists investigating Morgellons Disease claim to have found that the related fibers contain a source of polymer and also fungi/yeast plays a significant role.

source: http://www.ncbi.nlm.nih.gov/pubmed/9499079

Another article according to the protein p10:

source:

http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=bacvir&part=ch02

Fibrous p10-containing material aligned image

excerpt:

P10 (Ac137). Although p10 does not appear to be a major occlusion body protein, it colocalizes with the PE protein and appears to be required for the proper formation of the polyhedron envelope. When p10 is phosphorylated, it becomes associated with microtubules (15). This could be related to the structures it forms that include microtubule-associated filaments, and tube-like structures that surround the nuclei of infected cells.

Us, Morgellons Sufferers, have discussed the effects of Global Warming and its effects on nature.  We are assuming that this condition is responsible for the enhancement and over population of insects causing this disease.

http://jvi.asm.org/cgi/content/full/72/3/2213?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=mating&searchid=1&FIRSTINDEX=1390&resourcetype=HWFIG

Entomopoxviruses and baculoviruses are pathogens of insects which replicate in the cytoplasm and nuclei of their host cells, respectively.

During the late stages of infection, both groups of viruses produce occlusion bodies which serve to protect virions from the external environment.

Immunofluorescence and electron microscopy studies have shown that large bundles of filaments are associated with these occlusion bodies.

Entomopoxviruses produce cytoplasmic fibrils which appear to be composed of the filament-associated late protein of entomopoxviruses (FALPE).

Baculoviruses, on the other hand, yield filaments in the nuclei and cytoplasm of the infected cell which are composed of a protein called p10.

Evidence that FALPE and p10 could produce filaments in the absence of other viral proteins is presented.

When FALPE was expressed in insect cells from a recombinant baculovirus, filaments similar to those produced by the wild-type Amsacta moorei entomopoxvirus were observed.

In addition, when expression plasmids containing FALPE or p10 genes were transfected into Vero monkey kidney cells, filament structures similar to those found in infected insect cells were produced.

The manner in which FALPE and p10 subunits interact to form polymers was investigated through deletion and site-specific mutagenesis in conjunction with immunofluorescence microscopy, yeast two-hybrid protein interaction analysis, and chemical cross-linking of adjacent molecules.

These studies indicated that the amino termini of FALPE and p10 were essential for subunit interaction.

Although deletion of the carboxy termini did not affect this interaction, it did inhibit filament formation.

In addition, modification of several potential sites for phosphorylation also abolished filament assembly.

We concluded that although the sequences of FALPE and p10 were different, the structural and functional properties of the two polypeptides appeared to be similar.

Cytoskeletal elements have previously been demonstrated to be involved in several aspects of virus assembly.

For example, vaccinia virus has been shown to associate with actin during its release from the plasma membrane , while adenovirus is transported through the cytoplasm to the nucleus through its interaction with microtubules.

Actin has been implicated in the transport of baculovirus nucleocapsids to the nucleus.

Other viruses contain actin in their envelopes along with viral surface glycoproteins, implying some role in the budding process.

In addition, cytochalasin D, a disruptor of microfilaments, has been shown to impair the assembly of a number of different viruses.

Most viruses use preexisting microtubule or microfilament proteins derived from host cells in these processes.

However, we have recently demonstrated that insect poxviruses establish their own filament network during the later stages of infection, using a protein encoded by the viral genome.

AmEPV derives its name from the Indian red army worm, a larva from the Lepidoptera family and the host from which the virus was originally isolated.

Lepidoptera
Lepidoptera – Wikipedia, the free encyclopedia

Baculoviruses also infect Lepidoptera larvae but instead replicate in the nuclei of their host cells .

A number of baculoviruses have been studied, but knowledge of Autographa californica nuclear polyhedrosis virus (AcNPV), which infects a wide variety of larvae including that of the alfalfa leaf hopper, is most extensive.

This virus is used routinely to produce recombinant proteins in insect virus expression systems.

A common property of EPVs and baculoviruses is the formation of large intracellular structures known as occlusion bodies which assemble during the late stages of viral infection.

Virions are embedded within these occlusion bodies, and the process serves to protect the virus from the external environment.

In the case of baculoviruses, the occlusion bodies are called polyhedra and are composed predominantly of a 31-kDa protein called polyhedrin .

The occlusion bodies of EPVs are known as spheroids and consist mainly of a 110-kDa protein known as spheroidin.

Spheroidin and polyhedrin do not appear to exhibit sequence homology.

A multilamellar envelope also appears to surround both polyhedra and spheroids and may help to stabilize these structures during assembly.

During the late phases of AmEPV and baculovirus infections, large bundles of filaments also appear to accumulate in the infected insect cells.

In the case of AmEPV, these structures are present in the cytoplasm, while those found in cells infected with baculoviruses reside both in the cytoplasm and in the nucleus .

Baculovirus fibrils are composed primarily of a 10-kDa protein called p10.

Deletion mutagenesis of AcNPV p10 has demonstrated that both the amino- and carboxy-terminal regions of this protein are necessary for the formation of filaments in the infected cell.

Our laboratory recently demonstrated that the cytoplasmic filaments, which characterize the late stages of infection by AmEPV, are composed primarily of a 156-amino-acid protein called FALPE (filament-associated late protein of EPVs).


These filaments are closely associated with the spheroids and their membrane envelopes.

FALPE is a phosphoprotein which migrates on sodium dodecyl sulfate (SDS)-polyacrylamide gels as a 25/27-kDa doublet.

This protein also contains an unusual proline-glutamic acid repeat region spanning 20 residues in the carboxy terminus of the polypeptide.

The ultrastructure and close association of this protein with the occlusion bodies of AmEPV suggested that FALPE and p10 played analogous roles during infections by the respective viruses.

This article addresses the structural and functional similarities between FALPE and p10.

These two viral proteins are known to be major components of filamentous structures, but it is not known whether additional viral or cellular proteins cooperate during the polymerization process.

In this report, we provide insight into the mechanisms which produce filaments in cells infected with either baculoviruses or EPVs. We demonstrate that p10 and FALPE can produce filaments in the absence of other viral gene products. Using the yeast two-hybrid system and a chemical cross-linking agent, we obtained evidence for self-association of either FALPE or p10. Finally, the polypeptide regions of FALPE and p10 which are required for self-association and subsequent filament formation are mapped.

image

showing filaments associated with FALPE in Sf9 insect cells infected with AmEPV and a FALPE recombinant baculovirus. Sf9 cells were mock infected (A), infected with AmEPV (B), infected with wild-type AcNPV (C), or inoculated with a recombinant AcNPV expressing the FALPE gene (D). At 72 h postinfection, cells were incubated with MAb CLP001, and bound antibody was detected with goat antimouse antibody conjugated to fluorescein. Labeled proteins were visualized with a Leitz fluorescence microscope. Nuclear DNA in panels A and D was also stained with Hoescht dye, and the two panels represent double exposures from the fluorescein and Hoescht signals. Panels B and D show the filaments formed by FALPE when expressed by AmEPV and AcNPV, respectively, while panel C illustrates background fluorescence found in the nuclei of cells infected with wild-type AcNPV.

image

Immunofluorescence microscopy of Vero monkey kidney cells transfected with a FALPE or p10 expression plasmid. Vero cells were transiently transfected with either the expression plasmid pRBK (A), plasmids expressing the p10 gene of AcNPV (B and D), or a plasmid expressing FALPE (C). FALPE was visualized by using MAb CLP001 (C), while p10 was detected with a rabbit polyclonal antibody directed against the p10 protein of AcNPV (B and D). Control cells in panel A were stained with the p10 polyclonal antibody; nuclei in panel D were also stained with Hoescht dye. Clearly both p10 and FALPE formed filament networks following transient transfections of their genes into mammalian cells.

image

Immunofluorescence microscopy showing intracellular localization of FALPE mutant proteins produced by recombinant baculoviruses. Sf9 insect cells were infected with baculoviruses expressing N45.FALPE (A), 8098.FALPE (B), Ct.FALPE (C), or the phosphorylation mutant P Mut.FALPE (D) as described for Fig. 2. MAb CLP001 and fluorescein isothiocyanate-coupled goat anti-mouse antibody were used to detect FALPE variants in panels A, B, and D. The mutant Ct.FALPE was detected by using a rabbit polyclonal primary antibody directed against FALPE and rhodamine-coupled goat anti-rabbit secondary antibody. Nuclear DNA in panels A and B was also stained with Hoescht dye, and the photographs represent double exposures of fluorescein and DNA signals. All mutations abolished the ability of FALPE to produce filaments in cells infected with the different recombinant baculoviruses. The bars in panels A and C represent 10 µm, while those in panels B and D indicate 15 µm.

~ by k&k on September 7, 2009.

2 Responses to “Insect virus proteins (FALPE and p10) self-associate to form filaments in infected cells”

  1. Hey very nice blog!!….I’m an instant fan, I have bookmarked you and I’ll be checking back on a regular….See ya

  2. thanks, Bill..glad you like it..stop by soon again

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