Extracellular Virus-like Particles Research Articles

Targeting of Moloney murine leukemia virus gag precursor to the site of virus budding.

Retrovirus Moloney murine leukemia virus (M-MuLV) matures by budding at the cell surface. Central to the budding process is the myristoylated viral core protein precursor Gag which, even in the absence of all other viral components, is capable of associating with the cytoplasmic leaflet of the plasma membrane and assembling into extracellular virus-like particles. In this paper we have used heterologous, Semliki Forest virus-driven, expression of M-MuLV Gag to study the mechanism by which this protein is targeted to the cell surface. In pulse-chase experiments, BFA, monensin, and 20 degrees C block did not affect incorporation of Gag into extracellular particles thereby indicating that the secretory pathway is not involved in targeting of Gag to the cell surface. Subcellular fractionation studies demonstrated that newly synthesized Gag became rapidly and efficiently associated with membranes which had a density similar to that of plasma membrane-derived vesicles. Protease-protection studies confirmed that the Gag-containing membranes were of plasma membrane origin, since in crude cell homogenates, the bulk of newly synthesized Gag was protease-resistant as expected of a protein that binds to the cytoplasmic leaflet of the plasma membrane. Taken together these data indicate that targeting of M-MuLV Gag to the cell surface proceeds via direct insertion of the protein to the cytoplasmic side of the plasma membrane. Furthermore, since the membrane insertion reaction is highly efficient and specific, this suggests that the reaction is dependent on as-yet-unidentified cellular factors.

Production of infectious recombinant Moloney murine leukemia virus particles in BHK cells using Semliki Forest virus-derived RNA expression vectors.

We describe a heterologous, Semliki Forest virus (SFV)-driven packaging system for the production of infectious recombinant Moloney murine leukemia virus particles. The gag-pol and env genes, as well as a recombinant retrovirus genome (LTR-psi (+)-neoR-LTR), were inserted into individual SFV1 expression plasmids. Replication-competent RNAs were transcribed in vitro and introduced into the cytoplasm of BHK-21 cells using electroporation. The expressed Moloney murine leukemia virus structural proteins produced extracellular virus-like particles. In these particles the gag precursor was processed into mature products, indicating that the particles contained an active protease. The protease of the gag-pol fusion protein was also shown to be active in a trans-complementation assay using a large excess of Pr65gag. Moreover, the particles possessed reverse transcriptase (RT) activity as measured in an in vitro assay. Cotransfection of BHK-21 cells by all three SFV1 constructs resulted in the production of transduction-competent particles at 4 x 10(6) colony-forming units (cfu)/ml during a 5-hr incubation period. Altogether, 2.9 x 10(7) transduction-competent particles were obtained from about 4 x 10(6) transfected cells. Thus, this system represents the first RNA-based packaging system for the production of infectious retroviral particles. The facts that no helper virus could be detected in the virus stocks and that particles carrying the amphotropic envelope could be produced with similar efficiency as those that carry the ecotropic envelope make the system very interesting for gene therapy.

Chehalis River Disease: A Unique Gill Disease of Salmonids

Epidemic mortalities of chinook salmon alevins (Oncorhynchus tshawytscha) have occurred annually at a freshwater hatchery at the Chehalis River, British Columbia, Canada, since 1982. Previous work has identified branchial epithelial hyperplasia as the predominant pathological change. Our initial work lead us to test the hypothesis that Chehalis River disease (CRD) was actually a novel form of bacterial gill disease (BGD). Histological and ultrastructural observations indicated that CRD-affected gills harboured a bacterial biofilm apparently complexed with particulate iron prior to and during clinical CRD. In one instance, extracellular viruslike particles were observed. Large numbers of a mixed bacterial population were recovered from diseased gills. Flavobacterium sp. and Pseudomonas fluorescens were considered the predominant isolates from the 1990–91 outbreak. However, indirect immunofluorescence of gills with anti-Flavobacterium branchophila serum failed to detect antigenically similar bacteria on the gills of CRD-affected fish. Chemotherapeutic treatment of both naturally and experimentally infected fish was successful, thereby confirming the infectious nature of the disease. We conclude that CRD is a form of BGD, but the significance of the branchial viruslike particles and the presence of particulate iron has not yet been established.

Virus-like particles induced in guinea pig cells by 5-bromo-2'-deoxyuridine are morphologically similar to murine B-type virus.

VIRUS-LIKE particles in tissues of leukaemic and normal guinea pigs have been demonstrated repeatedly1–5. Tumour cells contain intracisternal particles resembling mouse intracisternal A particles3, and extracellular virus-like particles are found in tissues of leukaemic guinea pigs. To the best of our knowledge, a convincing micrograph of a virus-like particle budding from the surface of a guinea pig tumour cell (in vivo) has never been published. In a separate study (J.E.D. and K.P., in preparation), it has been concluded that the guinea pig leukaemia virus1–3 results from the release of intracisternal A particles from dying tumour cells.

Isolation and characterization of extracellular virus-like particles from BHK cells. Brief report.

Isolation and characterization of extracellular virus-like particles from BHK cells. Brief report.