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.

Forest losses predict bird extinctions in eastern North America.

Claims that there will be a massive loss of species as tropical forests are cleared are based on the relationship between habitat area and the number of species. Few studies calibrate extinction with habitat reduction. Critics raise doubts about this calibration, noting that there has been extensive clearing of the eastern North American forest, yet only 4 of its approximately 200 bird species have gone extinct. We analyze the distribution of bird species and the timing and extent of forest loss. The forest losses were not concurrent across the region. Based on the maximum extent of forest losses, our calculations predict fewer extinctions than the number observed. At most, there are 28 species of birds restricted to the region. Only these species would be at risk even if all the forests were cleared. Far from providing comfort to those who argue that the current rapid rate of tropical deforestation might cause fewer extinctions than often claimed, our results suggest that the losses may be worse. In contrast to eastern North America, small regions of tropical forest often hold hundreds of endemic bird species.