No description available.

No description available.

No description available.

Foamy viruses (FV) represent a distinct genus in the retrovirus family and separate themselves from the large group of orthoretroviruses by various distinct features in their replication cycle (reviewed in Lindemann & Rethwilm, 2011). In gene therapy retroviruses are commonly used as vectors to deliver genetic information into target cells and also FV has been successfully used for example in a canine genetic disease model (Trobridge et al., 2009). Here we investigated the interactions between the FV capsid-forming protein ‘Gag’ and nucleic acids. We found that prototype FV (PFV) Gag binds various cellular mRNAs, incorporates them into the nascent particle and thereby enables their transfer into the cytosol of target cells. There these mRNAs can serve as template for protein translation. This feature seems uniquely efficient for PFV and we developed it further into a “RNA transfer vector system” allowing efficient transgene mRNA transfer into target cells, as showed in proof-of-principle experiments in vitro and in vivo (Hamann et al., 2014a). In parallel we started investigating the specificity in viral RNA genome packaging (Hamann et al., 2014b). To date little is known how PFV selects its RNA genome over the vast excess of cellular RNAs present in the cytosol. Elevated fundamental knowledge of this mechanism could help to make the “RNA transfer vector system” even more efficient since it would allow enrichment of certain specific “designer-RNAs” in virus particles.

Despite recent advances in antiretroviral therapy, nearly 37 million people continue to live with human immunodeficiency virus (HIV). Basic and applied research on the assembly of HIV could be enhanced by using a genetically tractable organism, such as yeast, rather than mammalian cells. While previous studies showed that expression of the HIV Gag polyprotein in Saccharomyces cerevisiae spheroplasts resulted in the production of virus-like particles (VLPs), many questions regarding the utility of yeast in HIV assembly remain uninvestigated. Here, we report use of S. cerevisiae for both the production of VLPs with selectively packaged RNA and to evaluate the human Y-box-binding protein 1 (YB-1) in selective RNA packaging into VLPs. Our data reveal: (1) When co-expressed alongside HIV-1 Gag, an RNA mammalian expression cassette is selectively encapsidated and released in VLPs produced from spheroplasts; (2) Inclusion of the 5’UTR-5’Gag RNA upstream of the mammalian expression cassette greatly increased the selectivity to which non-viral RNA was packaged into VLPs; and (3) heterologous expression of the human YB-1 protein in S. cerevisiae did not facilitate the selective packaging of viral RNA into VLPs, likely due to inability to bind upstream elements in the HIV-1 viral RNA. Overall, this research provides a key first step in the use of yeast for the production of viral vectors used in gene therapy, and lays a foundation for further experiments investigating the role of YB-1 and other host proteins in selective RNA packaging.

Experiments show that MgSO4 salt has a non-monotonic effect as a function of MgSO4 concentration on the ejection of DNA from bacteriophage lambda. There is a concentration, N0, at which the minimum amount of DNA is ejected. At lower or higher concentrations, more DNA is ejected. We propose that this non-monotonic behavior is due to the overcharging of DNA at high concentration of Mg⁺² counterions. As the Mg⁺² concentration increases from zero, the net charge of ejected DNA changes its sign from negative to positive. N0 corresponds to the concentration at which DNA is neutral. Our theory fits experimental data well. The DNA-DNA electrostatic attraction is found to be -0.004 kBT/nucleotide. Simulations of DNA-DNA interaction of a hexagonal DNA bundle support our theory. They also show the non-monotonic DNA-DNA interaction and reentrant behavior of DNA condensation by divalent counterions. Three problems in understanding the capsid assembly for a retrovirus are studied: First, the way in which the viral membrane affects the structure of in vivo assembled HIV-1 capsid is studied. We show that conical and cylindrical capsids have similar energy at high surface tension of the viral membrane, which leads to the various shapes of HIV-1 capsids. Secondly, the problem of RNA genome packaging inside spherical viruses is studied using RNA condensation theory. For weak adsorption strength of capsid protein, most RNA genomes are located at the center of the capsid. For strong adsorption strength, RNA genomes peak near the capsid surface and the amount of RNA packaged is proportional to the capsid area instead its volume. Theory fits experimental data reasonably well. Thirdly, the condensation of RNA molecules by nucleocapsid (NC) protein is studied. The interaction between RNA molecules and NC proteins is important for the reverse transcription of viral RNA which relates to the viral infectivity. For strong adsorption strength of the NC protein, there is a screening effect by RNA molecules around a single NC protein.