Sample carryover and cleaning procedures for asymmetrical flow field-flow fractionation instrument.

Asymmetrical flow field-flow (AF4) fractionation aims in separation of sample components to yield elution of homogenous fractions identified as well-defined peaks in the chromatograms. Separation that occurs in matrix-free open channel potentiates high recovery that can be close to 100%. However, sample properties and separation conditions may induce carryover of sample components during AF4 analysis and in sample sequences. This compromises the quality of the data collected from the online detectors and the downstream offline analytics of the collected fractions. In this study, we followed sample carryover in AF4 using model viruses and analyzed various cleaning solutions and rinse methods to reduce carryover. We introduce an SDS-NaOH -based rinsing and decontamination protocol for the AF4 instrument enabling high-quality data collection.

Self-assembly of a viral molecular machine from purified protein and RNA constituents.

We present the assembly of the polymerase complex (procapsid) of a dsRNA virus from purified recombinant proteins. This molecular machine packages and replicates viral ssRNA genomic precursors in vitro. After addition of an external protein shell, these in vitro self-assembled viral core particles can penetrate the host plasma membrane and initiate a productive infection. Thus, a viral procapsid has been assembled and rendered infectious using purified components. Using this system, we have studied the mechanism of assembly of the common dsRNA virus shell and the incorporation of a symmetry mismatch within an icosahedral capsid. Our work demonstrates that this molecular machine, self-assembled under defined conditions in vitro, can function in its natural environment, the cell cytoplasm.

A novel virus-host cell membrane interaction. Membrane voltage-dependent endocytic-like entry of bacteriophage straight phi6 nucleocapsid.

Studies on the virus-cell interactions have proven valuable in elucidating vital cellular processes. Interestingly, certain virus-host membrane interactions found in eukaryotic systems seem also to operate in prokaryotes (Bamford, D.H., M. Romantschuk, and P. J. Somerharju, 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1467-1473; Romantschuk, M., V.M. Olkkonen, and D.H. Bamford. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1821-1829). straight phi6 is an enveloped double-stranded RNA virus infecting a gram-negative bacterium. The viral entry is initiated by fusion between the virus membrane and host outer membrane, followed by delivery of the viral nucleocapsid (RNA polymerase complex covered with a protein shell) into the host cytosol via an endocytic-like route. In this study, we analyze the interaction of the nucleocapsid with the host plasma membrane and demonstrate a novel approach for dissecting the early events of the nucleocapsid entry process. The initial binding of the nucleocapsid to the plasma membrane is independent of membrane voltage (DeltaPsi) and the K(+) and H(+) gradients. However, the following internalization is dependent on plasma membrane voltage (DeltaPsi), but does not require a high ATP level or K(+) and H(+) gradients. Moreover, the nucleocapsid shell protein, P8, is the viral component mediating the membrane-nucleocapsid interaction.

Packaging and replication regulation revealed by chimeric genome segments of double-stranded RNA bacteriophage phi6.

Bacteriophage phi6 has a double-stranded RNA genome composed of three linear segments, L, M, and S. The innermost particle in the virion of phi6, like in the other dsRNA viruses, is an RNA-dependent RNA polymerase complex, which carries out all the functions needed for the replication of the viral genome. Empty polymerase complexes can package the single-stranded copies of the viral genome segments, replicate the packaged segments into double-stranded form (minus strand synthesis), and then produce new plus strands (transcripts) from the double-stranded RNA templates. The three viral genomic segments contain unique packaging signals at their 5' ends, and minus strand synthesis initiation is dependent on the sequence at the 3' end. Here we have constructed chimeric segments that have the packaging signal from one segment and the minus strand synthesis initiation signal from another segment. Using purified recombinant polymerase complexes and single-stranded/chimeric and original RNA segments, we have analyzed the packaging and replication regulation operating in in vitro conditions. We show that the 5' end of the L genome segment in single-stranded form is needed to switch from the packaging to the minus strand synthesis and the same sequence is required in double-stranded form to switch on plus strand synthesis. In addition we have constructed deletions to the M segment to analyze the possible regulatory role of the internal noncoding area of this segment.