War against viruses

A team of UK researchers is the first to observe what happens when a strand of RNA compresses into the coat protein envelope. According to the lead researcher, Professor Peter Stockley, the study results could lead to a completely new class of anti-virals, writes S Ananthanarayanan

The main thing a virus does is to reproduce. It is equipped with the genetic code to make copies of itself and little else. The virus evolves to have the surface features that allow it to enter a host cell. Once it has entered, it uses the resources of the host to multiply. This prevents the host from doing its usual work and also creates numbers of virus to infect other cells.

The usual defense against a virus attack has been to block the matching surface feature of the virus, or the host cell, to prevent entry. As the virus can evolve and get through anyway, the defense has to be developed afresh for every new strain. An alternate strategy has been to prevent the virus from reproducing after it has entered the cell. Alexander Borodavka, Roman Tuma and Peter G Stockley at the University of Leeds, UK, report in the journal, Proceedings of the National Academy of Sciences (PNAS), their work on the reproduction process of the virus, which could show a longer lasting way to stop the virus.

Reproduction

The genetic blueprint of cells is in the DNA, which is a millions-of-units-long molecule that contains the codes for the myriad of proteins that the organism needs to produce. The DNA is in the form of a pair of interlocking segments. At the time of reproduction, the segments decouple and each one re-creates its complement from the environment. The new DNA can then move out as a new cell, again to replicate, and so on.

Except that this act of replication is not something that is simply stated. With millions of units, the DNA would normally be many times the size of the cell in which it resides. But it is able to be there, in a small pocket of space, because it is ‘folded’ and wound up into a ball or egg shape and kept in place by an envelope of proteins produced by the cell. For reproduction, the proteins that initiate the process need to ‘open’ specific parts of the envelope and allow segments of DNA to emerge and replicate. The segments then need to ‘fold’ and wind themselves up and then merge, to form a new DNA ball.

All this action takes place in the fervent activity of the cell environment, with proteins, bits of DNA, fat, sugars in constant motion, bumping, twisting and bending, many times each split second.

But success of replication is important for the virus and the stages of the process are of interest to researchers, to find a place where they could step in and block the progress.

Virus genome

The way the DNA acts to create different proteins that define the cell is by sending out portions of the code in the form of segments called RNA. In some cases, the virus don’t have DNA or a set of DNA but only a single strand of the simpler RNA. Many of the viruses that cause significant human disease are of this kind. The usual vaccination strategy is unlikely to control more than a small portion of them.

There has been some work on using synthetic virus-like particles, which contain a bit of RNA, to attack disease causing organisms, including viruses implicated in cancers, but this approach has been found to have potential pitfalls in case of error in the RNA used. Detailed information of what happens at the molecular level when viral RNA replicate is necessary to develop new ways to control viral infections.The way the virus RNA coils and bends to get packed and compacted is with the help of specific proteins.

These proteins affect specific portions of the RNA by creating electrostatic forces that cause the RNA to bend at those points. The RNA thus ‘coils’ and further action by proteins causes it to ‘supercoil’, either in the same sense or in the sense opposite to the direction of the helix form of the RNA itself. Once the RNA has been condensed, there are other proteins that hold it in that way. Till the time comes for the DNA, in the case of cells, to send out portions of code in the form of RNA for creating proteins, or for the RNA in viruses that are replicating to go out and reproduce. At this time, the proteins that maintain the shape of the DNA envelope are modified.

This allows portions of DNA to project into the interior of the cell for proteins to form. In the case of viruses with RNA, the segments that have replicated become well compressed and enveloped by a coat of protein (coat protein or CP) by a spontaneous process, as the segments are short, unlike DNA strands. The view has been that this takes place as a result of the CP neutralising electrostatic forces that stiffen RNA strands, thus bringing about bends and folds.

RNA compression

The Leeds group used a method called single molecule fluorescent correlation spectroscopy (smFCS) to catch a glimpse of what happens when a strand of RNA compresses into the CP envelope. The method uses statistical analysis of the flashes that are seen when molecules under observation move in and out of a very small window, in the ceaseless motion of tiny particles in solution. Given the concentration of the particles, the number of flashes seen indicates the size and by varying the concentration, it is possible to monitor changes in size and the interactions between proteins and RNA at the single molecule level.

The  observations revealed an important difference between non-viral RNA and single strand RNA viruses. On addition of the CP that correspond to RNA fragments, the RNA rapidly reduces in volume as the different CP form small envelopes of compressed RNA sections. After this collapse and formation of sub-units, there is consolidation to form larger units of folded RNA.

This process does not take place with fragments of other RNA. In other cases, there is no formation of smaller units and the units formed are not uniform and suited for consolidation, as in the case of viral RNA. This indicates that the process with viral RNA depends on specific RNA-protein interactions.

The view so far, based on many CP being able to assemble even without RNA being there, around other kinds of RNA-like strands or even very small particles, has been that it is the proteins that are central to the assembly process.

Therapeutic efforts have also been directed at the protein components. But the discovery that viral RNA can be packaged into defined units suggests that there is a mechanism that depends on the RNA structure and sequence.

The Leeds team deduce that coat proteins and RNA act as ‘mutual chaperones’ to enable the protein shell enclosing the folded RNA to grow and complete the replication process with economy and efficiency. This action, of the RNA strand that is being packed, influencing the action of the proteins that are the outer cover, has been likened to clothes folding and packing themselves into a suitcase.

“It seems that viral RNAs have evolved a self-folding mechanism that makes closing the ‘viral suitcase’ very efficient. It’s as though ‘the suitcase and the clothes’ work together to close the lid and protect the content,” says team member Tuma.

Lead researcher Professor Peter Stockley said their results overturn accepted thinking about the process and could open a chink in the armour of a wide range of viruses.