The Science of Engineering Viruses

Introduction

Have you ever wondered how viruses are engineered in a lab setting? Although it may sound like complex wizardry, the process is quite approachable with a bit of molecular biology under your belt. This blog post aims to demystify the creation of viruses, making it accessible to anyone curious about the steps involved.

Understanding Virus Structure

Before diving into virus creation, it’s essential to understand what a virus consists of, in this case specifically lytic RNA viruses, as these are the ones we are engineering. Imagine a virus as a tiny, well-organized package as shown in the schematic drawing. 

At its core, there’s a strand of RNA containing several genes, each coding for specific proteins. Surrounding the RNA is a protective layer called the capsid. The capsid covered RNA and a few essential proteins are packaged inside the envelope. Covering the envelope are glycoproteins, which are crucial for the virus to latch onto and infect host cells. This ensemble of components is what we need to replicate in the lab to create a virus.

The Virus Life Cycle

To understand how we make viruses, let’s look at their lifecycle, focusing on a typical lytic RNA virus. It starts when a viral glycoprotein binds to a receptor on a host cell — think of it as a key fitting into a lock. For vesicular stomatitis virus, this receptor is LDLR on host cells. Once bound, the virus enters the cell, sheds its glycoproteins and envelope, and releases RNA and polymerase into the cytoplasm. Here, the polymerase springs into action, using the RNA to produce mRNA. These mRNA strands are then translated into viral proteins by the cell’s ribosomes. Once enough proteins are assembled, the polymerase will start making full length copies of the RNA. The matrix proteins will coat the inside of the cell membrane and the glycoproteins will be exported to the outside of the cell membrane. Finally the full length RNA with the polymerase and these proteins will form new virus particles that eventually burst out of the cell, rupturing the cell membrane and killing it in the process.

Making a Virus in the Lab

The detailed life cycle of a virus underscores the complexity involved in replicating this process in the laboratory. To begin, we introduce a vaccinia virus carrying the T7 gene into HEK 293T host cells, which provides the T7 polymerase necessary for transcription. This polymerase facilitates the transcription of DNA plasmids into mRNA and the viral DNA into RNA. After infecting the cells with the vaccinia virus, we transfect them with helper DNA plasmids. These plasmids provide essential proteins for replication, such as the polymerase, P, and N proteins. Finally, we transfect the cells with the viral genome DNA.

Successfully combining these components in a single cell requires precise control over the quantities introduced to avoid overwhelming the cell. If not enough plasmids are provided, the chance that all necessary components are present in large enough quantities becomes too small. Conversely, providing too much can lead to cell death due to DNA toxicity. This balance makes the task sophisticated yet achievable within our lab setting. Through this process, we are able to construct viruses in a controlled environment, paving the way for the development of oncolytic viral therapies.

Optimizing the Process

This method, while intricate, is ready for optimization. For instance, using a mammalian promoter to transcribe the viral genome can bypass the need for the vaccinia virus and T7 polymerase. 

Understanding and replicating the viral lifecycle not only advances our basic knowledge but also enhances our ability to develop targeted therapies, such as oncolytic viruses that selectively attack cancer cells. By manipulating the viral components, we can design viruses that are specific to certain cells, minimizing damage to healthy cells and maximizing therapeutic potential. We make viruses specific by engineering selective infection and selective replication into their genome. We have covered how we do that in other blog posts.

In summary, though the process of making viruses in a lab is complex, it’s built on basic principles of molecular biology that, with the right tools and knowledge, can be understood and applied by scientists to create new forms of treatment and study.