Viruses as Microscopic Trojan Horses: Part 1 Lecture Notes
Key words and terms
Virus, host cell, virus entry, virus replication, genome, capsid, lipid bilayer envelope, binding, endocytosis, uncoating, glycolipid, filopodia, signal transduction, Simian virus 40, Human Papilloma Virus-16.
What is a virus?
Viruses are obligate intracellular parasites. This means that they cannot replicate on their own but instead have to rely on host cells and the cellular biosynthetic machinery for reproduction. They enter the cytosol or nucleus of a host cell, and force the cell to synthesize new virus particles according to instructions that they provide in the form of RNA or DNA.
A virus particle is generally very small and structurally quite simple. In addition to proteins and sometimes also lipids, it contains as an essential component one or more molecules of DNA or RNA. These constitute the genome of the virus that carries genes necessary for the production of new viruses in the host cell. The number of genes varies from a handful to a few hundred depending on the virus.
In a virus particle isolated from the extra cellular space, the genome is present in a highly condensed form and protected by a coat of proteins in the so called viral capsid. Capsids take the form of a helix or they have an icosahedral protein shell that encloses the genome. In enveloped viruses, the capsid is further covered by a lipid bilayer membrane, the viral envelope. The membrane contains additional viral proteins that are usually glycosylated and often form spike-like projections required for cell attachment and penetration into the host cell.
Virus particles serve as carriers for the viral genome and accessory proteins from the infected host cell to an uninfected cell. Importantly, the particle must also protect the genome in transit so that it can be delivered in a replication competent form. The target cell can be a neighboring cell in the same tissue and organism, or a cell in another organism. The lifecycle of a typical virus occurs in four stages: Entry and uncoating, replication, assembly, and release. Once infected, the new host cell produces and releases thousands of new virus particles. The cell often suffers from the infection and eventually dies. Many viruses are therefore dangerous pathogens.
There are different types of animal viruses
There are many different types of viruses. Here we focus on animal viruses. They differ in size, shape, composition, and in the type of cells and host animals that they infect. Both DNA and RNA viruses come in both enveloped and non-enveloped forms.
Viruses as a health risk
Infectious diseases are the second most common cause of human deaths globally, and about half these are attributed to viruses. There are a number of long established viruses in the human populations that cause diseases such as polio and measles. In addition, there are re-emerging viral disease already known as human pathogens but expanding due to local and global changes in the world. Finally, there are emerging viruses, such as SARS virus and HIV-1, which have their origin in other species and are new to the human population.
Viruses are infectious and spread from organism to organism. The most common routes of virus transmission are directly via contact, via aerosols, through contaminated surfaces, by exchange of body fluids, etc. Viruses can also be transferred by insects, and as contaminants in food and water. There are many factors that contribute to the rate and mode of transmission, including population density and fitness, trade and travel, hygiene, and effective vaccination and other counter measures.
Studying virus entry
The study of virus entry requires an interdisciplinary approach including, in addition to virological methods, approaches from cell biology, molecular biology, biochemistry, systems biology, etc. Light and electron microscopy are important tools, together with in vitro systems, and a variety of perturbations through inhibitors, mutant viruses and mutant cells, expression of dominant and constitutively active proteins, and siRNA silencing. Most studies so far have been performed in tissue culture cells.
Example 1: Entry of Semliki forest virus (SFV)
SFV is a simple enveloped RNA virus that is transmitted by mosquitoes. It serves as an important model virus for analyzing entry. A variety of experiments including scanning electron microscopy have demonstrated that after attaching to the cell surface, the SFV particles are internalized by endocytosis in clathrin-coated vesicles and delivered to the lumen of endosomal vacuoles called endosomes. Here they are exposed to lowered pH, and this triggers a conformational change in the spike glycoproteins. This change activates a membrane fusion activity in the viral proteins, and the virus fuses its envelope membrane with the limiting membrane of the endosome. As a result, the viral capsid is delivered to the cytosol where the RNA is rapidly uncoated and used as an mRNA to translate viral proteins. Within a few hours, the single particle that entered manages to convert the host cell into a virus factory.
The entry program of a typical animal virus
Virus entry occurs in multiple stages beginning with virus binding to cell surface receptors. Different viruses use different cell surface molecules as their receptors. Following binding, the most viruses move laterally on the cell surface. During this process, the may activate cellular signaling pathways to prime the host cell for uptake. Although some viruses can penetrate directly through the plasma membrane, the majority undergo endocytosis first. The primary endocytic vesicles deliver the viruses to secondary organelles such as endosomes whose conditions favor virus penetration. Once in the cytosol, the capsids are often transported on microtubules to the location of uncoating and replication. For most DNA viruses the target is the nuclear pore complex through which the genome is transported into the nucleus where replication is initiated.
Of course there are exceptions. Some viruses fuse directly with the cell surface by-passing the need for endocytosis. Others fail to initiate cellular signaling pathways and enter pre-formed endocytic carriers. Viruses that replicate in the cytosol are transported to specific cytosolic locations for uncoating and replication rather than to the nuclear pore complex.
Entry involves multiple steps where the movement of the virus deeper into the cell is coupled to step-wise uncoating of the particle according to a built-in disassembly program. Almost all steps depend on cellular factors. The viruses respond to cues such as low pH by undergoing changes made possible by the metastable structure of the capsids or spike glycoproteins. The incoming virus and the cell engage in a sort of biochemical dialogue that allows the critical events in the program to occur in the right place at the right time. For all of this to be possible, the viruses must speak the language of the cell, and the cell has to inadvertently support the entry program.
Differences between enveloped and non-enveloped virus infection strategies
For transmission of the genome form cell to cell, enveloped viruses use the same mechanism as cells to move macromolecules between membrane-bounded compartments, i.e. vesicle-mediated transfer. The virus envelope serves as the transport vesicle. The cargo, in this case the viral capsid, is packaged into the vesicle during budding and membrane fission from the plasma membrane or the limiting membrane of an intracellular transport vesicle. The envelope membrane thus formed later fuses with a membrane in the uninfected cell releasing the capsid into the cytosol. Thus transfer is achieved without the capsid having to pass directly through a hydrophobic interior of a membrane.
Non-enveloped viruses work differently. They are typically released from infected cells by a lytic event. The released particles are then endocytosed and penetrate into the cytosol of another cell either by lysing an endosomal membrane, or by generating a channel in a cell membrane through which the genome can slip into the cytosol.
Viruses use many different types of cell surface receptor molecules. Their identity and tissue distribution dictates which cell-type and species can be infected, and ultimately what type of disease the virus causes. The receptors are normal cellular proteins, lipids, or carbohydrates on the cell surface that end up inadvertently playing a role in assisting a pathogen. Often viruses use multiple receptors, and receptor binding is multivalent. Molecules that serve merely to bind the viruses and concentrate them on the cell surface are called attachment factors, whereas the receptors mediate in addition conformational changes in the virus, trigger signaling, or induce endocytosis.
Example 2: Simian Virus 40 (SV40)
SV40 is a non-enveloped DNA virus that replicates in the nucleus. It is a member of the polyoma virus family. It has an icosahedral capsid - 42nm in diameter - composed of 72 homo-pentamers of the surface protein VP1. Binding to the cell surface is mediated by attachment of VP1 pentamers to the carbohydrate moiety of GM1, a ganglioside. Gangliosides and other glycolipids are components of the plasma membrane. The crystal structure of VP1 in complex with GM1 shows that each VP1 in the pentamer can bind one GM1 receptor molecule.
Cell free systems can be used to study viruses
Viruses on the surface of cells display multiple modes of movement ranging from random diffusion to directed movement (‘surfing’) along surface specializations such as filopodia. In some instances, artificial membranes can be used to investigate particular aspects of movement. Using this artificial system it was recently shown that the SV40 bound to GM1 receptors moves along the membrane by sliding and wobbling rather than rolling.
Example 3: Human papilloma virus 16 (HPV16)
HPV16 is a member of the papillomaviridae. It is a non-enveloped DNA virus that binds to cell surface heparansulfates, enters cells by endocytosis, and penetrates after acid-activation. HPV16 replicates in the nucleus, and is a major causative agent of cervical cancer. Using electron microscopy virus it was found to bind to cellular filopodia, long motile finger-like projections from the cell body. When visualized by fluorescence live cell imaging, it was determined that HPV16 particles are moving or “surfing” on the filopodia. Movement occurs in a directed fashion towards the cell body, and is dependent upon retrograde movement of actin within the filopodia. Endocytosis of the virus particles appears to occur at the base of filopodia.
Summary of events during virus binding
Virus binding occurs by multivalent association of the virus with its receptor. This results in receptor clustering, which in turn activates a trans-bilayer coupling (outside-in) initiating cellular signaling pathway(s). This activation informs the cell of the presence of the virus particle, resulting in an endocytic “reflex” by the cell and the uptake of the virus particle. This is followed by transport of the particle into cytoplasmic vacuoles followed by virus penetration.
Words you should be able to define
Virus, host cell, entry, uncoating, replication, virus genome, capsid, non-enveloped virus, enveloped virus, envelope glycoprotein, attachment factor, virus receptor, signal transduction, endocytosis, endosome, pH-activation, fusion, fission, filopodia.