Endocytosis and Penetration: Part 2 Lecture Notes
Key Words and Terms
Virus, host cell machinery, virus entry, receptor mediated endocytosis, pinocytosis, phagocytosis, early endosome, late endosome, lysosomes, penetration, cytoskeleton, GTPases, fusion machinery.
Binding of viruses to the cell surface is in most cases followed by endocytosis and penetration of the viral capsids into the cytosol from an intracellular organelle such as an endosome. Once inside the cytosol, the viruses or their capsids move to their site of replication and uncoat. For most DNA viruses this is the nucleus. Most RNA viruses replicate in different cytoplasmic locations.
The entry program of a typical animal virus
The major stages in the entry of a typical virus are as follows:
The entry of viruses thus involves multiple, consecutive steps. Note that while some viruses can penetrate directly through the plasma membrane, the endocytic entry is the rule.
Endocytosis is a process by which fluid, solutes, membrane, and particles are internalized by invagination of the plasma membrane and formation of closed vesicles or vacuoles.
Receptor-mediated endocytosis is a special category of endocytic processes that depends on binding of the cargo (ligand) to cell surface molecules (receptors). The ligand/receptor complexes are then internalized together. This is very important because the receptors help to concentrate specific ligands on the cell surface even if these are present in very low concentrations, and to mediate and control the uptake process. The actual mechanisms by which endocytosis occurs can involve clathrin-coated pits, but other mechanisms are also possible.
Cellular endocytic pathways
Cells support many endocytic mechanisms. They are typically divided into phagocytosis (cell eating) and pinocytosis (cell drinking). Phagocytosis is the uptake of large particles into large tight-fitting vacuoles (ex. bacterial uptake by macrophages). It is restricted to specialized cell types such as certain immune cells. Pinocytosis includes a wide spectrum of mechanisms and cellular machinery. Clathrin-mediated endocytosis is the best characterized and is used by many viruses (see lecture 1 for examples). Some viruses can make use of different endocytic pathways in the same cell. Others use cell-type specific endocytic mechanisms.
After release from the plasma membrane, the cargo-containing endocytic vesicles and vacuoles usually fuse with endosomes. These are components of a highly complex and dynamic network of organelles forming the endocytic pathway. In addition to early endosomes, the pathway contains late endosomes, recycling endosomes, and lysosomes.
Early endosome: Almost all incoming-cargo is first delivered to these peripherally located complex organelles that serve as an important sorting station for incoming cargo. They have globular domains and long tubular extensions. Most of the incoming membrane components are recycled back from early endosomes to the plasma membrane either via the recycling endosomes or directly. A large fraction of the fluid, solutes, and particles (such as viruses) are directed to late endosomes.
Late endosome: These arise from the globular domains of early endosomes that detach and mature into late endosomes through loss/gain of factors, formation of internal vesicles, and movement into the perinuclear space.
Lysosome: Lysosomes are highly acidic vacuoles filled hydrolases. They fuse with late endosomes to form endolysosomes in which the incoming cargo is digested.
Importantly, the pH of each compartment in the pathway decrease as one progresses from early endosomes to lysosomes. In early endosomes the pH is 6-6.3, while in lysosomes it is lower than 5.0. The pH threshold for activation of the penetration process varies between 6.5 and 5.2 or so depending on the virus type. This dictates in which organelle penetration occurs.
The primary endocytic organelles of macropinocytosis and phagocytosis (macropinosomes and phagosomes) are unique. However they too can feed into the classic endosomal system. The trafficking of these specialized organelles depends on the cell-type and cargo.
Which endocytic mechanisms are used?
It is important to realize that endocytosis displays great complexity with several different mechanisms working in parallel. Each endocytic mechanism relies on a distinct subset of cellular factors. As mentioned in lecture 1, there are a variety of biochemical and genetic tools that one can use to perturb the cell. By determining a perturbation sensitivity profile one can define the endocytic mechanism that a particular virus it is using.
The cellular factors used to define endocytic pathways include:
Example 1: Endocytosis of HPV16
HPV16 uses non-coated vesicles for endocytosis, and moves to late endosomes before penetration. With a half time of particle internalization of 3 hours, and a half time of acidification of 10 hours, entry of HPV16 is unusually slow. The cellular factors required were determined using the perturbation mechanisms described in lecture 1. The profile showed that this virus uses a novel endocytic mechanism; a non clathrin, non caveolin pathway. It is likely that other viruses utilize this mechanism as well.
After endocytosis, viruses are still separated from the cytosol by a membrane. To transfer their capsids into the cytosol they have to penetrate this membrane. What follows represents one of a few stages in the entry program where the virus actively participates. In response to low pH or other cues, the spike glycoproteins of enveloped viruses undergo a conformational change and become membrane fusion-active. They then mediate the fusion of the viral envelope with the limiting membrane of the organelle in the lumen of which they are located. Non- enveloped viruses respond to the cues by lysing the membrane or by generating a pore through which the genome can be translocated into the cytosol.
The site of virus penetration varies depending on the virus:
Example 1: Non-enveloped virus penetration by endosome lysis
Adenoviruses 2 and 5 are non-enveloped viruses that cause common cold symptoms. The viruses are endocytosed by a clathrin-mediated process. Low endosomal pH causes a change in the virus particle. This makes components of the capsids capable of inducing lysis of the endosomal membrane. The virus then escapes from the ruptured endosome, and moves to the nuclear pore complexes where it is uncoated. As the lysis is compartmentalized and transient, it apparently does not damage the cell.
Example 2: Non-enveloped virus penetration by pore formation
Poliovirus uses a pore forming mechanism to deliver its genome into the host cell cytosol. Upon activation via receptor binding, capsid components insert into the endosomal membrane forming a pore through which the RNA genome passes. The empty viral capsid remains in the endosomal compartment.
Example 3: Enveloped virus fusion at the plasma membrane
In the case of HIV, the fusion activity of the surface spike glycoproteins is activated upon consecutive binding to two receptors. This triggers a major conformational change that results in fusion of the virus envelope with the plasma membrane. HIV can also enter by endocytosis and fuse in endosomes.
Example 4: Enveloped virus fusion in endosomes
The Influenza virus hemagglutinin, a spike glycoprotein, is responsible for both virus binding to cell surface receptors and for penetration by membrane fusion. Influenza is endocytosed by a clathrin-mediated and by a non-clathrin mediated endocytosis mechanism. Upon reaching the late endosome, the hemagglutinin undergoes an acid-triggered conformational change (pH threshold about 5.5). This leads to insertion of the fusion peptide of the glycoprotein into the endosomal membrane, approach of the two membranes, and eventually to a membrane fusion reaction. The viral capsids are delivered into the cytosol, and the viral membrane becomes part of the endosomal membrane.
Viral glycoproteins: Complex membrane fusion machines
Several viral glycoproteins have been crystallized. The structures show that while they share the overall functional principles, they are structurally different. Importantly, the energy necessary to mediate fusion is provided by the conformational change, and therefore does not requires energy in the form of ATP. The reactions are, as a rule, irreversible, which means that unlike cellular fusion machines, viral fusogens can only induce a single fusion event.
Mechanism of class II viral fusion
The fusion reaction mediated by a so-called class II viral fusion protein is discussed here as an example. It involves is a multistep process beginning with the low pH-triggered conformational change in the SFV spike glycoprotein complex that leads to a change in oligomeric structure of the fusion protein E1 subunits. In the homo-trimeric structure generated, each E1 subunit exposes a fusion peptide, a hydrophobic loop previously hidden in the structure. The fusion peptides insert into the target membrane, and several trimers together then undergo a cooperative change pulling the viral and host membranes into close proximity, which results in the fusion of the outer bilayer leaflets. This is known as hemifusion. Fusion between the two remaining leaflets occurs, next and in the final step the fusion pore expands to let the viral capsid pass through.
Example: Visualization of influenza virus fusion in a live cell
To visualize the fusion of an incoming influenza virus in live cells, investigators used a self-quenching fluorescent dye to label the envelope. After endocytosis, the virus could be seen to enter the perinuclear region of a cell. Fusion was seen as a “flash” of fluorescence caused by de-quenching the dye as it is diluted out in the late endosome membrane.
Intracellular transport of viral capsids
After penetration viral capsids delivered into the cytosol need to move to the site of genome uncoating and replication. For many viruses this means moving long distances through the cytosol to the nucleus. Once again, viruses take advantage of existing cellular machinery to achieve this. In many cases, microtubules and dynein-based motors (Discussed in Ron Vale’s Lecture 1: Introduction to Motor Proteins) are hijacked to move the capsids to the nuclear pore complex.
Nuclear import of viral genomes
To transport their genome into the nucleus most viruses use the nuclear pore complexes (NPCs). There are several different ways in which viruses ensure genome delivery into the nucleoplasm.
Words you should be able to define
Virus, virus entry, virus uncoating, virus replication, virus genome, capsid, spike glycoprotein, endocytosis, early endosome, late endosome, lysosomes, macropinosome, phagosome, fusion, fusion peptide, hemifusion, penetration, intracellular transport, nuclear pore complex.