Specific Aims
The overall goal of this proposal is to investigate the mechanism of virus-mediated formation of double membrane vesicles within the endoplasmic reticulum (ER). The ER is considered the origin of induced intracellular structures for a number of viruses.1 It has been shown that the formation of double-membrane vesicles (DMVs) within the ER is a critical step in viral infectivity. Thus far, there are two conventional models that attempt to describe the formation of DMVs: protrusion detachment and double budding.1 Albeit different, both models assume that the integral membrane proteins of viruses attach to the membranes in localized areas, which results in budding and subsequently, formation of tubules from the ER membrane. We know that viral proteins induce membrane curvature. Previous studies have hypothesized that the five primary mechanisms employed by viral proteins are: intrinsic membrane protein shape, amphipathic helix insertion, membrane protein oligomerization, scaffolding by peripheral protein, and lipid composition; however, the specific mechanism has yet to be determined. Additionally, the general mechanism for the stabilization of large double-membrane sheet-like structures like the ER and other organelles such as, the Golgi Apparatus, is unknown.2 The ubiquity of protein-localization in all proposed mechanisms underscores the importance of understanding the viral protein- membrane interface from a fundamental perspective. While conventional models for membrane curvature generation have been discovered, a number of key details pertaining to the problem still remain unresolved. What role does membrane phase structure and dynamics play in regulating the membrane shape? How do membrane shape changes affect viral infectivity, and subsequently, other integral cellular processes? Addressing these questions can greatly aid in elucidating the mechanism of viral infection that will ultimately lead to the development of novel therapeutic modalities for infections and diseases associated with viruses and ER dysfunction.
- Aim 1: Determine the mechanism of double-membrane vesicle formation within the endoplasmic reticulum.
- Aim 2: Determine how the luminal domains of viral membrane proteins affect ER membrane curvature induction and detachment.
- Aim 3: Determine how curvature asymmetry in endoplasmic reticulum sheets affect overall membrane structure stability.
II. Significance and background
Viruses. Viruses are small, parasitic agents that survive by infecting host cells with genetic material and, utilizing that cell’s machinery, producing viral protein to promote each step of virus genome replication. In a typical viral infection pathway, the virus is first invaginated into the host cell via receptor-mediated endocytosis. The genetic material of the virus is translated and transcribed, leading to viral protein production and assembly. The resulting progeny undergo additional structural changes before leaving the cell to infect other hosts. Despite the large variations among viruses, the viral life cycle – entry, translation, replication, assembly, morphogenesis, and egress – is employed by nearly all viruses.6
Viruses have developed the ability to hijack every intercellular organelle and while most organelles are only involved in a limited number of steps, the endoplasmic reticulum is involved throughout the life cycle.6 Methods of ER exploitation include manipulating Ca2+ homeostasis, activating IRE-1, and using the native, endocytosis pathways for progeny to egress.
Endoplasmic reticulum. The endoplasmic reticulum is an organelle found in eukaryotic cells that is a key player in many cellular processes such as, protein synthesis and transport. Unlike other organelles with simple, spherical structures, the ER is complex; it consists of a dynamic tubular and cisternal network that connects to the outer nuclear envelope. Membrane tubules are continuously forming, fusing, and moving with one another. Previous studies have found that virus-induced membrane deformations occur at nearly every step but a wide range of virus. For example, Rubella, Semliki Forest virus, Flock house virus, and Kunjin virus have modification sites in the lysosomal, mitochondrial, and Golgi membrane. However, the large majority of viruses have modification sites in the ER lipid membrane; E.g. equine arteritis virus, hepatitis C virus, murine hepatitis virus, Poliovirus, SARS-coronavirus, and tobacco mosaic virus.1 The ER appears to be the preferential site for membrane deformation due to the unique physical structure and the abundance of adaptive lipid membrane.
Intracellular membrane modification. In the replication and assembly steps of the virus life cycle, endocytosed viral protein is delivered to organelles in transport liposomes. These vesicles function by budding from the donor liposomes and fusing to the acceptor compartment. Vesicle membrane dynamics are thought to be primarily controlled by coat proteins; e.g. Coatomer protein complex I and II (COP I and II), and clathrin coat proteins. Research by Miller and Krijnse-Locker (2008) give the putative explanation for why these vesicles form:
“…to help to increase the local concentration of components required for replication; provide a scaffold for anchoring the [viral protein replication complex]; confine the process of RNA replication to a specific cytoplasmic location; aid in preventing the activation of certain host defense mechanisms that can be triggered by dsRNA intermediates of RNA-virus replication; tether viral RNA during unwinding; and provide certain lipids that are required for genome synthesis.”
Figure 1. Proposed models for virus-induced liposome formation. Figure adapted from “Modification of intracellular membrane structures for virus replication,” by S. Miller and J. Krijnse-Locker, 2008, Nature Review Microbiology, 6, 369, Copyright (1999) American Society for Microbiology.
It is evident that these vesicles are vital to viral infection but the specific mechanism of vesicle formation still remains unknown. Two models have been proposed, protrusion detachment and double budding which are shown in Figure 1. In the protrusion detachment model, a tubule extends from the ER cisternae, bends to form a loop, and detaches forming the double membrane liposome. In the double budding model, a single membrane liposome buds into the ER and forms a double membrane upon budding out.1 Previous studies have described the mechanism in which viruses induce membrane curvature: insertion of conical-shaped membrane proteins, insertion of proteins with amphipathic alpha helices, oligomerization of native integral-bound membrane proteins, formation of protein scaffolds, and modification of lipid composition.1 Using these findings and various imaging techniques, the mechanism of DMV formation for all organelles involved in the viral infection pathway can be elucidated.
III. Innovation
There is currently a wide gap in our knowledge about the specific mechanisms of viral infection. In the field of cellular biology, virus-mediated membrane deformation is not well understood. The function of the induced membrane vesicles and their origins within the cell are still unclear for a number of viruses. From this study, we can not only learn about the ER but also other double membrane organelles during viral infection. Over time, these findings can then be used to develop new therapies for people suffering from life-threatening viral infections and diseases associated with ER dysfunction.
IV. Approach
Methodology: To investigate my questions, I will begin by isolating ERs in an in vitro system and conduct experiments that quantitatively and qualitatively monitor the changes in the membrane morphology.
I. Synthesis and characterization of endoplasmic reticulum using Xenopus laevis eggs.3
In vitro systems that allow for the synthesis of ER networks have been developed.3 From these studies, it has been shown that microtubules are not required for the development of this system. Instead, other cytosolic factors produce a fusion reaction that causes round vesicles to become tubular networks.3 These cytosolic factors include Ca2+ concentrations and the presence of proteins. Thus, I will conduct experiments modifying each of these factors. Using electron microscopy, I will observe the changes in tubule formation as the concentration of cytosolic Ca2+ changes. I will also use Dynamic Light Scattering (DLS) to quantify these membrane changes. Rather than using proteinase K, a classic model system, I will use Green fluorescent protein
(GFP) and super-charged GFP mutants (+36 and -30) as positive controls. Because GFP is a naturally fluorescent protein, it would be much easier to use rather than using charged proteins that will require additional modification. I will use supercharged mutants because both super- positively charged and super-negatively charged proteins have a unique ability to withstand thermally or chemically induced aggregation.4 Using confocal fluorescence microscopy, I will monitor the changes in vesicle morphology as the respective cytosolic GFP concentrations change. Ensemble characterization of vesicle morphology will be performed by DLS for quantification of these changes.
II. Observing the effects of clathrin-coated vesicles on ER membrane morphology.
Clathrin-mediated endocytosis is a universal endocytic mechanism that involves the uptake of a pathogen into the cell from the surface using clathrin-coated vesicles. Clathrin is a self- polymerizing protein comprised of six parts: three heavy chains and three light chains that form triskelion structures that can polymerize into flat lattices or cages.5 Clathrin is one of three coat proteins that are used in viral infection and is employed specifically by many common viruses such as, poliovirus, Hepatitis C, and influenza. For this reason, I will use this protein to conduct my experiments. Additionally, this process is very similar to caveolin-mediated endocytosis so the results of this experiment can give insight to other coat proteins. First, I will synthesize GFP tagged SUVs (~100-400nm) with integrally bound clathrin light chains. Then I will observe the ER membrane morphology changes when various concentrations of these clathrin-coated liposomes are mixed using fluorescence microscopy. From these observations, I will determine which model for forming DMVs, protrusion detachment or double budding, is occurring.
References:
- Miller, S.; Krijnse-Locker, J., Modification of intracellular membrane structures for virus replication. Nat Rev Micro 2008, 6 (5), 363-374.
- Knorr, R. L.; Dimova, R.; Lipowsky, R., Curvature of Double-Membrane Organelles Generated by Changes in Membrane Size and Composition. PLoS ONE 2012, 7 (3), e32753.
- Dreier, L.; Rapoport, T. A., In Vitro Formation of the Endoplasmic Reticulum Occurs Independently of Microtubules by a Controlled Fusion Reaction. The Journal of Cell Biology 2000, 148 (5), 883-898.
- Thompson, D. B.; Cronican, J. J.; Liu, D. R., Engineering and Identifying Supercharged Proteins for Macromolecule Delivery into Mammalian Cells. Methods in enzymology 2012, 503, 293-319.
- McMahon, H. T.; Boucrot, E., Molecular mechanism and physiological functions of clathrin- mediated endocytosis. Nat Rev Mol Cell Biol 2011, 12 (8), 517-533.
- Ravindran, Madhu Sudhan, et al. “Opportunistic Intruders: How Viruses Orchestrate Er Functions to Infect Cells.” Nat Rev Micro 14.7 (2016): 407-20. Print.
- Dreier, Lars, and Tom A. Rapoport. “In Vitro Formation of the Endoplasmic Reticulum Occurs Independently of Microtubules by a Controlled Fusion Reaction.” The Journal of Cell Biology 148.5 (2000): 883-98. Print.