Characterisation of Viral Membrane Assembly Proteins A6 and A11 of Vaccinia Virus
(2025) KEMR20 20251Department of Chemistry
Computational Chemistry
- Abstract
- Poxviruses are large, double-stranded DNA viruses that replicate directly in the cytoplasm of the host eukaryotic cell. During assembly of viral particle progeny, viral membrane precursors derived from the endoplasmic reticulum, exhibit stabilised open ends inside intermediate structures called crescents. The process of viral membrane biogenesis during poxvirus assembly remains poorly understood. In particular, the key role of five essential Viral Membrane Assembly Proteins (VMAPs) remains enigmatic, including the contribution of A6 and A11 to the early stages of virion assembly, how they interact with one another and cell-derived membranes.
This study aims at characterising A6 and A11 using a combination of biophysical and biochemical... (More) - Poxviruses are large, double-stranded DNA viruses that replicate directly in the cytoplasm of the host eukaryotic cell. During assembly of viral particle progeny, viral membrane precursors derived from the endoplasmic reticulum, exhibit stabilised open ends inside intermediate structures called crescents. The process of viral membrane biogenesis during poxvirus assembly remains poorly understood. In particular, the key role of five essential Viral Membrane Assembly Proteins (VMAPs) remains enigmatic, including the contribution of A6 and A11 to the early stages of virion assembly, how they interact with one another and cell-derived membranes.
This study aims at characterising A6 and A11 using a combination of biophysical and biochemical approaches to investigate their potential interaction and assess A11 interaction with lipids in vitro.
We expressed and purified recombinant A6 and A11 using E. coli and wheat germ extract in cell-free expression systems, respectively. A6 was analysed by analytical ultracentrifugation, SEC-MALS, and mass photometry to study its oligomeric state. In parallel, the extent of A11 reconstitution in proteoliposomes was assessed by flotation assays. Finally, protein-protein interactions were examined using pull-down and western-blotting.
For A6, there is a concentration-dependent shift from monomer to higher-order assemblies. While A11 appeared to be difficult to purify and unstable in solution, flotation assays suggested it may associate with liposomes under some conditions. First results of pull-down also showed a possible weak or transient interaction between A6 and A11.
On one hand, A6 is a highly dynamic protein with multiple oligomeric states. On the other, A11 shows potential membrane-binding capacity and interaction with A6, though more work is required to confirm and further characterise these interactions. (Less) - Popular Abstract
- Poxviruses (a family of viruses that includes Variola virus, the causative agent of smallpox) are fascinating because of their unique ability to build their own walls and structures. Most viruses hijack the host cell’s machinery to do this, but poxviruses bring their own tools. If we can understand how they construct themselves inside human cells, we might be able to design better vaccines or treatments that stop them before they cause disease.
In this project, we studied two of the virus’s "construction workers"—proteins called A6 and A11—that help build the virus’s outer shell. Without them, the shell doesn’t form properly, and the virus cannot go on to infect.
To study these proteins, we recreated parts of the virus assembly... (More) - Poxviruses (a family of viruses that includes Variola virus, the causative agent of smallpox) are fascinating because of their unique ability to build their own walls and structures. Most viruses hijack the host cell’s machinery to do this, but poxviruses bring their own tools. If we can understand how they construct themselves inside human cells, we might be able to design better vaccines or treatments that stop them before they cause disease.
In this project, we studied two of the virus’s "construction workers"—proteins called A6 and A11—that help build the virus’s outer shell. Without them, the shell doesn’t form properly, and the virus cannot go on to infect.
To study these proteins, we recreated parts of the virus assembly process in test tubes. We produced and purified the A6 and A11 proteins and examined their behaviour using different scientific techniques. We found that A6 is very dynamic and forms flexible clusters, while A11 is much less stable and trickier to work with.
We also mimicked the human cell membrane using artificial membrane-like structures to see how A11 would interact with them. In addition, we tested whether A6 and A11 interact with each other by mixing them in a test tube and checking if they stuck together. The results suggest that there might be an interaction—but more work is needed to be sure. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9198770
- author
- Inam, Hadiqa LU
- supervisor
- organization
- course
- KEMR20 20251
- year
- 2025
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- viral assembly, membrane remodelling, poxvirus, VMAPs, biochemistry
- language
- English
- id
- 9198770
- date added to LUP
- 2025-06-13 14:48:15
- date last changed
- 2025-06-13 14:48:15
@misc{9198770,
abstract = {{Poxviruses are large, double-stranded DNA viruses that replicate directly in the cytoplasm of the host eukaryotic cell. During assembly of viral particle progeny, viral membrane precursors derived from the endoplasmic reticulum, exhibit stabilised open ends inside intermediate structures called crescents. The process of viral membrane biogenesis during poxvirus assembly remains poorly understood. In particular, the key role of five essential Viral Membrane Assembly Proteins (VMAPs) remains enigmatic, including the contribution of A6 and A11 to the early stages of virion assembly, how they interact with one another and cell-derived membranes.
This study aims at characterising A6 and A11 using a combination of biophysical and biochemical approaches to investigate their potential interaction and assess A11 interaction with lipids in vitro.
We expressed and purified recombinant A6 and A11 using E. coli and wheat germ extract in cell-free expression systems, respectively. A6 was analysed by analytical ultracentrifugation, SEC-MALS, and mass photometry to study its oligomeric state. In parallel, the extent of A11 reconstitution in proteoliposomes was assessed by flotation assays. Finally, protein-protein interactions were examined using pull-down and western-blotting.
For A6, there is a concentration-dependent shift from monomer to higher-order assemblies. While A11 appeared to be difficult to purify and unstable in solution, flotation assays suggested it may associate with liposomes under some conditions. First results of pull-down also showed a possible weak or transient interaction between A6 and A11.
On one hand, A6 is a highly dynamic protein with multiple oligomeric states. On the other, A11 shows potential membrane-binding capacity and interaction with A6, though more work is required to confirm and further characterise these interactions.}},
author = {{Inam, Hadiqa}},
language = {{eng}},
note = {{Student Paper}},
title = {{Characterisation of Viral Membrane Assembly Proteins A6 and A11 of Vaccinia Virus}},
year = {{2025}},
}