Uukuniemi virus-like particles : a model system for bunyaviral assembly
Author: Överby, Anna K
Date: 2007-06-08
Location: Cell- och molekylärbiologiska institutionens auditorium, Berzelius väg 21, Karolinska Institutet, Solna
Time: 09.30
Department: Institutionen för cell- och molekylärbiologi (CMB) / Department of Cell and Molecular Biology
View/ Open:
Thesis (877.2Kb)
Abstract
Viruses are intracellular parasites that are unable to multiply except
when inside a living cell of a host. Outside their host they remain
inert. There are many different types of viruses that are classified into
different virus families according to their size, nucleic acid
composition (RNA or DNA), or the composition of their outer shell (naked
or enveloped). Following entry, negative stranded viruses release their
genome which is subsequently replicated, transcribed, and the different
viral components are assembled into new virus particles, which are
released from the host cell and ready to infect new cells. This thesis
focuses on how the different viral parts assemble into an infectious
particle inside the cell as well as on the identification of parts in the
RNA genome important for viral replication.
Uukuniemi virus (UUKV), a prototypic member of the Bunyaviridae family, has a segmented RNA genome with negative polarity. The three segments encode four structural proteins, two glycoproteins (GN and GC) located in the envelope, one nucleoprotein (N protein), and a RNA-dependent RNA polymerase (L protein). Flanking these open reading frames (ORFs) are non-coding regions (NCRs) containing cis-acting signals important for viral transcription, replication, encapsidation and packaging. These NCRs encompass one variable region, and one highly conserved region located in both the 5 and 3 terminal ends of the three segments, which are complementary to each other.
To study the function of the NCRs we used a minigenome system developed for UUKV. In this system the viral protein coding sequence is replaced by sequences encoding a reporter protein. This minigenome is transfected into cells together with the N and L protein necessary for replication and transcription, after which reporter protein expression can be measured. In the first paper we studied the variable region of the NCRs and its effect on promoter strength and packaging efficiency. We performed this by comparing the activity of minigenomes that contained the NCRs derived from all three different segments. We found that the variable region is not only important for the regulation of promoter activity but also for packaging efficiency, since the three different minigenomes all showed different reporter activity.
Next the assembly of the UUKV inside the cell was examined. In order to study packaging and assembly, we first developed a virus-like particle (VLP) system for UUKV. With the addition of the glycoprotein precursor encoding both glycoproteins GN and GC to the minigenome system, VLPs containing the minigenome are produced and released into the supernatant. These particles are able to infect new cells where reporter protein expression can be detected. Characterization of these VLPs revealed that they have similar size and surface morphology as wild-type UUKV. Moreover we demonstrated that only the two glycoproteins are required for generating VLPs, suggesting that UUKV budding is driven by the two glycoproteins.
We further analyzed the importance of specific amino acids in the cytoplasmic tail of both glycoproteins, GN and GC, for packaging of the RNA genome into VLPs and the budding of UUKV. This was done by performing an alanine scan of the GN cytoplasmic tail (81 amino acids) and the GC cytoplasmic tail (5 amino acids), and analyzing the effect of these mutations on particle formation in our VLP system. We identified three regions in the GN tail (amino acids 21-25, 46-50 and 71-81) that are important for minigenome transfer of the VLPs. A more detailed analysis showed, four amino acids in the GN cytoplasmic tail involved in the packaging interaction with the ribonucleoproteins while two other amino acids are important for the budding into the Golgi compartment. Finally three amino acids in the GN and two in the GC cytoplasmic tail are important for the correct localization of the two glycoproteins in the cell.
In conclusion, we show a mechanism of UUKV assembly and demonstrate the usefulness of our experimental system. We also postulate that the VLP system is a useful tool for analyzing packaging, assembly, and budding for other members of the Bunyavidae family.
Uukuniemi virus (UUKV), a prototypic member of the Bunyaviridae family, has a segmented RNA genome with negative polarity. The three segments encode four structural proteins, two glycoproteins (GN and GC) located in the envelope, one nucleoprotein (N protein), and a RNA-dependent RNA polymerase (L protein). Flanking these open reading frames (ORFs) are non-coding regions (NCRs) containing cis-acting signals important for viral transcription, replication, encapsidation and packaging. These NCRs encompass one variable region, and one highly conserved region located in both the 5 and 3 terminal ends of the three segments, which are complementary to each other.
To study the function of the NCRs we used a minigenome system developed for UUKV. In this system the viral protein coding sequence is replaced by sequences encoding a reporter protein. This minigenome is transfected into cells together with the N and L protein necessary for replication and transcription, after which reporter protein expression can be measured. In the first paper we studied the variable region of the NCRs and its effect on promoter strength and packaging efficiency. We performed this by comparing the activity of minigenomes that contained the NCRs derived from all three different segments. We found that the variable region is not only important for the regulation of promoter activity but also for packaging efficiency, since the three different minigenomes all showed different reporter activity.
Next the assembly of the UUKV inside the cell was examined. In order to study packaging and assembly, we first developed a virus-like particle (VLP) system for UUKV. With the addition of the glycoprotein precursor encoding both glycoproteins GN and GC to the minigenome system, VLPs containing the minigenome are produced and released into the supernatant. These particles are able to infect new cells where reporter protein expression can be detected. Characterization of these VLPs revealed that they have similar size and surface morphology as wild-type UUKV. Moreover we demonstrated that only the two glycoproteins are required for generating VLPs, suggesting that UUKV budding is driven by the two glycoproteins.
We further analyzed the importance of specific amino acids in the cytoplasmic tail of both glycoproteins, GN and GC, for packaging of the RNA genome into VLPs and the budding of UUKV. This was done by performing an alanine scan of the GN cytoplasmic tail (81 amino acids) and the GC cytoplasmic tail (5 amino acids), and analyzing the effect of these mutations on particle formation in our VLP system. We identified three regions in the GN tail (amino acids 21-25, 46-50 and 71-81) that are important for minigenome transfer of the VLPs. A more detailed analysis showed, four amino acids in the GN cytoplasmic tail involved in the packaging interaction with the ribonucleoproteins while two other amino acids are important for the budding into the Golgi compartment. Finally three amino acids in the GN and two in the GC cytoplasmic tail are important for the correct localization of the two glycoproteins in the cell.
In conclusion, we show a mechanism of UUKV assembly and demonstrate the usefulness of our experimental system. We also postulate that the VLP system is a useful tool for analyzing packaging, assembly, and budding for other members of the Bunyavidae family.
List of papers:
I. Flick K, Katz A, Overby A, Feldmann H, Pettersson RF, Flick R. (2004). "Functional analysis of the noncoding regions of the Uukuniemi virus (Bunyaviridae) RNA segments." J Virol 78(21): 11726-38
Pubmed
II. Overby AK, Popov V, Neve EP, Pettersson RF. (2006). "Generation and analysis of infectious virus-like particles of uukuniemi virus (bunyaviridae): a useful system for studying bunyaviral packaging and budding." J Virol 80(21): 10428-35
Pubmed
III. Overby AK, Pettersson RF, Neve EP. (2007). "The glycoprotein cytoplasmic tail of Uukuniemi virus (Bunyaviridae) interacts with ribonucleoproteins and is critical for genome packaging." J Virol 81(7): 3198-205
Pubmed
IV. Överby AK, Popov V, Pettersson RF, Neve EPA. (1970). "The cytoplasmic tails of Uukuniemi virus (Bunyaviridae) GN and GC glycoproteins are important for intracellular localization and budding of VLPs." (Submitted)
I. Flick K, Katz A, Overby A, Feldmann H, Pettersson RF, Flick R. (2004). "Functional analysis of the noncoding regions of the Uukuniemi virus (Bunyaviridae) RNA segments." J Virol 78(21): 11726-38
Pubmed
II. Overby AK, Popov V, Neve EP, Pettersson RF. (2006). "Generation and analysis of infectious virus-like particles of uukuniemi virus (bunyaviridae): a useful system for studying bunyaviral packaging and budding." J Virol 80(21): 10428-35
Pubmed
III. Overby AK, Pettersson RF, Neve EP. (2007). "The glycoprotein cytoplasmic tail of Uukuniemi virus (Bunyaviridae) interacts with ribonucleoproteins and is critical for genome packaging." J Virol 81(7): 3198-205
Pubmed
IV. Överby AK, Popov V, Pettersson RF, Neve EPA. (1970). "The cytoplasmic tails of Uukuniemi virus (Bunyaviridae) GN and GC glycoproteins are important for intracellular localization and budding of VLPs." (Submitted)
Issue date: 2007-05-18
Rights:
Publication year: 2007
ISBN: 978-91-7357-238-5
Statistics
Total Visits
Views | |
---|---|
Uukuniemi ...(legacy) | 1264 |
Uukuniemi ... | 133 |
Total Visits Per Month
September 2023 | October 2023 | November 2023 | December 2023 | January 2024 | February 2024 | March 2024 | |
---|---|---|---|---|---|---|---|
Uukuniemi ... | 2 | 2 | 0 | 0 | 1 | 1 | 1 |
File Visits
Views | |
---|---|
thesis.pdf(legacy) | 581 |
thesis.pdf | 180 |
thesis.pdf.txt(legacy) | 2 |
Top country views
Views | |
---|---|
United States | 510 |
Sweden | 168 |
China | 114 |
Germany | 110 |
United Kingdom | 23 |
Denmark | 20 |
Italy | 18 |
South Korea | 18 |
Russia | 16 |
Finland | 13 |
Top cities views
Views | |
---|---|
Stockholm | 62 |
Romeo | 43 |
Sunnyvale | 39 |
Beijing | 32 |
Nürnberg | 32 |
Kiez | 29 |
Seoul | 18 |
Shenzhen | 16 |
London | 13 |
Ballerup | 12 |