Sculpting Membranes: Electron Tomography of Virus-Infected Cells

  • Electron Tomography of virus-infected cells allow us to better understand how viruses remodel intracellular membranes to build up their replication factories.Electron Tomography of virus-infected cells allow us to better understand how viruses remodel intracellular membranes to build up their replication factories.
  • Electron Tomography of virus-infected cells allow us to better understand how viruses remodel intracellular membranes to build up their replication factories.
  • Fig. 1: Electron tomography (ET) analysis of the membrane rearrangements induced by different members of the genus Flavivirus on endoplasmic reticulum (ER) membranes: (A) Dengue virus (DENV), (B) Tick-borne encephalitis virus (TBEV). Slices through tomograms of infected cells (on the left) and 3D top and lateral (90° rotation) views of the same tomograms (on the right) are depicted, showing the characteristic Flavivirus-induced replication vesicles (Ve). These vesicles are formed as invaginations of ER membranes that remain connected to the cytosol via 10 nm-pores (white arrows). (C) Schematic diagram of vesicle formation. Negative membrane curvature results in the formation of invaginations towards the ER lumen, generating arrays of vesicles that are known as vesicle packets (VP). Panels A and B are reproduced with permission from [6].
  •  Fig. 2: Electron tomography (ET) analysis of the membrane rearrangements induced by Hepatitis C virus (HCV, genus Hepacivivus). (A) A slice through a tomogram of infected cells (on the left) and 3D top and lateral (90° rotation) views of the same tomogram (on the right) are depicted, showing the characteristic HCV-induced double membrane vesicles (DMVs). These DMVs remain connected to ER membranes via neck-like structures (white arrows). (B and C) Schematic diagrams of hypothetical DMV formation mechanisms. (B) HCV proteins might induce positive membrane curvature generating exvaginations of ER membranes towards the cytosol, giving rise to cytoplasmic vesicles. These vesicles might undergo a secondary invagination and subsequent double membrane wrapping, originating DMVs. (C) By analogy to flaviviruses, HCV proteins might induce invaginations of the ER membrane. Extensive invagination leads to a local ‘shrinking’ of the ER lumen and membrane pairing. Panel A is reproduced with permission from [6]. Panels B and C are adapted from [4].

Viruses are obligate intracellular parasites. In order to replicate their genomes and produce new virions, viruses need to reprogram the cell machineries. This includes remodeling of cell membranes to form the so-called replication factories (RFs). Imaging cells infected with several members of the family Flaviviridae by means of electron tomography (ET) revealed the different 3D architectures of their RFs.

Life Cycle of Viruses Belonging to the Family Flaviviridae

In order to perform a successful infection cycle, viruses need to enter into their host cells. Upon entry and uncoating the viral genome is released into the cytosol, where it will be subsequently copied and packaged into new viral particles.
Members of the family Flaviviridae have a single-stranded RNA genome with a positive (+) polarity, mimicking a messenger RNA (mRNA). Upon release into the cytosol, the (+)-strand RNA can be directly translated on ribosomes, giving rise to a viral polyprotein, which is cleavage to generate the structural and nonstructural (NS) proteins. These proteins, mainly the NS proteins, also called ‘replicase’, induce dramatic changes at the endoplasmic reticulum (ER) membranes.

These modified membranous structures, known as replication factories (RFs), represent a ‘safe’ environment, where replication of the viral genome can happen, shielded from the threatening activity of nucleases or from the recognition by the host immune system. The formation of RFs helps also to locally concentrate all the components required for RNA amplification at specific cytoplasmic sites and might enable a spatio-temporal coordination of the different steps of the viral life cycle (RNA translation, replication and assembly).

In the protected lumen of the RFs the (+) RNA genome is converted into a (−) RNA copy that serves as a template for the production in excess amounts of new (+) RNA genomes. These (+) progeny RNAs are then released to the cytosol, where they are used for the assembly of infectious progeny virus. 

3D Architecture of the RFs of Members of the Family Flaviviridae

The use of high resolution microscopy techniques has enormously contributed to increase our knowledge about how viruses interact with their cellular host and, as a result of such an interaction, how the cell landscape and its membranes are being extensively remodeled (reviewed in [1]).
Electron tomography (ET) analysis of cells infected with members of the genus Flavivirus, e.g., Dengue virus (DENV) and Tick-borne encephalitis virus (TBEV), has shown that both induce invaginations of ER membranes, leading to the formation of vesicles towards the ER lumen, that remain connected to the cytosol via  ~10 nm pores ([2,3], respectively)  (fig.

1A and 1B). Several vesicles are formed per ER tube, generating swollen ER sacs filled with vesicles, also known as vesicle packets (VPs) (fig. 1C). We have also applied ET to elucidate the architecture of the RFs of Hepatitis C Virus (HCV), the prototype member of the genus Hepacivirus. Our tomographic analysis revealed that HCV induces the formation of double membrane vesicles (DMVs) that remain connected to the ER via neck-like structures [4] (fig. 2A).

Thus far, our findings revealed that viruses belonging to the family Flaviviridae seem to follow a quite different strategy to build up their RFs, being the different morphology of the RFs likely genus-dependent. Thus, while members of the genus Flavivirus induce the formation of structures with negative curvature (fig. 1C), HCV modifies the ER in a more complex fashion (fig. 2B and 2C), most likely involving the formation of positive curvature structures (fig. 2B). Interestingly, only a minority (~10%) of the DMVs have an opening connecting the interior of the vesicles with the cytosol [4], whereas pore-like openings could be detected in approximately 50% of the Flavivirus-induced vesicles [2,3]. This pore likely allows recruitment of cellular factors to active sites of virus replication, as well as for the release of newly synthesized viral RNA to be used for translation and assembly. Therefore the presence of more pores in Flavivirus-induced vesicles might reflect their higher level of replication in comparison to HCV.

Strategies to Build Up RFs Among (+)-strand RNA Viruses

Similarly to members of the genus Flavivirus, other (+)-strand RNA viruses induce invaginations of ER membranes, e.g., plant viruses of the families Bromoviridae and Tombusviridae. Although the ER is one of the most usurped cellular organelles (reviewed in [5]), membranes of other organelles, e.g., mitochondria and lysosomes are also used as scaffold to build up viral RFs, involving also the formation of invaginations at these targeted organelles (reviewed in [6]).

Formation of DMVs is also a strategy frequently used by other (+)-strand RNA viruses such as corona-, arteri- and picornaviruses. So far their biogenesis mechanism has only been elucidated for picornaviruses. Thus, recent findings revealed that DMVs originate from single-membrane tubules that undergo secondary invaginations to achieve their double-membrane morphology [7,8] (similar to fig. 2C). However, in absence of any observation of similar single-membrane precursors (‘pre-DMV’), the origin of the DMVs induced by other viruses, including HCV, remains to be elucidated.

Conclusions

ET analysis of cells infected with members of the family Flaviviridae provided us with unprecedented ultrastructural information about the 3D architecture of their RFs. In this regard, ET has opened the door to the direct visualization of virus-induced intracellular modifications. However, although very detailed, the obtained information comes from only ~300 nm thick cell sections. To overcome this volume limitation, application of novel microscopy techniques that enable the reconstruction of complete cell volumes (reviewed in [1]), will reveal the morphology of not only a part, but of the entire virus-remodeled cell organelles.
Of course, the challenge will remain the time-consuming analysis of all these data to reconstruct, with help of molecular and biochemical assays, the large puzzle of virus-host interactions.

References
[1]  Romero-Brey, I.; Bartenschlager, R. Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells. Viruses. 2015, 7 (12), 6316-6345. DOI: 10.3390/v7122940.
[2]  Welsch, S.; Miller, S.; Romero-Brey, I.; Merz, A.; Bleck, C. K.; Walther, P.; Fuller, S. D.; Antony, C.; Krijnse-Locker, J.; Bartenschlager, R. Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host. Microbe 2009, 5 (4), 365-375. DOI: 10.1016/j.chom.2009.03.007.
[3]  Miorin, L.; Romero-Brey, I.; Maiuri, P.; Hoppe, S.; Krijnse-Locker, J.; Bartenschlager, R.; Marcello, A. Three-dimensional architecture of tick-borne encephalitis virus replication sites and trafficking of the replicated RNA. J. Virol. 2013, 87 (11), 6469-6481. DOI: 10.1128/JVI.03456-12.
[4]  Romero-Brey, I.; Merz, A.; Chiramel, A.; Lee, J. Y.; Chlanda, P.; Haselman, U.; Santarella-Mellwig, R.; Habermann, A.; Hoppe, S.; Kallis, S.; Walther, P.; Antony, C.; Krijnse-Locker, J.; Bartenschlager, R. Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLoS. Pathog. 2012, 8 (12), e1003056. DOI: 10.1371/journal.ppat.1003056.
[5]  Romero-Brey, I.; Bartenschlager, R. Endoplasmic reticulum: the favourite intracellular niche for viral replication and assembly. Viruses 2016, 8 (6), 160-186. DOI:10.3390/v8060160
[6]  Romero-Brey, I.; Bartenschlager, R. Membranous replication factories induced by plus-strand RNA viruses. Viruses. 2014, 6 (7), 2826-2857. DOI:  10.3390/v6072826
[7]  Belov, G. A.; Nair, V.; Hansen, B. T.; Hoyt, F. H.; Fischer, E. R.; Ehrenfeld, E. Complex dynamic development of poliovirus membranous replication complexes. J. Virol. 2012, 86 (1), 302-312. DOI: 10.1128/JVI.05937-11
[8]  Limpens, R. W.; van der Schaar, H. M.; Kumar, D.; Koster, A. J.; Snijder, E. J.; van Kuppeveld, F. J.; Barcena, M. The transformation of enterovirus replication structures: a three-dimensional study of single- and double-membrane compartments. MBio. 2011, 2 (5). DOI: 10.1128/mBio.00166-11

Author
Dr. Inés Romero-Brey

University of Heidelberg
Department Department of Infectious Diseases-Molecular Virology
Heidelberg, Germany
www.molecular-virology.uni-hd.de

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