Coupling of replication and assembly in flaviviruses
Introduction
Flaviviruses (Family — Flaviviridae) are one of the major causes of arthropod-borne illness in humans. Flavivirus pathogenesis ranges from mild illness such as fever, rash and joint pain, to more severe symptoms such as hemorrhagic fever and fatal encephalitis. The flavivirus genus consists of more than 70 enveloped, positive-strand RNA viruses including yellow fever virus (YFV), dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV) and tick-borne encephalitis virus (TBEV). Altogether flaviviruses affect hundreds of millions of individuals every year. Despite the availability of vaccines against YFV, JEV and TBEV, diseases resulting from these viruses are still prevalent worldwide. Protective vaccines or therapies are not yet available against the more pathogenic flaviviruses and prevention from insect bites remains the major defense against some of these viruses. Therefore, a better understanding of the flavivirus life cycle is essential in order to develop effective strategies for antiviral intervention and for development of novel vaccines.
The virus enters host cells by receptor-mediated endocytosis and the ∼11 kb positive-sense RNA genome gains entry into the cytoplasm by viral glycoprotein-mediated membrane fusion. Flavivirus replication begins when the genome is recognized as messenger RNA and translated by host cell machinery to yield a single polyprotein. The polyprotein is co-translationally and post-translationally cleaved by viral and cellular proteases into 10 gene products (Figure 1). The structural proteins capsid (C), precursor membrane (prM/M) and envelope (E) are incorporated into the virion, whereas the non-structural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 serve to coordinate the intracellular aspects of virus replication, assembly and modulation of host defense mechanisms. NS1 is essential for virus replication and inhibition of complement-mediated immune response [1]. NS3 contains serine protease, Nucleoside 5′ triphosphatase (NTPase), RNA helicase, and 5′ RNA triphosphatase (RTPase) activities, while NS2B serves as a cofactor for the protease activity of NS3. NS5 contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains required for genome replication and capping of nascent RNA [2]. Three non-enzymatic, integral membrane proteins NS2A, NS4A and NS4B are poorly understood. NS2A is required for virus replication and assembly [3, 4, 5]. NS4A induces membrane rearrangement [6, 7] and autophagy to enhance viral replication [8], whereas NS4B modulates host immune response by suppressing the α/β interferon signaling and the helicase activity of NS3 [9, 10].
Polyprotein processing into component proteins takes place on ER membranes (Figure 1). The replication complex (RC) is formed on modified ER membranes where negative-sense RNA is copied from the genomic RNA template. The process of RNA synthesis is asymmetric favoring positive-sense RNA [2]. The RNA genome then interacts with C protein, which buds through the glycoprotein-containing ER membranes as immature virus particles into the lumen. The immature virus particles then go through maturation steps in the ER and Golgi complex and are released as mature particles from the cell. The biogenesis of the RC and its assembly involve modifications of cellular metabolic and structural components. This review presents an overview of the current knowledge of interactions between the viral and cellular components to promote cellular changes required for replication and assembly and how these two processes are coupled. Replication and assembly are important aspects of the virus life cycle that are targeted for future antiviral development.
Section snippets
Virus-induced membrane rearrangements
It is now established that flavivirus replication and viral RNA synthesis occurs on an extended network of modified ER membranes. At least three distinct membranous structures are found in flavivirus-infected cells: membranous sacs or vesicle packets (Vp), membrane vesicles (Ve) and convoluted membranes (CM) (Figure 2) [11, 12, 13, 14••, 15•, 16•]. Vps are small clusters of Ve formed by modification of ER membranes and used as sites of replication by the virus [13, 14••]. Ve are open to the
Flavivirus assembly
We understand exceedingly little about the assembly process of flaviviruses. A packaging sequence on the genome of flaviviruses has not been identified with C-RNA interactions believed to be non-specific and electrostatic [32, 33], yet the genomic RNA is specifically packaged into virions. It has been suggested that this specificity is achieved by coupling genomic replication and encapsidation. There is evidence to indicate that only actively synthesized RNAs are packaged, hinting that
Roles of cellular lipids in replication and assembly
On the basis of studies so far, it appears that the high demand for lipids due to membrane remodeling and budding virions in flavivirus-infected cells is met in several ways. Reabsorption of LDs, the natural reservoirs and processing centers of lipids in the cell by the ER membrane was observed in DENV-infected cells [51]. Redistribution of fatty acid synthase (FASN), in a Rab18-dependent manner, by DENV NS3 at the sites of replication appears to be a mechanism utilized by the virus to maintain
Conclusions
A common feature of flaviviruses is to remodel ER membranes to generate distinct organelle-like structures. These structures may be utilized by the virus for specific steps of its life cycle to ensure efficient replication, protection of viral RNA from cellular defense mechanisms and well-coordinated assembly and budding processes. Although there is evidence indicating the role of viral and host factors, the exact molecular mechanisms behind these membrane modifications are still poorly
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We acknowledge support from the NIH R21 AI083984 and NIAID R01 AI76331 to R.J.K.
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