# Sevgisu Eğilmez - Flaviviruses
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> [name=Sevgisu Eğilmez]

> [time=Tue, Jun 01 2021 4:16PM]

**FLAVIVIRUSES: [Wang-Shick Ryu - Molecular Virology of Human Pathogenic Viruses-Academic Press (2017)]**
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Virion structure
Genome
Attachment and Entry
Translation
Replication
Assembly
Release
Replication Cycle
Fundamental Concepts

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### FLAVIVIRUSES


The Flaviviridae are a family of positive-strand RNA viruses that includes several notable human pathogens. Among these are yellow fever virus, dengue hemorrhagic fever virus, Japanese encephalitis virus, St. Louis encephalitis virus, West Nile encephalitis virus, the several tick borne encephalitis viruses, and hepatitis C virus.The name “flavivirus” is derived from the Latin word flavus, meaning yellow, signifying the jaundice caused by yellow fever virus.

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**Table 1: Members of the Flaviviridae family**
Source: Fundamentals of Molecular Virology

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## Structure

**Figure 1: Virion structure of Flaviviruses**
Source [ http://www.expasy.org]

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Family --> Flaviviridae
Enveloped, icosahedral viruses
Capsids that are 37 to 50 nm in diameter
Contain positive-strand RNA genomes (9-12kb)
Genomes at about 11,000 nucleotides in length
Have genomic RNAs that contain a 5′ cap.

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**Figure 2: The distinct arrangement of E proteins on immature (left) and mature (right) forms of the virion are depicted**
Source: Pierson, T. C., & Diamond, M. S. (2020). The continued threat of emerging flaviviruses. Nature Microbiology.

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**Figure 3: Membrane topology of the flavivirus structural proteins.**
Source: Fundamentals of Molecular Virology

The flavivirus envelope contains two virus-encoded proteins. The major and larger one is the envelope (E) protein, and the smaller one is the membrane (M) protein. The function of the E protein is to bind to the receptor and to mediate envelope fusion to a cellular membrane. Since the E protein is the major envelope protein, it is also the major target on the virus for neutralizing antibodies. M protein is generated from a precursor polypeptide known as prM. There is experimental evidence suggesting that prM is a chaperone, which ensures that E folds and interacts properly during intracellular virion assembly and maturation. This function of prM is discussed further below.

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Source: ResearchGate

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## Genome

Virus genomes are positive-stranded, non-segmented RNA of approximately 9.0–13 kb. They contain a single, long ORF flanked by 5'- and 3' terminal non-coding regions, which form specific secondary structures required for genome replication and translation.



**Figure 3: Genom organization of the Flaviviruses**
Source: Fundamentals of Molecular Virology

There is one translation initiation site and one open reading frame on the positive-strand RNA genome in each instance. Processing of the flavivirus precursor polyprotein is carried out either by the host signal peptidase or by the viral protease, NS3.

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## Attachment and Entry

Once attached to cells, flaviviruses are taken up by clathrindependent endocytic vesicles. Flavivirus membrane fusion occurs in the low pH compartments of the endosome and is catalysed by conformational changes in the E protein that involve the formation of E protein trimers, penetration of the highly conserved E-DII fusion loop into the adjacent host membranes and the folding of the E protein helical stem against the exterior surface of the newly formed E protein trimer.

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**Figure 4: Attachment and entry to the cell of Flavivirus**
Source: Pierson, T. C., & Diamond, M. S. (2020). The continued threat of emerging flaviviruses. Nature Microbiology.

Flaviviruses bind to an array of mammalian cell types through interactions of multiple C-type lectins. The role of specific host proteins in the attachment and entry of viruses into cells varies. Host proteins classically defined as receptors are essential for the entry of viruses because they catalyse critical conformational events. Flaviviruses bound to synthetic lipid membranes devoid of host proteins are capable of stimulating E protein-mediated fusion once exposed to an acidic environment.

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## Translation

**Figure 5: Targeting secretory proteins to the ER**

**Step 1:** As the signal sequence emerges from the ribosome, it is recognized and bound by the signal recognition particle (SRP).
**Step 2:** The SRP escorts the complex to the ER membrane, where it binds to the SRP receptor.

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**Figure 6: Targeting secretory proteins to the ER**

**Step 3:** The SRP is released, the ribosome binds to a membrane translocation complex, and the signal sequence is inserted into a membrane channel.
**Step 4:** Translation resumes, and the growing polypeptide chain is translocated across the membrane.
**Step 5:** Cleavage of the signal sequence by signal peptidase releases the polypeptide into the lumen of the ER.

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If the protein were to contain a hydrophobic stop-transfer (transmembrane) domain (not shown), then it would become an integral membrane protein. If not, it would be released into the lumen of the ER.

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As the translation of the flavivirus nascent polyprotein ensues on a free ribosome in the cytoplasm, an internal signal sequence, located at the C terminus of the C protein, acts to target the still-ribosome-bound, nascent polyprotein to a translocation complex in the ER membrane. The ribosome binds to the translocation complex, and the signal sequence on the nascent polyprotein inserts into a translocation channel in the membrane. As the remainder of the nascent polyprotein, which contains the amino acid sequences of both prM and E, is translated, it crosses the membrane of the ER multiple times.

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**Figure 7: Predicted orientation of the structural proteins across the ER membrane**

During translation of the flavivirus polyprotein, the envelope proteins are translocated and anchored in the ER membrane by various signal sequences and membrane anchor domains. The C protein contains a hydrophobic signal sequence at its C terminus, which translocates prM into the lumen of the ER from its site of synthesis at a translocation complex. The prM protein has two transmembrane- spanning domains, which contain a stop-transfer sequence and a sig- nal sequence.

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As a result, the E protein is also translocated into the lumen of the ER. After the indicated proteolytic cleavages, the C protein and viral RNA are localized in the cytoplasm, where the capsid protein remains associated with the ER. On the luminal side of the ER, the prM and E proteins form a stable heterodimer within a few minutes of translation.

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Transmembrane helices are indicated by cylinders, arrows indicate the sites of posttranslational cleavage, and the cleavage sites of specific enzymes are indicated by different colors.

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## Replication

**Figure 8: The flavivirus replication**
Source: Pierson, T. C., & Diamond, M. S. (2020). The continued threat of emerging flaviviruses. Nature Microbiology.

Viral RNA replication occurs on membranes of the host reorganized through the actions of the non-structural proteins. These virus induced membrane structures spatially coordinate viral genomic RNA replication and virion morphogenesis, and shield replication products from host innate immune sensors.

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## Assembly

**Figure 9: Flavivirus Assamble**
Source: Fundamentals of Molecular Virology

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Flaviviruses assemble their virions inside the cell by using intracellular membranes for envelope formation.virions can be found in the lumen (interior) of the endoplasmic reticulum. Assembly of the nucleocapsid takes place at the cytoplasmic face of membranes with which prM and E transmembrane proteins are associated.

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The carboxy- terminal signal sequence of the precursor to the C protein anchors that protein to the membrane **(Figure 3)** this sequence penetrates into the lumen of the endoplasmic reticulum or smooth vesicles. This enables interactions between the C protein and the envelope proteins, which are also anchored to the membrane but reside within the lumen. Multiple copies of the C protein condense on the genome RNA to form the nucleocapsid. Subsequent cleavage of the membrane anchor of the C protein by the viral proteinase (see above) releases the capsid from direct contact with the membrane.

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**Figure 10: Flavivirus assamble**
Source: Pierson, T. C., & Diamond, M. S. (2020). The continued threat of emerging flaviviruses. Nature Microbiology.

The coupling of protein synthesis, RNA synthesis, and virion assembly on membranous structures assures that newly synthesized genome RNA can associate with C protein and initiate the assembly process. Encapsidation of the RNA initiates the budding of particles into the endoplasmic reticulum. Particles that have budded into the endoplasmic reticulum are then processed by car- bohydrate addition and modification as they proceed through the Golgi membrane system. It is likely that transport to the Golgi and into the trans-Golgi network requires the presence of the glycosylated prM protein.

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## Release


**Figure 11: Flavivirus releasing**
Source: Fundamentals of Molecular Virology

Virions follow the exocytosis pathway and are released to the extracellular space by fusion of vesicles containing virions with the plasma membrane The cleavage of the prM protein by host-encoded furin occurs just prior to virion release and converts the immature particle to its mature form.

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Recent structural and biochemical data demonstrate that the immature virus undergoes a significant rearrangement of its surface glycoproteins as it travels through the low pH of the trans-Golgi network

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## Replication Cycle

**Figure 12: Flavivirus Replication Cycle**
Source: Pierson, T. C., & Diamond, M. S. (2020). The continued threat of emerging flaviviruses. Nature Microbiology.

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## FUNDAMENTAL CONCEPTS

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• Flaviviruses cause severe human diseases such as yellow fever, Japanese encephalitis, dengue fever, and hepatitis.
• Most flaviviruses (but not hepatitis C virus) are transmitted by mosquitoes or ticks.
• West Nile virus entered the United States in 1999 and has progressively expanded its presence into most of the United States and parts of Canada by virtue of its mosquito-to-bird transmission cycle.
• The flavivirus particle is covered with a flat layer of envelope proteins arranged with icosahedral symmetry, giving the virus an appearance of a golf ball.
• Flaviviruses enter the cell by uptake into endosomal vesicles; at low pH the envelope protein rearranges from a dimer into a fusion-active trimer.
• All flavivirus proteins are synthesized as a single polyprotein that is cleaved into 10 functional viral proteins by viral and cellular proteinases.
• Like other positive-strand viruses, flavivirus RNA synthesis takes place in association with cytoplasmic membranes.
• Assembly of flavivirus virions takes place on membranes of the endoplasmic reticulum, and virions mature by cleavage of the premembrane protein as they pass through the trans-Golgi network.

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