Dengue Virus and Viral Proteins

Dengue Virus

Dengue Virus Overview

  • Dengue virus is a flavivirus.

  • It is an enveloped virus with a structural protein coat.

  • Related viruses include Zika virus, Yellow Fever, St. Louis encephalitis, Japanese encephalitis, and West Nile virus.

Viral Life Cycle

  • The virus infection cycle involves several key steps:

    • Fusion and virus disassembly

    • Viral genome replication

    • Polyprotein translation, transit to ER and processing

    • Virus assembly

    • Virus maturation

Viral Architecture

  • The mature virion has an icosahedral protein coat and a lipid bilayer envelope.The envelope is embedded with viral proteins that play crucial roles in host cell recognition and infection.

Polyprotein Cleavage

  • The viral polyprotein is processed by proteolysis.

  • The polyprotein is cleaved into 13 chains.

E Protein

  • E protein forms dimers in the mature virus.

  • There are 180 copies of E protein arranged as 90 dimers, but not in a regular T=3 arrangement.

E Protein Structures and Conformational Changes

  • Lipid triggers change in quaternary structure.

  • E protein is a Class II glycoprotein-mediated membrane fusion protein.

  • At pH > 7, E exists as a prefusion, dimeric form.

  • At low pH, E transitions to a postfusion, trimeric form.

  • This pH-induced conformational change involves relative movement of domains I, II, and III.

  • Solubilized E protein (sE) forms trimers in the presence of lipid.

  • In the absence of lipid and cross-linking, sE remains a monomer.

Fusion Peptide

  • The fusion peptide binds in the host cell membrane.

  • Stem peptides fit into a groove formed at the trimer interface.

  • These peptides are proposed to enter the outer leaflet of the bilipid membrane.

  • Three fusion peptides around the trimeric axis are rich in hydrophobic amino acids.

Mechanism of Viral Attachment and Fusion

  • Viral attachment to the host cell occurs via host cell glycoproteins.

  • Low pH in the endosome causes domain II to swing out.

  • Lipid promotes trimerization of E protein at the host cell surface.

  • Trimerization spreads to the base of the E protein, inducing bending of the membrane.

  • The stem participates in trimer contacts, leading to hemifusion intermediate and full pore formation.

  • E protein adopts the same conformation as the post-fusion crystal structure.

Antiviral Development Targeting E Protein

  • Developing antivirals targetting E protein.

  • Developing antivirals targetting methyl transferase.

  • Overcoming poor vaccine efficacy.

Targeting Stem-Loop-Binding Groove

  • Synthetic peptides matching the amino acid sequence of the stem polypeptide bind to trimer grooves in vitro.

  • In model systems, externally applied synthetic peptides were carried into the endosome by the virus through non-specific binding to the membrane.

  • Following conformational changes, synthetic peptides bound to the trimer grooves blocked membrane fusion.

  • Peptide mimetics are being explored to develop novel antivirals against Dengue and related viruses by targeting the stem-loop-binding groove.

Targeting NS5-NTD Methyltransferase

  • NS5-NTD methyltransferase is also a potential antiviral target.

  • Fragment-based drug design is used to discover novel dengue virus NS5 methyltransferase non-nucleoside inhibitors.

  • 7’ Methylation step is essential.

  • 2’O Methylation is not essential, but defects induce a strong antiviral response.

NS5 Methyltransferase and RNA Capping

  • The structure of NS5 includes:

    • NS5-ntd Methyltransferase (MTase)

    • NS5-ctd RNA-dependent RNA polymerase

  • The process involves:

    • ss RNA(+) genome capped

  • The product is:

    • 7MeGpppA2’OMe−RNA7MeGpppA2’OMe-RNA

Dengue Structure, Temperature, and Maturity

  • Dengue structure depends on temperature of culture (ToC) and maturity.

  • The most advanced tetravalent live-attenuated DENV vaccine candidate showed a poor overall efficacy rate of 30%.

  • Human and mosquito hosts have different temperatures: 37°C and ambient, respectively.

  • The virus changes structure at 33°C to a “bumpy form,” which is the predominant form observed in humans.

  • Vaccines should be raised against the “bumpy” form.

Forms of Dengue Virus

  • A mature, “bumpy” form at > 33°C

  • An immature, “bumpy” form at 37°C containing E and prM capsid

Difficulties in Vaccine Development

  • Multiple forms of Dengue virus hamper the development of an effective vaccine.

  • These forms include:

    • 4 serotypes of Dengue virus

    • several structural forms: immature, partially mature, compact mature, and expanded mature

Maturation of Dengue Virus

  • Virion assembled in the endoplasmic reticulum - 60 trimeric spikes on capsid surface

  • each spike is a trimer of the E:prM complex

  • low pH of the trans-Golgi triggers trimer prM:E to dimer prM:E transformation

  • cleavage site on the prM now exposed and cleaved by furin protease

  • cleaved pr remains bound and caps the fusion peptide of the E protein

  • prevents the virus from fusing immediately back into the cell

  • upon release into the cytosol, neutral pH causes dissociation of the pr molecule

Classes of Glycoprotein-Mediated Membrane Fusion

  • Comparing Classes I, II, and III glycoprotein-mediated membrane fusion.

Structural Comparisons and Evolutionary Constraints

  • Class II fusion proteins from alphaviruses and flaviviruses are structurally closer to each other than they are to rubella virus E1.

  • This suggests that the constraints on arboviruses imposed by alternating cycles between vertebrates and arthropods resulted in more conservative evolution.

  • Rubella virus, being strictly human, seems to have drifted into a unique niche.

  • Rubella virus E1 protein is a Class II fusion protein with:

    • Significantly larger fusion loops presenting a “fusion surface”

    • More stem peptide visible

    • Metal atom incorporated into the fusion surface.

Fusion Surface Area

  • Rubella: 8,000A˚28,000 Å^2

  • Flaviviruses: 3,000A˚23,000 Å^2

  • Alpha-viruses: 5,000A˚25,000 Å^2

Sequence Inserts

  • Rubella: 40 AA sequence “inserts”

Class III Fusion Proteins

  • Glycoprotein G of the vesicular stomatitis virus is a Class III fusion protein.

Key Steps of Membrane Fusion

  • The key steps of membrane fusion include:

    • Pre-fusion

    • Extended Intermediate

    • Collapse of Intermediate

    • Hemi-fusion

    • Fusion pore

Comparison of Fusion Classes

Feature

Class I

Class II

Class III

Prefusion

Trimer

Dimer

Trimer

Extended

Trimer

Trimer

Trimer

Virus Example

Influenza HA

Dengue E

VSV G

Fusion Peptide

Buried linear fusion peptide generated post cleavage

Buried fusion loop

Buried fusion loop

Dengue Virus

The Dengue virus is a flavivirus, characterized as an enveloped virus with a structural protein coat. It shares similarities with other viruses such as Zika virus, Yellow Fever, St. Louis encephalitis, Japanese encephalitis, and West Nile virus.

Viral Life Cycle

The viral infection cycle consists of fusion and virus disassembly, followed by viral genome replication, polyprotein translation, transit to the endoplasmic reticulum (ER), and processing, culminating in virus assembly and maturation.

Viral Architecture

The mature virion features an icosahedral protein coat and a lipid bilayer envelope, which is embedded with viral proteins essential for host cell recognition and infection.

Polyprotein Cleavage

During its life cycle, the viral polyprotein undergoes proteolysis, where it is cleaved into 13 chains.

E Protein

The E protein forms dimers in the mature virus, with 180 copies arranged as 90 dimers, not in a regular T=3 arrangement.

E Protein Structures and Conformational Changes

The quaternary structure of the E protein changes with lipid triggers. Functioning as a Class II glycoprotein-mediated membrane fusion protein, E exists as a prefusion, dimeric form at pH > 7 and transitions to a postfusion, trimeric form at low pH. This pH-induced conformational change involves the movement of domains I, II, and III. Solubilized E protein (sE) forms trimers in the presence of lipid but remains a monomer without lipid and cross-linking.

Fusion Peptide

The fusion peptide binds in the host cell membrane, with stem peptides fitting into a groove formed at the trimer interface. These peptides enter the outer leaflet of the bilipid membrane, and three fusion peptides around the trimeric axis are rich in hydrophobic amino acids.

Mechanism of Viral Attachment and Fusion

Viral attachment to the host cell occurs through host cell glycoproteins. Low pH in the endosome causes domain II to swing out, and lipid promotes trimerization of E protein at the host cell surface. This trimerization induces bending of the membrane, involving stem participation in trimer contacts, leading to hemifusion intermediate and full pore formation, with the E protein adopting the same conformation as the post-fusion crystal structure.

Antiviral Development Targeting E Protein

Antiviral development focuses on targeting the E protein, methyl transferase, and overcoming poor vaccine efficacy.

Targeting Stem-Loop-Binding Groove

Synthetic peptides that match the amino acid sequence of the stem polypeptide bind to trimer grooves in vitro. In model systems, externally applied synthetic peptides are carried into the endosome by the virus through non-specific binding to the membrane. Conformational changes lead to these synthetic peptides binding to the trimer grooves, blocking membrane fusion. Peptide mimetics are being explored to develop novel antivirals against Dengue and related viruses by targeting the stem-loop-binding groove.

Targeting NS5-NTD Methyltransferase

NS5-NTD methyltransferase is a potential antiviral target. Fragment-based drug design is used to discover novel dengue virus NS5 methyltransferase non-nucleoside inhibitors. The 7’ Methylation step is essential, while 2’O Methylation is not essential, but defects induce a strong antiviral response.

NS5 Methyltransferase and RNA Capping

The structure of NS5 includes NS5-ntd Methyltransferase (MTase) and NS5-ctd RNA-dependent RNA polymerase. The process involves ss RNA(+) genome capping, resulting in 7MeGpppA2’OMe−RNA7MeGpppA2’OMe-RNA

Dengue Structure, Temperature, and Maturity

Dengue structure depends on the temperature of culture (ToC) and maturity. The most advanced tetravalent live-attenuated DENV vaccine candidate showed a poor overall efficacy rate of 30%. Human and mosquito hosts have different temperatures: 37°C and ambient, respectively. The virus changes structure at 33°C to a “bumpy form,” which is the predominant form observed in humans. Vaccines should be raised against the “bumpy” form. Mature forms exist at > 33°C, and immature forms containing E and prM capsid are present at 37°C.

Difficulties in Vaccine Development

Multiple forms of Dengue virus hamper the development of an effective vaccine. These forms include 4 serotypes of Dengue virus and several structural forms: immature, partially mature, compact mature, and expanded mature.

Maturation of Dengue Virus

Virion assembly occurs in the endoplasmic reticulum with 60 trimeric spikes on the capsid surface, each spike being a trimer of the E:prM complex. The low pH of the trans-Golgi triggers trimer prM:E to dimer prM:E transformation, exposing the cleavage site on the prM, which is then cleaved by furin protease. The cleaved pr remains bound and caps the fusion peptide of the E protein, preventing the virus from fusing immediately back into the cell. Upon release into the cytosol, neutral pH causes dissociation of the pr molecule.

Classes of Glycoprotein-Mediated Membrane Fusion

Classes I, II, and III glycoprotein-mediated membrane fusion are compared regarding structure and function.

Structural Comparisons and Evolutionary Constraints

Class II fusion proteins from alphaviruses and flaviviruses are structurally closer to each other than they are to rubella virus E1. This suggests that the constraints on arboviruses imposed by alternating cycles between vertebrates and arthropods resulted in more conservative evolution. Rubella virus, being strictly human, seems to have drifted into a unique niche. Rubella virus E1 protein is a Class II fusion protein with significantly larger fusion loops presenting a “fusion surface,” more stem peptide visible, and a metal atom incorporated into the fusion surface. Rubella’s fusion surface area is 8,000A˚28,000 Å^2, compared to 3,000A˚23,000 Å^2 for flaviviruses and 5,000A˚25,000 Å^2 for alpha-viruses. Rubella also has 40 AA sequence “inserts.”

Class III Fusion Proteins

Glycoprotein G of the vesicular stomatitis virus is a Class III fusion protein.

Key Steps of Membrane Fusion

The key steps of membrane fusion include pre-fusion, extended intermediate, collapse of intermediate, hemi-fusion, and fusion pore.

Comparison of Fusion Classes

A table comparing fusion classes is included to summarize differences in structure and mechanisms.