2010 Biochemistry Protein Projects


Biochemistry I CHM 340
Spring Semester
Science Building Room 106
MWF, 1:20p.m.–2:30p.m.
Contact: Dr. Matthew Saderholm
CPO 2191
Berea College
Berea KY 40404
859-985-3322


Kathryn A Massana

CHM 345

Dengue Fever: Is An Antiviral Feasible?

Introduction

Dengue fever is a tropical viral disease that affects millions of people every year. It is vectored by mosquitoes and currently there is no vaccine or antiviral for this virus. This poses as a significant problem because this is an expanding disease that harms many people. Dengue is a flavivirus that has four serotypes, one of which is the Dengue-2-virus. A serotype refers to a variation of cell surface antigens within a viral subspecies. This paper will focus on the Dengue-2-virus serotype that has a NS5 methyltransferase protein. The NS5 protein is crucial to viral replication, without it, the virus will not be able to proliferate. This protein also plays a role in the pathogenesis of the Dengue-2-virus. It is important that scientists understand the protein structure and function of the Dengue-2 NS5 methyltransferase protein because it could help them discover a possible antiviral or vaccine. Furthermore, understanding this protein can also help with an antiviral development for other flaviviruses that have a similar protein as the Dengue-2 NS5 methyltransferase protein.

Background on Dengue Fever

Dengue is transmitted predominantly by the two mosquito species, Aedes aegypti and Aedes albopictus. This disease exists predominantly in Europe, Asia, South America and the Caribbean. In usual infections, people have several symptoms such as fever, severe headaches, muscle and joint pains, weakness and severe prostration. In uncomplicated cases, the recovery is usually rapid. However, if a "hemorrhagic complication" occurs there can be a series of symptoms ranging "from a rash and mottled skin to severe hemorrhaging in the lungs, digestive tract, and skin" (1). The spread of Dengue has increased due to aerial navigation (1), urbanization of people that are in "close association" with one of the mosquito vectors, and water storage practices that create a suitable living environment for the mosquitoes (2). Currently, there are a total of 30 million to 100 million cases of infected people a year and 2 billion people at risk (1). A problem with generating a vaccine is that all the dengue virus serotypes show genetic variation and studies provide evidence of genetic drift in different countries (2).

Flavivirus

As stated previously, the Dengue-2-virus is a flavivirus. Flaviviruses are relatively small enveloped RNA viruses and are a part of the Flaviviridae family (3). A flavivirus virus has a positive single stranded RNA structure. The structures of a flavivirus are shown in Figure 1. "The 5′ end of the genome contains a type 1 cap followed by a conserved dinucleotide sequence 5′-AG-3′. The single open reading frame of the flavivirus genome encodes a polyprotein which is processed by viral and cellular proteases into three structural proteins and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5)" (4).

Figure 1: Flavivirus Structure. A Single-Stranded RNA Strand is surrounded by a protein capsid. Both of these are encompassed by an envelope (5).

Flaviviral cap structure formation involves the proteins, NS3 and NS5 which are important in this process. This paper will focus on the NS5 protein. NS5 exhibits N-7 and 2-O-methyltransferase activities. Bioinformatic research has shown a probable methyltransferase domain in the initial 296 amino acids of Degue-2 NS5. Other examinations of methyltransferase activity using "short capped (GpppAC5 and m7GpppAC5) and non-capped (pppAC5) RNA substrates" have demonstrated that methyltransferase has 2'-O-methyltransferase activity (3). However, N-7 methyltransferase activity was not detected with these RNA substrates (3).

Methyltranferases in flaviviruses successively methylate the 5' viral RNA cap at guanine N-7 and ribose 2′-O positions. This is the mechanism that flaviviruses generally use to encode the NS5 methyltransferase with dual methylation for the synthesis of the viral RNA cap. A mutation in both of the methylations to the N-7 and 2'-O sites is lethal to the virus and will end the virus' life cycle (4). The life cycle of a flavivirus is shown in Figure 2.

Figure 2: The Life Cycle of a Flavivirus. In Section C, the capped single stranded RNA can be seen. In Section D, viral replication is taking place (6).

The NS5 methyltransferase domains have a dimeric relationship that permit the "sequential and coordinated" methylation of the N-7 and 2'-O groups caused by the Ado-Met co-factors of the methyltransferase domains (7). The RNA that has been capped will enter the groove between the two methyltransferase domains that have the Ado-Met co-factors. The Ado-Met1 co-factor will methylate the N-7 group of the inverted guanosine base. Furthermore, the Ado-Met2 co-factor will continue the process by methylating the ribose 2'-OH of the second base in the capped RNA strand. Figure 3 shows this methylation process (7).

Figure 3: A Model for the N-7 and 2'-O Methylation Process. The capped RNA strand enters the two methyltransferase domains. The Ado-Met co-factors further methylate the N-7 and 2'-O groups of the capped RNA strand (7).

Dengue-2 NS5 methyltransferase

Dengue-2 NS5 methyltransferase is one of the seven nonstructural proteins in the polyprotein encoded by the flavivirus genome's single open reading frame. This is the non-structural protein, NS5, which is the largest and most conserved protein in a flavivirus. The single open reading frame also encodes the capsid, membrane and envelope which are structural components of the virus (See Figure 1) (8). The Dengue-2-virus methyltransferase has an N-terminal subdomain, a core subdomain, a C-terminal subdomain (3) and a K-D-K-E motif (9). Dengue-2 NS5 methyltransferase also has an "S-adenosyl methionine-dependent methyltransferase fold" structure which is essentially a "sandwich" of αβα sheets in the N-terminal domain (4). The S-adenosyl methionine ligand as well as the αβ sheets are shown in Figure 4. [Click Bound SAH in Jmol Section] [Click Reset Button, Click Active Site in Jmol Section] [Click Reset, Click Alpha Helices in Jmol Section] [Click Reset, Click Beta Sheets in Jmol Section]

Figure 4: The Crystal Structure of Dengue-2-Virus NS5 Methyltransferase Complexed With S-Adenosyl-L-Homocysteine. The β-sheets are located near the center of the protein, while the α-helices are located on the outside of the structure (10).

The N-terminal domain has the capacity to bind GTP, hold the guanosine of the viral cap structure, and synthesize two different methylation reactions that are required for the formation of the RNA cap (3). A GTP-binding site in the N-terminal domain is suggested to be a cap-binding site for the Dengue-2-methyltransferase (4). The C-terminal subdomain is an RNA-dependent-RNA polymerase (RdRp) domain. The core subunit is responsible for Ado-Met binding and catalytic activity due to the GTP-binding pocket (3).

Interaction with Host

It has been shown from past experiments that seven host proteins interact with the viral replication complex. An example of one of these proteins is the La protein. NS5 is associated with membrane bound replication within the endoplasmic reticulum of the cell (8), but it also exists free in the cytoplasm and in some cases in the nucleus of infected cells. NS5 is associated with replication at the endoplasmic reticular membrane because this is the site where flaviviral replication occurs and NS5 in combination with a methyltransferase are needed­­­­­ for the methylation of the 5′-cap structure of the RNA (8). Since NS5 is known to be phosphorylated, this implies that NS5 interacts with host proteins that are involved in "intracellular trafficking and phosphorylation/dephosphorylation" (3). This NS5 protein could also disturb signaling of interferons and the production of cytokines in the host. For example, NS5 can disrupt type I Interferon signaling and has the capacity to induce the production of the cytokine Interleukin-8. Since this protein perturbs the host immune response, it plays a role in viral pathogenesis (3).

Mechanism

It is known that the S-adenosyl methionine ligand donates methyl groups to the N-7 and 2′-O positions of the viral RNA cap. The exact sequence of the two methylation processes is not currently known and is still being explored. Two sequences are possible, the N-7 methlyation requires the 2'-O methylation or the 2′-O methylation is dependent on the prior N-7 methylation. Some experiments have shown that the 2'-O methylation is not entirely dependent on the N-7 methylation. The 2′-O methylation requires a high pH which supports an SN2 mechanism of methyl transfer between the S-adenosyl methionine and the 2'-O of the viral RNA cap. After which, the target 2'-OH will nucleophilically attack the methyl group of the S-adenosyl methionine for methyl transfer (9).

Mutations

A study that incorporated mutations to different Dengue-2 NS5 methlyransferase residues that are involved in GTP binding showed that these mutations interfered with the methylation process, which is needed for viral replication. A mutation of Phe-25 to Ala eliminated GTP binding altogether, while a mutation of Asn-18, Lys-29, and Ser-150 to Ala decreased GTP binding, reassuring the significance of these residues in GTP binding (3) [Click Reset, Click Phe 25 in Jmol Section] [Click Reset, Click Asn 18 in Jmol Section] [Click Reset, Click Lys 29 in Jmol Section] [Click Reset, Click Ser 150 in Jmol Section]. It was also found that ribavirin triphosphate inhibits the 2'-O-methyltranferase activity by binding to the GTP binding site which suggests that the 2'-O-methylation "requires correct placement of capping the GTP-binding pocket" in order for replication to continue (3). A comparison of the structure of ribavirin triphosphate (Figure 5) to the structure of GTP (Figure 6) is shown in the figures below.

Figure 5: Structure of Ribavirin Triphosphate (11)

Figure 6: The Structure of Guanosine Triphosphate (GTP) (12)

Another type of mutation that can interfere with the replication of this virus is the phosphorylation of the methyltransferase. As stated previously, the methyltransferase is needed to methylate the viral cap so the viral replication process can continue, otherwise the virus cannot replicate. The 2'-O-methylation requires a hydroxyl side chain of the Serine-56 residue [Click Reset, Click Serine 56 in Jmol Section]. When this residue is phosphorylated, it will then have a negative charge which inhibits enzymatic activity and essentially stops the viral replication process. The "phosphorylation of NS5 affects NS5 interactions with the viral helicase NS3" (13). Also, a "hyperphosphorylated form of NS5 was found to localize to the nucleus away from the cytoplasmic sites of viral replication" (13). Therefore, phosphorylation has shown to be a good way to regulate protein activity because it interferes with the viral cycle by affecting NS5, and without NS5, the viral cycle could not exist.

As mentioned before, the Dengue-2 virus is subject to changes in its genome. This is an important problem to consider when generating a vaccine. The Dengue-2-virus NS5 methyltransferase protein is a good choice to use as a target to disrupt the proliferation of this virus because it is highly conserved. Therefore, even though the virus is prone to changes in its genome, it most likely will not have a mutation in the NS5 protein sequence, because this is a very highly conserved molecule involved in viral replication, and without it, the virus would cease to exist. Any slight mutation might disrupt the viral replication process and terminate it.

Conclusion

Many scientists agree that an antiviral and/or vaccine should be developed for this emerging disease. The Dengue-2 NS5 methyltransferase protein associated with this virus is relatively new, and specifics about its mechanism are not exactly known. However, the information that is known about this specific protein and virus can lead scientists to potentially develop a vaccine. Although this flavivirus has the ability to mutate like many other viruses and create other strains, there are different approaches to creating an antiviral or vaccine using the NS5 protein as a target. Some of these approaches are phosphorylating the NS5 protein, tampering with NS5 methyltransferase, and causing mutations to specific residues that are actively interacting in the methlyation of the viral RNA cap. Since viruses in the Flaviviridae family are somewhat similar to the NS5 protein of the Dengue-2-virus, then by understanding this virus's protein structure and function it can be used as a target protein when developing antivirals and/or vaccines for other flaviviruses.

Bibliography

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