Detailed computational study of the active site of the hepatitis C viral RNA polymerase to aid novel drug design.

The hepatitis C virus (HCV) RNA polymerase, NS5B, is a leading target for novel and selective HCV drug design. The enzyme has been the subject of intensive drug discovery aimed at developing direct acting antiviral (DAA) agents that inhibit its activity and hence prevent the virus from replicating its genome. In this study, we focus on one class of NS5B inhibitors, namely nucleos(t)ide mimetics. Forty-one distinct nucleotide structures have been modeled within the active site of NS5B for the six major HCV genotypes. Our comprehensive modeling protocol employed 287 different molecular dynamics simulations combined with the molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) methodology to rank and analyze these structures for all genotypes. The binding interactions of the individual compounds have been investigated and reduced to the atomic level. The present study significantly refines our understanding of the mode of action of NS5B-nucleotide-inhibitors, identifies the key structural elements necessary for their activity, and implements the tools for ranking the potential of additional much needed novel inhibitors of NS5B.

Progress towards a hepatitis C virus vaccine.

New drugs to treat hepatitis C are expected to be approved over the next few years which promise to cure nearly all patients. However, due to issues of expected drug resistance, suboptimal activity against diverse hepatitis C virus (HCV) genotypes and especially because of their extremely high cost, it is unlikely that these HCV drugs will substantially reduce the world's HCV carrier population of around 170 million in the near future or the estimated global incidence of millions of new HCV infections. For these reasons, there is an urgent need to develop a prophylactic HCV vaccine and also to determine if therapeutic vaccines can aid in the treatment of chronically infected patients. After much early pessimism on the prospects for an effective prophylactic HCV vaccine, our recent knowledge of immune correlates of protection combined with the demonstrated immunogenicity and protective animal efficacies of various HCV vaccine candidates now allows for realistic optimism. This review summarizes the current rationale and status of clinical and experimental HCV vaccine candidates based on the elicitation of cross-neutralizing antibodies and broad cellular immune responses to this highly diverse virus.

The antiviral activity and mechanism of action of (S)-[3-hydroxy-2-(phosphonomethoxy)propyl] (HPMP) nucleosides.

One class of compounds that has shown promise as antiviral agents are the (S)-[3-hydroxy-2-(phosphonomethoxy)propyl] (HPMP) nucleosides, members of the broader class of acyclic nucleoside phosphonates. These HPMP nucleosides are nucleotide analogs and have been shown to be effective inhibitors of a wide range of DNA viruses. Prodrugs of these compounds, which achieve higher levels of the active metabolites within the cell, have an expanded activity spectrum that also includes RNA viruses and retroviruses. Because they are analogs of natural nucleotide substrates, HPMP nucleosides are predicted to target polymerases (DNA polymerases, RNA polymerases and reverse transcriptases), resulting in the inhibition of viral genome replication. Previous work using the replicative enzymes of different viruses including human cytomegalovirus (HCMV) and vaccinia virus DNA polymerases and human immunodeficiency virus type 1 (HIV-1) reverse transcriptase has shown that the activated forms of these compounds are substrates for viral polymerases and that incorporation of these compounds into either the primer strand or the template strand inhibits, but does not necessarily terminate, further nucleic acid synthesis. The activity of these compounds against other viruses that do not encode their own polymerases, like polyoma viruses and papilloma viruses, suggests that host cell DNA polymerases are also targeted. This complex mechanism of action and broad activity spectrum has implications for the development of resistance and host cell genome replication, and suggests these compounds may be effective against other viruses such as influenza virus, respiratory syncytial virus and Dengue virus. This class of nucleotide analogs also points to a potential avenue for the development of newer antivirals.

Inhibition of HIV-1 by octadecyloxyethyl esters of (S)-[3-hydroxy-2-(phosphonomethoxy)propyl] nucleosides and evaluation of their mechanism of action.

(S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC [cidofovir]) and (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) are potent inhibitors of a variety of DNA viruses. These drugs possess a 3'-hydroxyl equivalent which could support chain extension from an incorporated drug molecule. HPMPC and HPMPA were initially reported to lack activity against human immunodeficiency virus type 1 (HIV-1); more recent results have shown that the octadecyloxyethyl (ODE) and hexadecyloxypropyl (HDP) esters of HPMPA are potent inhibitors of the virus. We have synthesized the ODE esters of a series of (S)-[3-hydroxy-2-(phosphonomethoxy)propyl] (HPMP) nucleosides, including HPMPC, HPMP-guanine (HPMPG), HPMP-thymine (HPMPT), and HPMP-diaminopurine (HPMPDAP), as well as the ODE ester of the obligate chain terminator (S)-9-[3-methoxy-2-(phosphonomethoxy)-propyl]adenine (MPMPA). All compounds except ODE-HPMPT were inhibitors of HIV-1 replication at low nanomolar concentrations. These compounds were also inhibitors of the replication of HIV-1 variants that are resistant to various nucleoside reverse transcriptase (RT) inhibitors at concentrations several times lower than would be expected to be achieved in vivo. To investigate the mechanism of the antiviral activity, the active metabolites of HPMPC and HPMPA were studied for their effects on reactions catalyzed by HIV-1 RT. Incorporation of HPMPC and HPMPA into a DNA primer strand resulted in multiple inhibitory effects exerted on the enzyme and showed that neither compound acts as an absolute chain terminator. Further, inhibition of HIV-1 RT also occurred when these drugs were located in the template strand. These results indicate that HPMPC and HPMPA inhibit HIV-1 by a complex mechanism and suggest that this class of drugs has a broader spectrum of activity than previously shown.

Solution structure of a DNA duplex containing the potent anti-poxvirus agent cidofovir.

Cidofovir (1(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine, CDV) is a potent inhibitor of orthopoxvirus DNA replication. Prior studies have shown that, when CDV is incorporated into a growing primer strand, it can inhibit both the 3'-to-5' exonuclease and the 5'-to-3' chain extension activities of vaccinia virus DNA polymerase. This drug can also be incorporated into DNA, creating a significant impediment to trans-lesion DNA synthesis in a manner resembling DNA damage. CDV and deoxycytidine share a common nucleobase, but CDV lacks the deoxyribose sugar. The acyclic phosphonate bears a hydroxyl moiety that is equivalent to the 3'-hydroxyl of dCMP and permits CDV incorporation into duplex DNA. To study the structural consequences of inserting CDV into DNA, we have used (1)H NMR to solve the solution structures of a dodecamer DNA duplex containing a CDV molecule at position 7 and of a control DNA duplex. The overall structures of both DNA duplexes were found to be very similar. We observed a decrease of intensity (>50%) for the imino protons neighboring the CDV (G6, T8) and the cognate base G18 and a large chemical shift change for G18. This indicates higher proton exchange rates for this region, which were confirmed using NMR-monitored melting experiments. DNA duplex melting experiments monitored by circular dichroism revealed a lower T(m) for the CDV DNA duplex (46 °C) compared to the control (58 °C) in 0.2 M salt. Our results suggest that the CDV drug is well accommodated and stable within the dodecamer DNA duplex, but the stability of the complex is less than that of the control, suggesting increased dynamics around the CDV.

Production and characterization of antibodies against vaccinia virus DNA polymerase.

Poxviruses are large DNA viruses that replicate in discrete locations in the cytoplasm of infected cells called viral factories. Because the host cell DNA replication machinery is located in the nucleus, poxviruses encode many of the proteins required for their own DNA replication, including a DNA polymerase. Although many if not all of the enzymes that are required for viral DNA replication have been identified, the actual mechanism of poxvirus DNA replication remains unclear. Two monoclonal antibodies and a polyclonal antibody against vaccinia virus DNA polymerase were produced and characterized for use as tools to investigate the mechanism of virus DNA replication. Although the monoclonal antibodies were not suitable for Western blotting, the polyclonal antibody was able to detect the protein in infected cell lysates using this method. In contrast, while the polyclonal antibody did not recognize the DNA polymerase when used for immunofluorescence microscopy, the monoclonal antibodies were able to detect the polymerase in vaccinia viral factories. In addition, one of these antibodies also stained viral factories produced by cowpox and ectromelia, two closely related viruses. Finally, all three antibodies were able to immunoprecipitate vaccinia DNA polymerase from infected cell lysates. These antibodies will be useful in experiments designed to describe more fully the role of the viral DNA polymerase in DNA replication of vaccinia virus.

Orthopoxviruses require a functional ubiquitin-proteasome system for productive replication.

Cellular homeostasis depends on an intricate balance of protein expression and degradation. The ubiquitin-proteasome pathway plays a crucial role in specifically targeting proteins tagged with ubiquitin for destruction. This degradation can be effectively blocked by both chemically synthesized and natural proteasome inhibitors. Poxviruses encode a number of proteins that exploit the ubiquitin-proteasome system, including virally encoded ubiquitin molecules and ubiquitin ligases, as well as BTB/kelch proteins and F-box proteins, which interact with cellular ubiquitin ligases. Here we show that poxvirus infection was dramatically affected by a range of proteasome inhibitors, including MG132, MG115, lactacystin, and bortezomib (Velcade). Confocal microscopy demonstrated that infected cells treated with MG132 or bortezomib lacked viral replication factories within the cytoplasm. This was accompanied by the absence of late gene expression and DNA replication; however, early gene expression occurred unabated. Proteasomal inhibition with MG132 or bortezomib also had dramatic effects on viral titers, severely blocking viral replication and propagation. The effects of MG132 on poxvirus infection were reversible upon washout, resulting in the production of late genes and viral replication factories. Significantly, the addition of an ubiquitin-activating enzyme (E1) inhibitor had a similar affect on late and early protein expression. Together, our data suggests that a functional ubiquitin-proteasome system is required during poxvirus infection.

Cidofovir and (S)-9-[3-hydroxy-(2-phosphonomethoxy)propyl]adenine are highly effective inhibitors of vaccinia virus DNA polymerase when incorporated into the template strand.

The acyclic nucleoside phosphonate drug (S)-9-[3-hydroxy-(2-phosphonomethoxy)propyl]adenine [(S)-HPMPA], is a broad-spectrum antiviral and antiparasitic agent. Previous work has shown that the active intracellular metabolite of this compound, (S)-HPMPA diphosphate [(S)-HPMPApp], is an analog of dATP and targets DNA polymerases. However, the mechanism by which (S)-HPMPA inhibits DNA polymerases remains elusive. Using vaccinia virus as a model system, we have previously shown that cidofovir diphosphate (CDVpp), an analog of dCTP and a related antiviral agent, is a poor substrate for the vaccinia virus DNA polymerase and acts to inhibit primer extension and block 3'-to-5' proofreading exonuclease activity. Based on structural similarities and the greater antiviral efficacy of (S)-HPMPA, we predicted that (S)-HPMPApp would have a similar, but more pronounced effect on vaccinia polymerase than CDVpp. Interestingly, we found that (S)-HPMPApp is a good substrate for the viral enzyme, exhibiting K(m) and V(max) parameters comparable to those of dATP, and certainly not behaving like CDVpp as a functional chain terminator. Metabolic experiments indicated that (S)-HPMPA is converted to (S)-HPMPApp to a much greater extent than CDV is converted to CDVpp, although both drugs cause identical effects on virus DNA replication at their 50% effective concentration. Subsequent studies showed that both compounds can be faithfully incorporated into DNA, but when CDV and (S)-HPMPA are incorporated into the template strand, both strongly inhibit trans-lesion DNA synthesis. It thus appears that nucleoside phosphonate drugs exhibit at least two different effects on DNA polymerases depending upon in what form the enzyme encounters the drug.

Mechanism of inhibition of vaccinia virus DNA polymerase by cidofovir diphosphate.

Cidofovir (CDV) is a broad-spectrum antiviral agent that has been approved for clinical use in the treatment of cytomegalovirus retinitis. It has also been used off label to treat a variety of other viral infections, including those caused by orf and molluscum contagiosum poxviruses. Because it is a dCMP analog, CDV is thought to act by inhibiting viral DNA polymerases. However, the details of the inhibitory mechanism are not well established and nothing is known about the mechanism by which the drug inhibits poxvirus DNA polymerases. To address this concern, we have studied the effect of the active intracellular metabolite of CDV, CDV diphosphate (CDVpp), on reactions catalyzed by vaccinia virus DNA polymerase. Using different primer-template pairs and purified vaccinia virus polymerase, we observed that CDV is incorporated into the growing DNA strand opposite template G's but the enzyme exhibits a lower catalytic efficiency compared with dCTP. CDV-terminated primers are also good substrates for the next deoxynucleoside monophosphate addition step, but these CDV + 1 reaction products are poor substrates for further rounds of synthesis. We also noted that although CDV can be excised from the primer 3' terminus by the 3'-to-5' proofreading exonuclease activity of vaccinia virus polymerase, DNAs bearing CDV as the penultimate 3' residue are completely resistant to exonuclease attack. These results show that vaccinia virus DNA polymerase can use CDVpp as a dCTP analog, albeit one that slows the rate of primer extension. By inhibiting the activity of the proofreading exonuclease, the misincorporation of CDV could also promote error-prone DNA synthesis during poxvirus replication.