Posts Tagged ‘Antivirals’

A new drug against MERS?

Friday, May 30th, 2014

MERS virus Before the emergence of the highly pathogenic severe acute respiratory syndrome-associated coronavirus (SARS-CoV) in 2003 only two circulating human coronaviruses (HCoVs), HCoV- 229E and HCoV-OC43 causing relatively mild common cold-like respiratory tract infections, were known, and coronaviruses have not been regarded as significant threat for human health. Now, more than ten years later, the emergence of another highly pathogenic coronavirus of zoonotic origin, the Middle East respiratory syndrome coronavirus (MERS-CoV) points to the need for effective drugs against coronaviruses. Viruses such as coronaviruses that replicate in the host cell cytoplasm have evolved to employ host cell-derived membranes to compartmentalize genome replication and transcription. Specifically for positive-stranded RNA viruses, accumulating knowledge concerning the involvement, rearrangement and requirement of cellular membranes for RNA synthesis specify the establishment of the viral replicase complex at host cell-derived membranes as an evolution- ary conserved and essential step in the early phase of the viral life cycle.

A new paper in PLoS Pathogens describe a small molecule inhibitor of coronavirus replication that specifically targets this membrane-bound RNA replication step and has broad antiviral activity against number of diverse coronaviruses including highly pathogenic SARS-CoV and MERS-CoV. Since resistance mutations appear in an integral membrane-spanning component of the coronavirus replicase complex, and since all positive stranded RNA viruses have very similar membrane-spanning or membrane-associated replicase components implicated in anchoring the viral replication complex to host cell-derived membranes, the data suggests that the membrane-bound replication step of the viral life cycle is a novel, vulnerable, and druggable target for antiviral intervention of a wide range of RNA virus infections.

Of course clinical trials are needed before such drugs could be used, so we’re still years away from this approach being put into practce. Just in time for the next emergent coronavirus maybe?


Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus. (2014) PLoS Pathog 10(5): e1004166. doi:10.1371/journal.ppat.1004166
Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS–CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.


Reasons to be cheeful: Influenza treatment

Friday, April 11th, 2014

Lung immunity against influenza virus As we find out that Tamiflu is no more effective than paracetamol or ibuprofen in treating influenza infection (NHS Choices: Effectiveness of Tamiflu and Relenza questioned) – giving Ben Goldacre the right to say I told you so – maybe there is some reason to be more optimistic about treating influenza.

A new paper in Immunity [subscription] shows that prostaglandin E2 (PGE2) is upregulated during influenza A virus infection, and this inhibits macrophage recruitment to the lungs as well as interferon production and apoptosis in influenza virus-infected macrophages. This results in impaired macrophage antigen presentation and reduced adaptive immunity against influenza virus. The good news is that suppression of PGE2 with prostaglandin inhibitors protects against influenza infection. And we’ve got lots of prostaglandin inhibitors, including ibuprofen and other nonsteroidal anti-inflammatory drugs (NSAIDs) that work by inhibiting a molecule called cyclooxygenase (COX). The lung innate immune system has a critical role in limiting respiratory viral infections, particularly in the case of the nastier strains of flu such as the 1918 Spanish Influenza virus (and those still to come). So this is potentially very good news.

The catch? Well this paper refers to studies in mice and clinical trials will need to be done in humans to show the same effects. Clinical trials will be easy to do as many COX- and PGE-inhibitors are already approved for human use. All we need to do is avoid Roche doing the trial, or we may never find out the results.

Targeted Prostaglandin E2 Inhibition Enhances Antiviral Immunity through Induction of Type I Interferon and Apoptosis in Macrophages. Immunity, 10 April 2014 doi:
Summary: Aspirin gained tremendous popularity during the 1918 Spanish Influenza virus pandemic, 50 years prior to the demonstration of their inhibitory action on prostaglandins. Here, we show that during influenza A virus (IAV) infection, prostaglandin E2 (PGE2) was upregulated, which led to the inhibition of type I interferon (IFN) production and apoptosis in macrophages, thereby causing an increase in virus replication. This inhibitory role of PGE2 was not limited to innate immunity, because both antigen presentation and T cell mediated immunity were also suppressed. Targeted PGE2 suppression via genetic ablation of microsomal prostaglandin E-synthase 1 (mPGES-1) or by the pharmacological inhibition of PGE2 receptors EP2 and EP4 substantially improved survival against lethal IAV infection whereas PGE2 administration reversed this phenotype. These data demonstrate that the mPGES-1-PGE2 pathway is targeted by IAV to evade host type I IFN-dependent antiviral immunity. We propose that specific inhibition of PGE2 signaling might serve as a treatment for IAV.

[Editorial comment: I can just imaging the authors and journal editors doing the happy dance that this paper came out on sthe same day as the Tamiflu news.]

HIV cure research – advances and prospects

Thursday, March 20th, 2014

HIV reservoirs Thirty years after the identification of HIV, a cure for HIV infection is still to be achieved. Advances of combined antiretroviral therapy (cART) (=HAART) in recent years have transformed HIV infection into a chronic disease when treatment is available. However, in spite of the favorable outcomes provided by the newer therapies, cART is not curative and patients are at risk of developing HIV-associated disorders. Moreover, universal access to antiretroviral treatment is restricted by financial obstacles. This review discusses the most recent strategies that have been developed in the search for an HIV cure and to improve life quality of people living with HIV.


  • Some cases of cure or remission of infection have boosted the search for an HIV cure.
  • cART intensification has not shown significant impact in the reservoirs, but early cART may limit them.
  • Strategies to purge the reservoirs face difficulties linked to the complexity of latency mechanisms and drug non-specificity.
  • Repression of reservoirs or cell manipulation to render them less permissive to HIV may facilitate HIV remission.
  • HIV cure/remission may require boosting immune responses while keeping inflammation in check.


HIV cure research: Advances and prospects. (2014) Virology pii: S0042-6822(14)00065-8. doi: 10.1016/j.virol.2014.02.021

Why mice don’t get MERS

Wednesday, November 13th, 2013

Mouse I’m interested in Middle East Respiratory Syndrome Coronavirus (MERS-CoV) for a number of reasons, and as a result I have a student currently doing a final year project with me on this topic – not chucking buckets of MERS-CoV around in the laboratory, but trying to figure out where this virus came from and what it is likely to do next. Both of these are interesting questions.

There has been a lot published about the origins of MERS-CoV recently. Only this week came the news that a camel in Saudi Arabia has tested positive for the virus. But which came first – the virus or the camel? Almost certainly the camel – there’s no reason to suppose that camels are the original source of the outbreak. MERS is almost certainly a zoonotic infection – arising in animals and transmitted to humans – but which animals? The closest relatives to MERS-CoV have been found in bats, and those viruses are pretty similar to the virus currently causing human deaths. However, these bat viruses have only been identified by nucleotide sequences and have never been isolated as live viruses from either bats or the environment, so the animal reservoir of MERS-CoV has still not been identified (Emergence of the Middle East Respiratory Syndrome Coronavirus. (2013) PLoS Pathog 9(9): e1003595. doi:10.1371/journal.ppat.1003595).

If we don’t know where MERS came from, we should all be interested in the question of what it is likely to do next. Since September 2012, there have been over 150 laboratory-confirmed cases of infection with MERS-CoV – not that many on a global scale. That’s because the virus is only weakly infectious in humans. As long as this remains the case we are OK, but if at some point it decides it likes being in humans and wants more of the same, then we’re in trouble. What are the odds of that happening? Right now, we simply don’t know. And that’s why I’m interested in MERS.

To answer the question of what MERS will do next, we need a lot more knowledge than we have right now. One of the key pieces of information is exactly how MERS-CoV gets inside a host cell, and specifically, why it finds it difficult to infect human cells. It was recently shown that the receptor MERS-CoV needs to infect cells is dipeptidyl peptidase 4, a cell surface protein which cleaves dipeptides from hormones and chemokines after a proline amino acid residue, regulating their bioactivity (Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. (2013) Nature 495, 251-254). Dipeptidyl peptidase 4 is similar to other known coronavirus receptors, but the use of these peptidases as receptors by coronaviruses could be more related to their abundance on epithelial and endothelial tissues – the primary tissues involved in coronavirus infection – rather than any inherent properties of the protein.

That’s where a new paper in the Journal of General Virology comes in. It’s difficult to study lethal viruses in humans, so like it or not animal models of infection still have their place in these life-threatening outbreaks. The study of SARS-CoV pathogenesis progressed rapidly due to the development of a mouse adapted variant of SARS-CoV that produced lethal lung disease in mice similar to SARS in humans. But MERS-CoV doesn’t like growing in mice and it turns out that this is because mice have low levels of dipeptidyl peptidase 4 mRNA in their lungs (Wild type and innate immune deficient mice are not susceptible to the Middle East Respiratory Syndrome Coronavirus. J Gen Virol. 06 Nov 2013 doi: 10.1099/vir.0.060640-0). Good news for the mice, but also of great interest when thinking about how the pathogenesis of MERS is shaped in humans. Most viruses are not able to switch from one receptro to another easily, so drugs which interfere with the binding of the virus to this protein or antibodies which block attachment could be the best way to treat MERS until we have a vaccine which is able to stop people becoming infected. And the search for those drugs and antibodies is going on right now.


HIV thirty years on

Thursday, August 29th, 2013

HIV It has been thirty years since human immunodeficiency virus (HIV) was first identified as the cause of acquired immunodeficiency syndrome (AIDS), a landmark discovery that has led to tremendous progress in understanding and combating infection by this life-threatening retrovirus. The most notable achievement during this time has been the development of antiretroviral therapies that substantially improve the quality and length of life in infected individuals. Nevertheless, to date neither a complete cure nor a protective vaccine have been found, and new infections continue to occur at a rate of 6,850 people per day.

This collection of articles takes stock of where we are now, with a collection of articles that discuss different aspects of HIV infection, the progress made towards eradicating the virus, and the challenges of fundamental science and clinical management that remain.

BioMed Central: HIV thirty years on (2013)

Getting rid of HIV for good

Wednesday, June 5th, 2013

HIV-infected cell The eradication of HIV-1 from infected individuals is prevented by the persistence of the virus in a stable reservoir of latently infected CD4+ T cells. Latently infected cells can be found in all HIV-1 infected individuals at a very low frequency and allow the virus to persist despite antiretroviral therapy for the lifetime of an infected patient. Current efforts are focused on identifying small molecules or immune strategies to eliminate these latently infected cells. To assess the efficacy of these elimination strategies in HIV-1 infected patients, we must be able to measure the size of the remaining latent reservoir. While a previous assay can measure the size of this latent reservoir, it is too laborious and costly to be utilized in large-scale HIV-1 eradication trials. A new paper in PLoS Pathogens describes a rapid assay to measure the size of the HIV-1 latent reservoir more amenable to eradication trials.


Rapid Quantification of the Latent Reservoir for HIV-1 Using a Viral Outgrowth Assay. (2013) PLoS Pathog 9(5): e1003398. doi:10.1371/journal.ppat.1003398
HIV-1 persists in infected individuals in a stable pool of resting CD4+ T cells as a latent but replication-competent provirus. This latent reservoir is the major barrier to the eradication of HIV-1. Clinical trials are currently underway investigating the effects of latency-disrupting compounds on the persistence of the latent reservoir in infected individuals. To accurately assess the effects of such compounds, accurate assays to measure the frequency of latently infected cells are essential. The development of a simpler assay for the latent reservoir has been identified as a major AIDS research priority. We report here the development and validation of a rapid viral outgrowth assay that quantifies the frequency of cells that can release replication-competent virus following cellular activation. This new assay utilizes bead and column-based purification of resting CD4+ T cells from the peripheral blood of HIV-1 infected patients rather than cell sorting to obtain comparable resting CD4+ T cell purity. This new assay also utilizes the MOLT-4/CCR5 cell line for viral expansion, producing statistically comparable measurements of the frequency of latent HIV-1 infection. Finally, this new assay employs a novel quantitative RT-PCR specific for polyadenylated HIV-1 RNA for virus detection, which we demonstrate is a more sensitive and cost-effective method to detect HIV-1 replication than expensive commercial ELISA detection methods. The reductions in both labor and cost make this assay suitable for quantifying the frequency of latently infected cells in clinical trials of HIV-1 eradication strategies.


Understanding Uncoating

Thursday, April 11th, 2013

Picornavirus Uncoating
Principles of Molecular Virology

I always tell my students that any stage of virus replication can be a target for antiviral therapy – as long as it is essential to replication and specific to the virus and therefore that inhibiting it does not damage the host cell. So far we have mostly limited ourselves to a very few stages of the replication cycle, and we only have one or two drugs (against influenza virus) that inhibit the vital uncoating step of the replication cycle. Understanding the processes involved is therefore of great importance in developing new drugs. A recent paper in PLOS Pathogens examines the uncoating of rhinovirus particles and makes some interesting findings.

Human rhinoviruses (HRV) are members of the picornavirus family, and are one of the major causative agents of the common cold. Additionally, they play important roles in the exacerbation of asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Similar to other picornaviruses, the rhinovirus particle are composed of 60 copies each of four capsid proteins, VP1, VP2, VP3 and VP4, arranged on an icosahedral lattice. The diameter of the particle is roughly 30 nm. The virus genome is a single-stranded RNA molecule of positive sense, about 7100 bases in length. It carries a covalently linked peptide (VPg) at its 5′-end and a poly-(A) tail of about 70 to 150 bases at its 3′-end. The 5′-nontranslated region is approximately 650 bases in length, highly structured, and involved in cap-independent translation initiation and RNA replication.

Minor group rhinoviruses, e.g. the prototype strain HRV2, bind members of the low-density lipoprotein receptor (LDLR) family including LDLR and LDLR-related protein for entry via clathrin-dependent endocytosis. Once in the endosome, the low pH leads to dissociation of the virus from the receptors as well as to structural changes in the viral capsid. The native virion sedimenting at 150S converts into the subviral A-particle sedimenting at 135S and devoid of the internal capsid protein VP4 and exposure of amphipathic N-terminal sequences of VP1 renders it hydrophobic, thus allowing its direct attachment to the inner endosomal membrane. These processes are accompanied by an expansion of the virus shell by about 4% along with the opening of symmetry-related channels. The largest channels are at the two-fold axes, whereas the smaller ones are located near the pseudo three-fold axes and at the base of the star-shaped vertices of the icosahedron. Finally, the RNA is released through one of these pores, most probably at a 2-fold axis. The final product of this uncoating process is the empty capsid (80S B-particle). Most enteroviruses undergo similar conformational changes; however, with the exception of minor receptor group rhinoviruses, the process appears to be triggered by receptor binding and possibly assisted by low pH.

These structural modifications can be mimicked, at least partially, in vitro. Exposure to pH <5.8 converts native HRV2 preferentially into A-particles whereas incubation at 50°C–56°C in low ionic strength buffers favours conversion into B-particles (empty capsids). In vivo, and in the presence of liposomes in vitro, both VP4 and N-terminal sequences of VP1 insert into lipid bilayers. They might contribute to formation of a pore connecting the virus interior with the cytosol of the host cell, thus allowing for the transit of RNA in its unfolded form.

The mechanism of RNA exit is poorly understood. Energy would be required for breaking the hydrogen bonds of the double-stranded regions in the encapsidated RNA genome in order to allow the RNA to thread through an opening only large enough to enable passage of a single strand. It appears likely that either the poly-(A) tail at the 3′-end or the VPg peptide linked to the 5 end of the RNA begins to emerge from the virion since other modes might be unproductive (e.g., simultaneous exit of both ends would be expected to impede complete uncoating and thus to be abortive). Directionality of this process may indicate that the RNA adopts a defined conformation inside the viral shell suggesting a well-organized process of assembly and uncoating.

The new paper shows that RNA exit does indeed occur in a specific and ordered manner, starting from the 3′-end. Ordered exit of RNA also suggests that the virus genome becomes organized during packaging or assembly, which may occur co-transcriptionally. Therefore, it is likely that the process of encapsidation begins when the 5′-end emerges from the replication complex or at least before the complete RNA has been synthesized. It is also possible that the same applies to other viruses with ssRNA of positive polarity. This would imply that in these viruses, the 3′-end becomes encapsidated last, remaining near the capsid wall presumably in close proximity to one of the holes poised to open upon uncoating, thus resulting in a ‘last-in-first-out’ process of assembly and uncoating.

So all we need now is a drug to inhibit this process, and we’ve cured the common cold. Well, some of them maybe :-)


Viral Uncoating Is Directional: Exit of the Genomic RNA in a Common Cold Virus Starts with the Poly-(A) Tail at the 3′-End. (2013) PLoS Pathog 9(4): e1003270. doi:10.1371/journal.ppat.1003270
Viral infection requires safe transfer of the viral genome from within the protective protein shell into the host cell’s cytosol. For many viruses this happens after uptake into endosomes, where receptor-binding and/or the acidic pH trigger conformational modifications or disassembly of the shell, allowing the nucleic acids to escape. For example, common cold viruses are converted into subviral particles still containing the single-stranded positive sense RNA genome; subsequently, the RNA escapes into the cytoplasm, leaving behind empty capsids. We triggered this process by heating HRV2 to 56°C and found that 3′- and 5′-end emerged with different kinetics. Crosslinking prevented complete RNA egress and upon nuclease digestion only sequences derived from the 5′-end were protected. Part of the RNA remaining within the viral shell adopted a rod-like shape pointing towards one of the two-fold axes where the RNA is presumed to exit in single-stranded form. Egress thus commences with the poly-(A) tail and not with the genome-linked peptide VPg. This suggests that assembly and uncoating are well-coordinated to avoid tangling, kinetic traps, and/or simultaneous exit of the two RNA ends at different sites.


Strategies to Develop Antivirals against Enterovirus 71 

Tuesday, January 22nd, 2013

Enterovirus 71 (EV71) is an important human pathogen which can cause severe neurological complications and death in children. The virus caused several outbreaks in the Asia-Pacific region during the past two decades and has been considered a significant public health problem in the post-poliovirus eradication era. Unlike poliovirus, there is no effective vaccine or approved antivirals against EV71. To explore anti-EV71 agents therefore is of vital importance. Several strategies have been employed to develop antivirals based on the molecular characteristics of the virus. Among these, some small molecules that were developed against human rhinoviruses and poliovirus are under evaluation. In this review, we discuss the recent development of such small molecules against EV71, known drug resistance and possible solutions to it, and animal models for evaluating the efficacy of these antivirals. Although further investigation is required for clinical applications of the existing candidates, the molecular mechanisms revealed for the inhibition of EV71 replication can be used for designing new molecules against this virus in the future. Virology Journal:


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HIV causes AIDS – and how to stop it

Monday, December 17th, 2012

HIV-1 Induced Bystander Apoptosis A couple of nice papers published recently on the mechanisms by which HIV infection causes AIDS. The first concerns the possible role of bystander apoptosis induction in HIV infection and its role in disease progression. It has become evident that the process is not as simple as previously thought:

HIV-1 Induced Bystander Apoptosis. (2012) Viruses 4(11): 3020-3043; doi:10.3390/v4113020
Apoptosis of uninfected bystander cells is a key element of HIV pathogenesis and believed to be the driving force behind the selective depletion of CD4+ T cells leading to immunodeficiency. While several viral proteins have been implicated in this process the complex interaction between Env glycoprotein expressed on the surface of infected cells and the receptor and co-receptor expressing bystander cells has been proposed as a major mechanism. HIV-1 utilizes CD4 as the primary receptor for entry into cells; however, it is the viral co-receptor usage that greatly influences CD4 decline and progression to AIDS. This phenomenon is relatively simple for X4 viruses, which arise later during the course of the disease, are considered to be highly fusogenic, and cause a rapid CD4+ T cell decline. However, in contrast, R5 viruses in general have a greater transmissibility, are encountered early during the disease and have a lesser pathogenic potential than the former. The above generalization gets complicated in numerous situations where R5 viruses persist throughout the disease and are capable of causing a rigorous CD4+ T cell decline. This review will discuss the multiple factors that are reported to influence HIV induced bystander apoptosis and pathogenesis including Env glycoprotein phenotype, virus tropism, disease stage, co-receptor expression on CD4+ T cells, immune activation and therapies targeting the viral envelope.

The second paper discusses the possibilites for new drugs which fight HIV infection in more subtle ways than simply blocking enzyes:

Back to the future: revisiting HIV-1 lethal mutagenesis. Trends Microbiol. 26 November 2012, doi: 10.1016/j.tim.2012.10.006
The concept of eliminating HIV-1 infectivity by elevating the viral mutation rate was first proposed over a decade ago, even though the general concept had been conceived earlier for RNA viruses. Lethal mutagenesis was originally viewed as a novel chemotherapeutic approach for treating HIV-1 infection in which use of a viral mutagen would over multiple rounds of replication lead to the lethal accumulation of mutations, rendering the virus population non infectious – known as the slow mutation accumulation model. There have been limitations in obtaining good efficacy data with drug leads, leaving some doubt on clinical translation. More recent studies of the apolipoprotein B mRNA editing complex 3 (APOBEC3) proteins as well as new progress in the use of nucleoside analogs for inducing lethal mutagenesis have helped to refocus attention on rapid induction of HIV-1 lethal mutagenesis in a single or limited number of replication cycles leading to a rapid mutation accumulation model.


This is all good stuff, but it’s frustrating that after all these years we still don’t have a better picture of how HIV causes AIDS – and how to stop it.