Cells fight back against virus factories

Rabies virus stress granules This week I’ve been talking to first year students about cell biology, discussing how much the environment of the cell varies from one site to another within the cell. Viruses “know” this and much virus replication is localized at particular sites within the cell, not just occurring haphazardly. The first example of this is to be discioved were Negri bodies, the virus factories induced in rabies virus-infected cells. But how does the cell respond?

Exposure of cells to environmental stresses, such as heat shock and viral infection, induces a cellular response leading to the formation of Stress Granules composed of stalled translation initiation complexes (RNA-binding proteins and mRNA). Subsequent inhibition of host translation contibutes to cell survival. Viruses modulate or interfere with Stress Granule formation to control virus replication and antiviral responses, but differences exist in the dynamics and outcome of the stress responses induced by various viruses. A new paper shows that Rabies virus (RABV) induces the formation of Stress Granules in infected cells. Stress Granules are highly dynamic structures that increase in size by fusion events, exhibit transient assembly or persist throughout infection. They localize close to viral factories, cytoplasmic structures characteristic of RABV infection involved in viral replication and transcription. Viral messenger RNAs, but not viral genomic RNA, are transported from the factories to Stress Granules, indicating the communication between both compartments. RABV-induced cellular stress is dependent on double-stranded RNA-activated protein kinase (PKR). PKR also participates in innate immune responses through the induction of the Interferon-B gene. These results give an insight on new and important aspects of RABV infection and host antiviral stress responses.


Rabies Virus Infection Induces the Formation of Stress Granules Closely Connected to the Viral Factories. (2016) PLoS Pathog 12(10): e1005942. doi: 10.1371/journal.ppat.1005942
Stress granules (SGs) are membrane-less dynamic structures consisting of mRNA and protein aggregates that form rapidly in response to a wide range of environmental cellular stresses and viral infections. They act as storage sites for translationally silenced mRNAs under stress conditions. During viral infection, SG formation results in the modulation of innate antiviral immune responses, and several viruses have the ability to either promote or prevent SG assembly. Here, we show that rabies virus (RABV) induces SG formation in infected cells, as revealed by the detection of SG-marker proteins Ras GTPase-activating protein-binding protein 1 (G3BP1), T-cell intracellular antigen 1 (TIA-1) and poly(A)-binding protein (PABP) in the RNA granules formed during viral infection. As shown by live cell imaging, RABV-induced SGs are highly dynamic structures that increase in number, grow in size by fusion events, and undergo assembly/disassembly cycles. Some SGs localize in close proximity to cytoplasmic viral factories, known as Negri bodies (NBs). Three dimensional reconstructions reveal that both structures remain distinct even when they are in close contact. In addition, viral mRNAs synthesized in NBs accumulate in the SGs during viral infection, revealing material exchange between both compartments. Although RABV-induced SG formation is not affected in MEFs lacking TIA-1, TIA-1 depletion promotes viral translation which results in an increase of viral replication indicating that TIA-1 has an antiviral effect. Inhibition of PKR expression significantly prevents RABV-SG formation and favors viral replication by increasing viral translation. This is correlated with a drastic inhibition of IFN-B gene expression indicating that SGs likely mediate an antiviral response which is however not sufficient to fully counteract RABV infection.

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Nanoparticle vaccines against dengue fever?

Dengue virus-infected cells Every year more than 350 million people in over 120 countries contact dengue fever, which can cause symptoms ranging from aching muscles and a skin rash to life-threatening haemorrhagic fever. Researchers have struggled to create effective vaccines against dengue virus, in part because four distinct serotypes of the virus cause dengue fever and a vaccine must immunize against all four individually. Attempts at using live dengue viruses to develop a dengue fever vaccine have often led to an imbalance in immunity to the four dengue serotypes. Previous infection with one serotype of dengue, or protection against just one serotype, can lead to more severe disease if a person contracts other serotypes, so it’s vital that vaccines are available that specifically target all four strains.

To create a new dengue virus vaccine researchers designed nanoparticles of various shapes and sizes. Each nanoparticle was studded with copies of DENV2-E protein, a key protein from serotype 2 of the virus. After immunization with the DENV2-E nanoparticles, mice had a specific antibody response to serotype 2 of the dengue virus, but not the other three serotypes. Compared to mice vaccinated with only the soluble DENV2-E proteins, the nanoparticle formulations led to a stronger immune response.

Clearly this enabling research is still a long way from an effective human vaccine against dengue fever. Future studies will be required to test whether the antibody levels prevent dengue infection as well as whether similar nanoparticles can be develop for all dengue serotypes. At the same time, it is difficult to imagine that the next few years will not bring a host of candidate vaccines based on nanoparticles.


Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses. (2016) PLoS Negl Trop Dis 10(10): e0005071. doi: 10.1371/journal.pntd.0005071
Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simulta- neous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial effi- cacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been pre- cisely molded using Particle Replication in Non-wetting Template (PRINT) technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE), purified and adsorbed to poly (lactic-co-glycolic acid) (PLGA) nanoparticles of dif- ferent sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT1 nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT© nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika.

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The skin is a reservoir for African trypanosomes

Trypanosome Human African trypanosomiasis – sleeping sickness – is a potentially fatal disease, which currently affects ~3,500 people in sub-Saharan Africa. The disease is caused by parasites called African trypanosomes and is spread by tsetse flies. Controlling these biting insects, combined with surveillance and treatment, reduces the impact of outbreaks of the disease and the World Health Organisation (WHO) hopes to eliminate sleeping sickness by 2020. A new paper suggests that this target might be overambitious.

Detection of trypanosomes in the skin is not well documented, although there are descriptions of cutaneous symptoms associated with African trypanosomiasis. This paper reports the investigation of a possible anatomical reservoir in the skin and provides evidence of T.b. brucei, (a causative agent of animal trypanosomiasis) and the human-infective trypanosome, T.b. gambiense, invading the extravascular tissue of the skin (including but not restricted to the adipose tissue) and undergoing onward transmission despite undetected vascular parasitaemia. It also provides evidence of localisation of trypanosomes within the skin of undiagnosed humans. The presence of a significant transmissible population of T. brucei in this anatomical compartment is likely to impact future control and elimination strategies for both animal and human trypanosomiases.

The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes. eLife 2016; 5: e17716 doi: 10.7554/eLife.17716
The role of mammalian skin in harbouring and transmitting arthropod-borne protozoan parasites has been overlooked for decades as these pathogens have been regarded primarily as blood-dwelling organisms. Intriguingly, infections with low or undetected blood parasites are common, particularly in the case of Human African Trypanosomiasis caused by Trypanosoma brucei gambiense. We hypothesise, therefore, the skin represents an anatomic reservoir of infection. Here we definitively show that substantial quantities of trypanosomes exist within the skin following experimental infection, which can be transmitted to the tsetse vector, even in the absence of detectable parasitaemia. Importantly, we demonstrate the presence of extravascular parasites in human skin biopsies from undiagnosed individuals. The identification of this novel reservoir requires a re-evaluation of current diagnostic methods and control policies. More broadly, our results indicate that transmission is a key evolutionary force driving parasite extravasation that could further result in tissue invasion-dependent pathology.

Trypanosomiasis: Skin deep

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Zoonotic Hepatitis E Virus

Zoonotic Hepatitis E Virus During the past ten years, several new hepatitis E viruses (HEVs) have been identified in various animal species. In parallel, the number of reports of indigenous hepatitis E in Western countries has increased as well, raising the question of what role these possible animal reservoirs play in human infections.

This review describes recent discoveries of animal HEVs and their classification within the Hepeviridae family, their zoonotic and species barrier crossing potential, possible use as models to study hepatitis E pathogenesis and transmission pathways identified from animal sources.

Zoonotic Hepatitis E Virus: Classification, Animal Reservoirs and Transmission Routes. (2016) Viruses 8(10): 270. doi:10.3390/v8100270.

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Herpesviruses as Pioneers in Cell Biology

HCMV Intracytoplasmic vesicular transport is well established; nucleo-cytoplasmic transport has so far been thought to be restricted to passage through the nuclear pore either passively, if size permits, or via karyopherin-mediated active transport. This limits transport in and out of the nucleus to particles of a maximum of 39 nm. With a diameter of 120 nm, herpesvirus capsids, which are assembled in the nucleus but mature to infectious virions in the cytosol, are unable to pass through the nuclear pore. It has become clear in the last decade that they leave the nucleus and traverse the nuclear envelope by a vesicle-mediated process that entails budding of nucleocapsids at the inner nuclear membrane, forming a primary enveloped virion in the perinuclear space. The primary envelope then fuses with the outer nuclear membrane.

Long thought to be specific for herpesviruses, this pathway has recently also been suggested to function in the export of large ribonucleoprotein (RNP) complexes during development of Drosophila. Common between the two is the involvement of kinases (viral, cellular, or both) to phosphorylate and soften the nuclear lamina allowing access of the ‘cargo’ (i.e., viral nucleocapsids or cellular RNPs) to the INM as well as morphological similarities [6]. The cellular AAA+ ATPase TorsinA has also been proposed to be involved in both processes. Thus the notion was developed that herpesviruses have actually co-opted a hitherto cryptic cellular transport pathway for their replication.


Vesicular Nucleo-Cytoplasmic Transport – Herpesviruses as Pioneers in Cell Biology. Viruses 2016, 8 (10): 266. doi:10.3390/v8100266
Herpesviruses use a vesicle-mediated transfer of nuclear-assembled nucleocapsids through the nuclear envelope for maturation in the cytoplasm. The molecular basis for this novel vesicular nucleo-cytoplasmic transport is beginning to be elucidated in detail. The heterodimeric viral nuclear egress complex, conserved within the classical herpesviruses, mediates vesicle formation from the inner nuclear membrane by polymerization into a hexagonal lattice followed by fusion of the vesicle membrane with the outer nuclear membrane. Mechanisms of capsid inclusion as well as vesicle-membrane fusion, however, are largely unclear. Interestingly, a similar transport mechanism through the nuclear envelope has been demonstrated in nuclear export of large ribonucleoprotein complexes during Drosophila neuromuscular junction formation, indicating a widespread presence of a novel concept of cellular nucleo-cytoplasmic transport.

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Fungal Super Glue – Biofilms

Candida albicans biofilm Biofilms are arguably the most common state of microbial growth found in nature and in patients infected with pathogenic organisms. A feature of prokaryotic and eukaryotic biofilms is their production of an extracellular matrix. The matrix provides a protective environment for biofilm cells, offering a three-dimensional framework for both surface adhesion and cell cohesion. In addition, this extracellular material controls cell dispersion from the biofilm and provides a nutrient source for the community. The physical barrier formed by the matrix is also clinically relevant, as it shields cells from environmental threats, including immune cells and antimicrobial drugs used for treatment. This defensive characteristic has been demonstrated for biofilms formed by diverse fungal pathogens, including Aspergillus, Candida, Cryptococcus, and Saccharomyces.

Biofilm-associated Candida infections are the fourth cause for nosocomial infections (predominantly infecting medical devices), which may lead to systemic infection associated with high mortality rates. Candida spp. are also the most common cause of mucosal infection of the oral and vaginal sites, where biofilm infection has been increasingly recognized. Despite the ubiquitous nature of the biofilm matrix, we are only beginning to understand the synthesis and composition of this material for a handful of species. This review discusses components of the extracellular matrix of fungal biofilms, including their synthesis, structure, and function.

Fungal Super Glue: The Biofilm Matrix and Its Composition, Assembly, and Functions. (2016) PLoS Pathog 12(9): e1005828. doi: 10.1371/journal.ppat.1005828

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Closing in on the causes of host shutoff

How influenza virus A drives virus-induced host shutoff When a virus enters a cell it relies on the molecular machinery of its host to help it replicate. In particular, the virus relies on the ribosomes in the host cell to translate viral messenger RNA (mRNA) into polypeptides. Many viruses also impair the translation of cellular mRNA, via a process termed “host shutoff”, in order to prevent the production of anti-viral, host defense proteins. For example, poliovirus does this by inactivating a translation factor that is required to load ribosomes onto host mRNAs, all of which have a type of “cap” called an “m7G cap”. Moreover, while the translation of these mRNAs is being suppressed, host ribosomes are involved in the translation of poliovirus mRNA, which does not have a cap: this is possible without the translation factor because poliovirus mRNA has an internal entry site for ribosomes. However, the mechanisms responsible for host shutoff in viruses that have mRNAs with m7G-caps, such as influenza A virus, have remained enigmatic.

A new paper in eLife shows that in Influenza virus-infected cells host and viral mRNAs were both translated with similar efficiencies, indicating that viral mRNAs were not preferentially translated relative to host mRNAs. Instead, Influenza virus-induced host shutoff primarily originates from a reduced abundance of cellular mRNA and from the high levels of viral mRNA in both the nucleus and cytoplasm. Fluorescence-based measurements confirmed these findings and revealed that the reduced abundance of cellular mRNA has its origins in the nucleus. This likely involves an RNA endoribonuclease called PA-X, which is encoded in the genome of Influenza virus, stimulating the decay of cellular mRNA.

A systematic view on influenza induced host shutoff. (2016) eLife 5: e18311. doi: 10.7554/eLife.18311
Host shutoff is a common strategy used by viruses to repress cellular mRNA translation and concomitantly allow the efficient translation of viral mRNAs. Here we use RNA-sequencing and ribosome profiling to explore the mechanisms that are being utilized by the Influenza A virus (IAV) to induce host shutoff. We show that viral transcripts are not preferentially translated and instead the decline in cellular protein synthesis is mediated by viral takeover on the mRNA pool. Our measurements also uncover strong variability in the levels of cellular transcripts reduction, revealing that short transcripts are less affected by IAV. Interestingly, these mRNAs that are refractory to IAV infection are enriched in cell maintenance processes such as oxidative phosphorylation. Furthermore, we show that the continuous oxidative phosphorylation activity is important for viral propagation. Our results advance our understanding of IAV-induced shutoff, and suggest a mechanism that facilitates the translation of genes with important housekeeping functions.

eLife: Virology: Closing in on the causes of host shutoff. doi: 10.7554/eLife.20755

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Latency in Herpes Simplex Virus 1

HSV-infected cell Herpes simplex virus 1 (HSV-1) is a neurotropic virus that establishes a life-long latent infection in the trigeminal ganglia (TG) of the infected human host. From time to time the virus asymptomatically or symptomatically reactivates from the latency stage producing epithelial lesions, most of the time on the face but also in the eye, inducing severe pathologies such as keratitis. HSV-1 infection is also associated with pathologies of the central nervous system such as encephalitis.

During latency the virus is in a transcriptionally restricted state. Of the about 80 genes transcribed during lytic infection, only a family of long non-coding RNAs is produced abundantly during latency. These latency associated transcripts (LATs) arise from the transcription of an 8.3 kb primary RNA that is processed in two major LATs of 1.5 kb and 2 kb and several microRNAs with cellular and viral targets. The precise role of LATs is a matter of debate; however, a point of convergence among the many studies of LATs is that their initial production would favor the survival of the infected neurons and the coordination of the infectious process towards the latency transcriptional program and reactivation. The lytic cycle is the alternative transcriptional program and is characterized by a temporarily regulated transcriptional program, which starts with the expression of immediate early (IE), then early (E), and finally late (L) genes.

In the virus particle, HSV-1 genome is a 150-kb double stranded naked linear DNA. Upon entry into the host nucleus, the viral genome does not integrate, remaining as an extrachromosomal plasmid-like molecule. Chromatinization of the viral genome during latency plays a major regulatory role, and post-translational modifications of histones associated to key viral promoters determines the fate of the latency/reactivation process. Latent viral genomes are present in multiple copies within the nucleus of infected neurons in mouse models and human, and little is known about the molecular determinants that enable one neuron rather than another to sustain reactivation.

This paper describes a fluorescent in situ hybridization (FISH) approach combined with immunofluorescence to investigate the interaction between viral genomes and nuclear proteins within TG neurons of latently infected mice and during the whole process of latency establishment (from 4 to 28 days post infection, dpi). Viral genomes were found in neurons and satellite cells at 4 and 6 dpi, but only in neurons at > 6 dpi. In satellite cells, viral genomes showed only replication compartment (RC) patterns, whereas in neurons both RC and “multiple-acute” patterns were detected. From 4 to 14 dpi both patterns progressively disappeared, and transformed from14dpi onwards to the latency-associated “single” and “multiple-latency” patterns. Immuno-FISH analyses of human TG showed a close spatial distribution between latent HSV-1 genomes and PML protein in neurons, which suggests that, similar to the situation in the mouse model, HSV-1 latency in human is probably tightly linked to the activity of PML-NBs.

We have been trying for decades to understand the biology of latency in HSV. The strength of this approach is to point out that this cannot be achieved by looking at the virus alone, the biology of the host cell also has to be considered. This study describes the nuclear architecture and nuclear distribution of viral genomes as major determinants of HSV-1 latency. It confirms the close interrelation between PML-NBs and HSV-1 genomes in the establishment of latency through the formation of vDCP-NBs. Finally, it confirms that the spatial organization of HSV-1 genomes and PML is conserved in latently infected neurons in human TG, which indicates PML-NBs to be major HSV-1 genome interactants during latency and probably reactivation.


Latency Entry of Herpes Simplex Virus 1 Is Determined by the Interaction of Its Genome with the Nuclear Environment. (2016) PLoS Pathog 12(9): e1005834. doi: 10.1371/journal.ppat.1005834
Herpes simplex virus 1 (HSV-1) establishes latency in trigeminal ganglia (TG) sensory neurons of infected individuals. The commitment of infected neurons toward the viral lytic or latent transcriptional program is likely to depend on both viral and cellular factors, and to differ among individual neurons. In this study, we used a mouse model of HSV-1 infection to investigate the relationship between viral genomes and the nuclear environment in terms of the establishment of latency. During acute infection, viral genomes show two major patterns: replication compartments or multiple spots distributed in the nucleoplasm (namely “multiple-acute”). Viral genomes in the “multiple-acute” pattern are systematically associated with the promyelocytic leukemia (PML) protein in structures designated viral DNA-containing PML nuclear bodies (vDCP-NBs). To investigate the viral and cellular features that favor the acquisition of the latency-associated viral genome patterns, we infected mouse primary TG neurons from wild type (wt) mice or knock-out mice for type 1 interferon (IFN) receptor with wt or a mutant HSV-1, which is unable to replicate due to the synthesis of a non-functional ICP4, the major virus transactivator. We found that the inability of the virus to initiate the lytic program combined to its inability to synthesize a functional ICP0, are the two viral features leading to the formation of vDCP-NBs. The formation of the “multiple-latency” pattern is favored by the type 1 IFN signaling pathway in the context of neurons infected by a virus able to replicate through the expression of a functional ICP4 but unable to express functional VP16 and ICP0. Analyses of TGs harvested from HSV-1 latently infected humans showed that viral genomes and PML occupy similar nuclear areas in infected neurons, eventually forming vDCP-NB-like structures. Overall our study designates PML protein and PML-NBs to be major cellular components involved in the control of HSV-1 latency, probably during the entire life of an individual.

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Human Hookworm Infection

Hookworms Hookworm affects approximately 500 million people worldwide, yet its global economic and health impact is not well understood. A new study suggests that the health and economic burden of hookworm infection is estimated to exceed those of a number of diseases receiving greater attention and investment. Human hookworm infection confers a substantial global health and economic burden through loss of productivity, and years of life living with disability due to infection outcomes. While hookworm infection rarely results in death, it can lead to iron-deficiency anemia and malnutrition. Chronic health problems resulting from these conditions include lethargy, impaired physical and cognitive development and adverse pregnancy outcomes.

The Global Economic and Health Burden of Human Hookworm Infection. (2016) PLoS Negl Trop Dis 10(9): e0004922. doi: 10.1371/journal.pntd.0004922
Even though human hookworm infection is highly endemic in many countries throughout the world, its global economic and health impact is not well known. Without a better understanding of hookworm’s economic burden worldwide, it is difficult for decision makers such as funders, policy makers, disease control officials, and intervention manufacturers to deter- mine how much time, energy, and resources to invest in hookworm control. We developed a computational simulation model to estimate the economic and health bur- den of hookworm infection in every country, WHO region, and globally, in 2016 from the societal perspective. Globally, hookworm infection resulted in a total 2,126,280 DALYs using 2004 disability weight estimates and 4,087,803 DALYs using 2010 disability weight estimates (excluding cognitive impairment outcomes). Including cognitive impairment did not significantly increase DALYs worldwide. Total productivity losses varied with the proba- bility of anemia and calculation method used, ranging from $7.5 billion to $138.9 billion annually using gross national income per capita as a proxy for annual wages and ranging from $2.5 billion to $43.9 billion using minimum wage as a proxy for annual wages. Even though hookworm is classified as a neglected tropical disease, its economic and health burden exceeded published estimates for a number of diseases that have received comparatively more attention than hookworm such as rotavirus. Additionally, certain large countries that are transitioning to higher income countries such as Brazil and China, still face considerable hookworm burden.

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