MicrobiologyBytes

NK cells have a prominent role in early virus control. Upon activation NK cells control infection either through the lysis of infected cells, or by release of antimicrobial cytokines. This latter function enables them to influence adaptive immune responses as well. NK cells survey their surroundings through numerous inhibitory and activating receptors – integration of these signals determines the activity of an NK cell. More recent data indicate that NK cells may even acquire memory-like function enabling them to respond differently upon recall challenge. The importance of NK cells in control of viral infection is best illustrated by the sheer number of viral evasion mechanisms. These mechanisms include regulation of apoptosis, interference with ADCC, modulation of cytokines and chemokines and function of APCs. Even more numerous are viral techniques dedicated to control of engagement of NK cell receptors. The viruses can downmodulate ligands for activating NK cell receptors, provide competitors and surrogates for cellular ligands, interfere with their translation or target the activating receptors directly.

Despite a remarkable increase in knowledge about relationship between NK cells and viruses there are still many outstanding issues. This review highlights recent progress and current understanding of most important viral immunosubversive mechanisms directed at NK cells with emphasis on receptor-ligand interactions and their impact on overall immunity.

Modulation of natural killer cell activity by viruses. Curr Opin Microbiol. Jun 15 2010

Related:

• Viruses and Apoptosis
• Viruses and Lymphomas

The innate immune system forms the first line of defence against invading micro-organisms such as viruses. It dampens initial virus replication and ensures survival of the host until specialized adaptive responses are developed. Type I interferons (IFNs) are secreted key cytokines on the innate immune axis that protect uninfected cells and stimulate leukocytes residing at the interface of innate and adaptive immunity, such as macrophages and dendritic cells. These cells prod the adaptive immune system to mount a full, specialized response against the invading microbe.

The ability to outrun innate immunity before adaptive immune responses are mounted is crucial for the survival of virtually all the mammalian viruses, regardless of their genome type and complexity. Relatively simple viruses such as RNA viruses from the Picornavirus family, as well as DNA viruses with large genomes, such as members from the Poxvirus family, have been shown to inhibit the IFN system. This review covers the latest insights into how virus-encoded antagonists sidetrack the IFN machinery and how this knowledge is currently used to generate second generation live vaccines and antiviral compounds.

Viral tricks to grid-lock the type I interferon system. Curr Opin Microbiol. Jun 9 2010
Type I interferons (IFNs) play a crucial role in the innate immune avant-garde against viral infections. Virtually all viruses have developed means to counteract the induction, signaling, or antiviral actions of the IFN circuit. Over 170 different virus-encoded IFN antagonists from 93 distinct viruses have been described up to now, indicating that most viruses interfere with multiple stages of the IFN response. Although every viral IFN antagonist is unique in its own right, four main mechanisms are employed to circumvent innate immune responses: (i) general inhibition of cellular gene expression, (ii) sequestration of molecules in the IFN circuit, (iii) proteolytic cleavage, and (iv) proteasomal degradation of key components of the IFN system. The increasing understanding of how different viral IFN antagonists function has been translated to the generation of viruses with mutant IFN antagonists as potential live vaccine candidates. Moreover, IFN antagonists are attractive targets for inhibition by small-molecule compounds.

Related:

• MicrobiologyBytes: Interferon
• Interferons and Viruses
• The Interferon Antiviral Response
• Interferon – 50 years on

Amazingly, all the phages on Earth, if placed end to end, would probably extend a distance equivalent to that of the nearest 60 galaxies. In this article in Microbiology Today, Eric Wommack explains how metagenomics is gradually revealing the amazing diversity and abundance of viruses in the biosphere:

Although the throughput and accuracy of methods for viral direct counting has improved since the 1989 report based on transmission electron microscopy, the ‘factor of 10’ ratio of virus to bacterial abundance within aquatic environments has remained a surprisingly common observation. Extrapolating the ‘factor of 10’ rule to the biosphere has lead to estimates that global viral abundance is in the order of 10³¹ individuals. Assuming an average length dimension of 100 nm, Curtis Suttle has proposed that, lined end-to-end, all the phages on earth would extend a distance equivalent to that of the nearest 60 galaxies (10 million light years, 10²⁴m).

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Related:

• New dimensions of the virus world discovered through metagenomics
• Genomes of unknown marine viruses

The replication cycle of viruses involves entry into host cells, synthesis of virus genes and proteins, assembly of progeny virus particles and their subsequent release. Along with the plasma membrane, viruses also have to interact with the endosomal and vesicular membranes during their replication in host cells. All cellular membranes are composed of lipids and proteins that are usually arranged in various micro domains. During infection of cells by enveloped viruses, the lipids present in both viral and cellular membranes mediate fusion and fission reactions to facilitate virus entry and release. Since non-enveloped viruses do not have a lipid envelope, it is generally believed that their entry mechanism does not involve membrane fusion activity and that these viruses are mainly released by cell lysis. Usually, non-enveloped viruses enter the cells by penetrating the membrane barrier, either via the endocytic pathway using clathrin-coated vesicles, or by the formation of a pore at the cell surface. Recent data obtained from biochemical and structural studies indicate that the overall mechanisms of both entry (Reoviridae) and release of certain non-enveloped viruses (e.g., members of the Picornaviridae and Reoviridae) are analogous to that of enveloped viruses, and that the capsid proteins can function in these activities in a similar manner to the membrane viral proteins. This review discusses the role of lipids in the entry, maturation and release of non-enveloped viruses, focusing mainly on Bluetongue virus (BTV).

Role of Lipids on Entry and Exit of Bluetongue Virus, a Complex Non-Enveloped Virus. Viruses 2010 2 (5) 1218-1235. doi:10.3390/v2051218

Related:

• Bluetongue virus
• Lipid Membranes in Poxvirus Replication
• How the antiviral protein viperin works

Most viruses are either helical or icosahedral in structure. The two highly symmetric shapes permit viruses to use the same component protein multiple times to create large structures from a minimum number of distinct protein species. The strategy conserves the amount of genetic material viruses need to encode structural proteins. Although the two basic shapes serve the needs of viruses more or less equally well, structural biologists have had a much easier time determining the structures of the icosahedra. For example, while more than one hundred high resolution structures of icosahedral viruses are now available, the number of comparable helical virus structures is limited to helical plant viruses such as tobacco mosaic virus and filamentous bacteriophage such as E. coli phage f1. It’s not as though helical animal and human viruses are of limited interest. Just the opposite. They include influenza virus plus members of the paramyxo-, rhabdo- bunya-, corona, filo- and arenavirus families, all of which contain important human pathogens. The problem is that structural analysis of these viruses is unusually difficult. The protein-RNA complex is often disordered or weakly ordered in the virion, and the viruses have a membrane, a structure that complicates both crystallization and electron microscopic analysis.

To advance our knowledge of helical virus structure, investigators have focused their attention on the rhabdoviruses, a family of bullet-shaped viruses that includes rabies and vesicular stomatitis viruses (VSV). Rhabdoviruses have a helical nucleocapsid that is well ordered over most of the virion length. Although a membrane is present, it is tightly wrapped around the nucleocapsid, and does not obscure the helix in electron micrographs of the virion. With such excellent images, one would think it would be a simple matter to compute a three-dimensional reconstruction of the particle. No structure has been forthcoming, however, despite the best efforts of many highly talented structural biologists – until now.

Helical Virus Structure: The Case of the Rhabdovirus Bullet. Viruses 2010 2(4), 995-1001; doi:10.3390/v2040995

Related:

• Cryo-EM Model of the Bullet-Shaped Vesicular Stomatitis Virus. 2010 Science 327 (5966) 689-693 doi:10.1126/science.1181766
  Assembly of a virus particle is generally presumed to be a stochastic process. However, assembly of VSV appears to follow a well-orchestrated program. It begins with RNA and N as a nucleocapsid ribbon. The ribbon curls into a tight ring and then is physically forced to curl into larger rings that eventually tile the helical trunk. M subunits bind on the outside of the nucleocapsid, rigidify the bullet tip and then the trunk, and create a triangularly packed platform for binding G trimers and envelope membrane, all in a coherent operation during budding.

Success of a virus infection requires that each infected cell delivers a sufficient number of infectious particles to allow new rounds of infection. In picornaviruses, virus replication is initiated by the viral polymerase and a viral-coded protein, termed VPg, that primes RNA synthesis. Foot-and-mouth disease virus (FMDV) is exceptional among picornaviruses in that its genome encodes three copies of VPg. Why FMDV encodes three VPgs is unknown.

Researchers constructed four mutant FMDVs that encode only one VPg: either VPg1, VPg3, or two chimeric versions containing part of VPg1 and VPg3. All mutants, except that encoding only VPg1, were replication-competent. Unexpectedly, despite being replication-competent, the mutants did not form plaques on BHK-21 cell monolayers. The one-VPg mutant FMDVs released lower amounts of encapsidated viral RNA to the extracellular environment than wild type FMDV, suggesting that deficient plaque formation was associated with insufficient release of infectious progeny. Mutant FMDVs subjected to serial passages in BHK-21 cells regained plaque-forming capacity without modification of the number of copies of VPg. Substitutions in non-structural proteins 2C, 3A and VPg were associated with restoration of plaque formation. Specifically, replacement R55W in protein 2C was repeatedly found in several mutant viruses that had regained competence in plaque development. The results link the VPg copies in the FMDV genome with the cytopathology capacity of the virus, and have unveiled yet another function of 2C: modulation of picornavirus cell-to-cell transmission. these data highlight the role of non–structural proteins in the adaptability to changing environments during picornavirus infections, with clear implications for viral pathogenesis.

Deletion Mutants of VPg Reveal New Cytopathology Determinants in a Picornavirus. 2010 PLoS ONE 5(5): e10735. doi:10.1371/journal.pone.0010735

Related:

• Foot and Mouth Disease

Human rhinoviruses (HRV) are the most frequent cause of respiratory infection in humans. These viruses belong to the Picornaviridae, one of the oldest and most diversified human virus family, characterized by a non-enveloped, single positive-stranded RNA genome. Although rhinovirus replication is often restricted to the upper respiratory tract leading to self-limited illnesses of short duration, such as the common cold, HRV can also invade the lower respiratory tract and lead to more serious infections. Similar to many other RNA viruses, the error-prone rhinoviral polymerase can accumulate a large number of nucleotide mutations over a very short period of time, a feature that favors viral adaptation. The error rate of picornavirus RNA polymerases has been estimated to range between 10⁻³ and 10⁻⁴ errors/nucleotide/cycle of replication. This variability is a driving force for virus evolution and results in a large genetic and phenotypic diversity illustrated by the very high number of different HRV serotypes identified to date.

By using ultra-deep sequencing technology, researchers were able to pinpoint HRV evolution at the level of a quasispecies population both in vivo and in vitro. The data illustrate the ability of rhinoviruses to produce several new variants as rapidly as 5 days’ post-infection.

Rhinovirus Genome Evolution during Experimental Human Infection. 2010 PLoS ONE 5(5): e10588. doi:10.1371/journal.pone.0010588
Human rhinoviruses (HRVs) evolve rapidly due in part to their error-prone RNA polymerase. Knowledge of the diversity of HRV populations emerging during the course of a natural infection is essential and represents a basis for the design of future potential vaccines and antiviral drugs. To evaluate HRV evolution in humans, nasal wash samples were collected daily for five days from 15 immunocompetent volunteers experimentally infected with a reference stock of HRV-39. In parallel, HeLa-OH cells were inoculated to compare HRV evolution in vitro. Nasal wash in vivo assessed by real-time PCR showed a viral load that peaked at 48–72 h. Ultra-deep sequencing was used to compare the low-frequency mutation populations present in the HRV-39 inoculum in two human subjects and one HeLa-OH supernatant collected 5 days post-infection. The analysis revealed hypervariable mutation locations in VP2, VP3, VP1, 2C and 3C genes and conserved regions in VP4, 2A, 2B, 3A, 3B and 3D genes. These results were confirmed by classical sequencing of additional samples, both from inoculated volunteers and independent cell infections, and suggest that HRV inter-host transmission is not associated with a strong bottleneck effect. A specific analysis of the VP1 capsid gene of 15 human cases confirmed the high mutation incidence in this capsid region, but not in the antiviral drug-binding pocket. We could also estimate a mutation frequency in vivo of 3.4×10⁻⁴ mutations/nucleotides and 3.1×10⁻⁴ over the entire ORF and VP1 gene, respectively. In vivo, HRV generate new variants rapidly during the course of an acute infection due to mutations that accumulate in hot spot regions located at the capsid level, as well as in 2C and 3C genes.

Related:

• Virus incubation periods
• Sleep and susceptibility to the common cold

The incidence and geographic range of dengue and dengue hemorrhagic fever has increased dramatically in recent decades. With 2.5 billion people now living in areas at risk for epidemic transmission, dengue has become the most important mosquito-borne viral disease affecting humans. Dengue virus (DENV) is a positive-strand RNA virus of the family Flaviviridae. It exists as four closely related but antigenically distinct serotypes, all of which have Aedes aegypti mosquitoes as their primary vector, with A. albopictus as a secondary vector.

Mosquitoes, like all insects, are exposed to a variety of microbes in their natural habitats, and possess an innate immune system that is capable of mounting a potent response against microbial challenge. The insect innate immune response is largely regulated by three main immune signaling pathways: the Toll, immune deficiency (IMD) and Janus kinase signal transducer and activator of transcription (JAK-STAT) pathways. The Toll pathway is involved in defense against fungi, Gram-positive bacteria, and viruses, and has been found to be specifically involved in the A. aegypti anti-DENV response.

In order to study the interaction of DENV with the A. aegypti immune response, researchers have characterized the DENV infection-responsive transcriptome of the immune-competent A. aegypti cell line. As in mosquitoes, DENV infection transcriptionally activated the cell line Toll pathway and a variety of cellular physiological systems. Most notably, however, DENV infection down-regulated the expression levels of numerous immune signaling molecules and antimicrobial peptides (AMPs). Functional assays showed that transcriptional induction of AMPs from the Toll and IMD pathways in response to bacterial challenge is impaired in DENV-infected cells. In addition, Escherichia coli, a Gram-negative bacteria species, grew better when co-cultured with DENV-infected cells than with uninfected cells, suggesting a decreased production of AMPs from the IMD pathway in virus-infected cells. Pre-stimulation of the cell line with Gram-positive bacteria prior to DENV infection had no effect on DENV titers, while pre-stimulation with Gram-negative bacteria resulted in an increase in DENV titers. These results indicate that DENV is capable of actively suppressing immune responses in the cells it infects, a phenomenon that may have important consequences for virus transmission and insect physiology.

Dengue Virus Inhibits Immune Responses in Aedes aegypti Cells. 2010 PLoS ONE 5(5): e10678. doi:10.1371/journal.pone.0010678

Related:

• The dengue vector Aedes aegypti
• Dengue Virus
• Rethinking dengue hemorrhagic fever

The interference of animal viruses with host translation was first documented in the 1960s in human fibroblasts infected with poliovirus. Further studies revealed that the halt of host translation (or “shut off”) was a general phenomenon observed in cells infected with lytic RNA and DNA viruses. Of the three steps in protein synthesis, viruses mainly affect the initiation step by hijacking or modifying the activity of key eukaryotic initiation factors (eIFs) to ensure an efficient translation of viral mRNAs and the simultaneous decline of host translation. The main targets of viruses are components of the cap-binding complex (eIF4F) that are required for the recruitment of ribosomes to mRNAs.

Infection of cultured cells with lytic animal viruses often results in “shut off”, but there is no direct or indirect evidence supporting the idea that it also should operate in whole animals infected with viruses. To address this issue, the authors of a new paper constructed a recombinant Sindbis virus (SV)-expressing reporter mRNA, the translation of which is sensitive or resistant to virus-induced shut off. As found in cultured cells, replication of SV in mouse brain was associated with a strong phosphorylation of eukaryotic initiation factor (eIF2) that prevented translation of reporter mRNA (luciferase and EGFP). Translation of these reporters was restored in vitro, in vivo, and ex vivo when a viral RNA structure, termed downstream hairpin loop, present in viral 26S mRNA, was placed at the 5′ end of reporter mRNAs.

By comparing the expression of shut off-sensitive and -resistant reporters, this work demonstrates that replication of SV in animal tissues is associated with a profound inhibition of nonviral mRNA translation. A strategy as simple as that followed here might be applicable to other viruses to evaluate their interference on host translation in infected animals.

Inhibition of host translation by virus infection in vivo. PNAS USA May 10 2010 doi: 10.1073/pnas.100411010