Communication between virus-infected cells

lysis or lysogenyYou might recall learning in high school biology that bacteriophage infection of a host can lead to either replication and cell lysis, or integration of the viral genome into the host (illustrated). The latter event, called lysogeny, spares the host from virus induced killing. For some phages, the decision between lysis and lysogeny appears to be communicated between cells by a small peptide (link to paper).

Evidence that virus-infected cells produce a substance that can regulate the lysis-lysogeny decision came from the observation that conditioned medium from Bacillus subtilis infected with the bacteriophage phi3T – prepared so that is was virus and cell free – protects cells from lysis. The protective component is destroyed by digestion with a proteinase and hence is a protein. Conditioned medium not only inhibits cell lysis, but increases lysogeny, measured by integration of viral DNA into the bacterial genome.

Examination of the genome sequence of phage phi3T suggested that a six amino acid peptide, Ser-Ala-Ile-Arg-Gly-Ala, was the component in conditioned medium that regulates the lytic-lysogenic decision. Addition of the synthetic peptide to infected cells decreased lysis. The levels of this peptide increase during each cycle of phage infection of the Bacillus host.

The authors call the communication peptide ‘arbitrium’ from the Latin word meaning ‘decision’. The gene encoding the peptide is aimP.

AimP appears to work by entering the bacterium through a transporter protein and binding a protein in the bacterial cell called AimR. The AimR protein in turn binds a sequence in the bacterial genome called aimX. When AimR is bound by the peptide, it cannot bind aimX and lysogeny occurs. In the absence of peptide, AimR binds aimX and lysis proceeds. The product of the aimX gene appears to be a regulatory RNA, but how it promotes lysis is not known.

Different phages of B. subtilis also encode peptides that regulate the lysis-lysogeny decision in a phage-specific manner.

These findings describe a viral communication system that determines whether a bacterial host is lysed or lysogenized. When viruses initially infect a host, the result is lysis because levels of peptide are low. After several cycles of infection the AimP concentrations increase, and upon entry of the peptide into bacteria they lead to lysogeny.

The authors of this work suggest that the arbitrium system is a way for the virus to sense the amount of previous infections to decide whether lysis or lysogeny should occur. If many previous infections have taken place, the host population could be too low to support lytic replication, hence lysogeny occurs.  Because lysogens can divide, the bacterial population can be restored to a level that can sustain virus infection.

Of course, the virus particle cannot sense anything – it is a bacterial protein that  binds AimP and another bacterial gene that controls lysis. In other words, the virus-infected cell, not the virus, can sense the amount of previous infections.

It should be straightforward to search the genome sequences of phages that infect other bacteria to determine if such a communication system is widespread. More interesting is whether viruses that infect eukaryotes also have  communication systems that guide decisions about lytic versus non-lytic or latent infection.

A viral nucleus

Cell typesA unique feature of eukaryotic cells, which distinguishes them from bacteria, is the presence of a membrane-bound nucleus that contains the chromosomal DNA (illustrated; image credit). Surprisingly, a nucleus-like structure that forms during viral infection of bacteria is the site of viral DNA replication (link to paper).

During infection of Pseudomonas bacteria with the phage 2O1phi2-1, a separate compartment forms in which viral DNA replication takes place. A phage protein, gp105, makes up the outer layer of this compartment, which initially forms near one end of the cell, and then migrates to the center. The migration of the compartment takes place on a spindle made up of the tubulin-like protein PhuZ.

In addition to viral DNA, certain proteins gain entry into this compartment, including viral proteins involved in DNA and mRNA synthesis, and at least one host cell protein. Other proteins, such as those involved in translation and nucleotide synthesis, are excluded. This compartmentalization very much resembles that of the nucleus of eukaryotic cells.

Packaging of the viral DNA takes place on the surface of the viral nucleus. Empty phage capsids form at the bacterial cytoplasmic membrane, then migrate to the compartment where they attach firmly to the surface. By an unknown mechanism, DNA moves from the compartment into the capsid. Then  capsids are released from the surface to further mature in the cytoplasm. The completed phages are released from the cell upon bacterial lysis.

These fascinating observations raise a number of unanswered questions. Does infection with other phages lead to assembly of a viral nucleus? How do molecules selectively move in and out of the structure?

Perhaps the most interesting question relates to the origin of viruses and cells. According to one hypothesis, self-replicating, virus-like nucleic acids might have first appeared on Earth, followed by cells without a nucleus. Was the nucleus a viral invention?

Giving your neighbor the gift of virus susceptibility

SiphoviridaeVirus infections initiate when virions bind to receptors on the cell surface. It is well known that cells can be made susceptible to infection by providing DNA encoding the virus receptor. For example, mice cannot be infected with poliovirus, but become susceptible if they are given the human poliovirus receptor gene. Now we have learned that providing the receptor protein is sufficient to make cells susceptible to infection (link to paper).

Bacteriophages determine the composition of microbial populations by killing some bacteria and sparing others. Bacteriophages are typically host specific, a property that is largely determined at the level of attachment to host cell receptors. How resistant and sensitive bacteria in mixed communities respond to phage infection has not been well studied.

Several phages (including SPP1, pictured) of the soil bacterium Bacillus subtilis first attach to poly-glycosylated teichoic acids (gTA), and then to the membrane protein YueB, leading to injection of DNA into the cell. Cells that lack the gene encoding either of these proteins are resistant to infection.

When a mixed culture of resistant and susceptible B. subtilis cells were infected with phage SPP1, both types of cells became infected and killed. Infection of resistant cells depended on the presence of susceptible cells, because no infection occurred in pure cultures of resistant cells.

Both infected and uninfected bacteria release small membrane vesicles that contain proteins, nucleic acids, and other molecules. Phage SPP1 can attach to  membrane vesicles released by susceptible strains of B. subtilis, showing that they contain viral receptor proteins. Furthermore, phage SPP1 can infect resistant cells that have been incubated with membrane vesicles from a susceptible strain – in the absence of intact susceptible cells.

These results show that membrane vesicles released by susceptible bacteria contain viral receptors that can be inserted into the membrane of a resistant cell, allowing infection. Because phage infection can lead to transfer of host DNA from one cell to another, the results have implications for the movement of genes for antibiotic resistance or virulence. It’s possible that such genes may move into bacteria that have only ‘temporarily’ received virus receptors via membrane vesicle transfer.

These findings should also be considered when designing phage therapy for infectious diseases. The idea is to utilize phages that are host specific and can only destroy the disease-producing bacteria. It’s possible that the host range of such phages could be expanded by receptor protein transfer. As a consequence, unwanted genes might make their way into ‘resistant’ bacteria.

I wonder if membrane vesicle mediated transfer of receptors also occurs in eukaryotic cells. They shed membrane vesicles called exosomes, which contain protein and RNA that are delivered to other cells. If exosomes bear receptors for viruses, they might be able to deliver the receptors to cells that would not normally be infected. The types of cells infected by a virus would thereby be expanded, potentially affecting the outcome of viral disease.

TWiV 412: WO, open the borders and rig the infection

The TWiVome reveal the first eukaryotic genes found in a bacteriophage of Wolbachia, and how DNA tumor virus oncogenes antagonize sensing of cytoplasmic DNA by the cell.

You can find TWiV #412 at microbe.tv/twiv, or listen below.

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Eukaryotic genes in a bacteriophage

Wolbachia

Wolbachia in an insect cell. Image credit: PLoS/Scott O’Neill.

Viruses are tidily categorized into three groups according to the hosts they infect – bacteriophages, eukaryotic viruses, and archaeal viruses. Viruses do not infect hosts in another domain of life, and therefore lateral gene transfer is limited (giant DNA viruses might be exceptions). Now there is evidence for lateral gene transfer between eukaryotes and bacteriophages.

Proof of this unusual movement of DNA comes from studies of the obligate intracellular bacteria Wolbachia, which infects 40% of arthropods (pictured). Wolbachia are in turn infected with a bacteriophage called WO; nearly all sequenced Wolbachia genomes contain integrated WO DNA. Analysis of complete WO genome sequences revealed the presence of mutiple eukaryotic genes (link to paper) that comprise about half of the phage genome!

Ten different protein domains were identified in the eukaryotic genes of WO phage with four functions: toxins, host-microbe interactions, host cell suicide, and protein secretion through membranes.

One eukaryotic gene in phage WO is a black widow spider toxin called latrotoxin-CTD. Sequence analysis suggests that the spider toxin gene was transferred to phage WO within a Wolbachia genome (these bacteria are known to infect widow spiders).

It is not surprising that a virus of a bacterium that infects a eukaryotic cell might acquire eukaryotic genes, but the exact mechanism of gene transfer is unknown. Eukaryotic DNA might enter the WO genome while the particles are in the insect cell cytoplasm, or during packaging of viral DNA in the presence of animal DNA. Another possibility is transfer of eukaryotic DNA to the Wolbachia genome, and then to phage WO.

The fact that eukaryotic-like DNA sequences make up half of the phage WO genome suggests that they serve important functions for the virus. The functions ascribed to these eukaryotic genes suggest roles in cell lysis, modification of host proteins, and toxicity.

There are other examples of phage-infected obligate intracellular bacteria of Chlamydia, aphids, and tsetse flies. A study of these viral genomes should reveal whether lateral gene transfer between metazoans and bacteriophages is a common mechansim for augmenting functions of the viral genome.

TWiV 405: All the world’s a phage

The TWiXers discuss a study on vertical transmission of Zika virus by Aedes mosquitoes, and uncovering Earth’s virome by mining existing metagenomic sequence data.

You can find TWiV #405 at microbe.tv/twiv, or listen below.

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TWiV 386: The dolphins did it

TWiVDid you know that the evolution of ancient retroviruses, millions of years ago, can be traced by studying their genomes in the chromosomes of contemporary animals? Ted Diehl and Welkin Johnson join the TWiV team to tell us how they did it with mammals. All without a single wet experiment! They also join in the discussion about virus dispersal by hand dryers.

You can find TWiV #386 at microbe.tv/twiv, or listen below.

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Viruses help form biofilms

Inoviridae virionBacteria frequently grow in communities called biofilms, which are aggregates of cells and polymers. An example of a biofilm is the dental plaque on your teeth. Biofilms are medically important as they can allow bacteria to persist in host tissues and on catheters, and confer increased resistance to antibiotics and dessication. Therefore understanding how biofilms form is crucial for controlling microbial infections. An advance in our understanding of biofilms formation is the observation that filamentous phages help them assemble, and contribute to their fundamental properties.

Pseudomonas aeruginosa is an important human pathogen which is a particular problem in patients with cystic fibrosis. The ability of this bacterium to form biofilms in the lung is linked to its ability to cause chronic infections. Pseudomonas aeruginosa biofilms contain large numbers of filamentous Pf bacteriophages (pictured). These viruses lyse cells and release DNA, which becomes one component of the biofilm matrix.

Mixing supernatants of P. aeruginosa cultures with hyaluronan, which is present in airways of cystic fibrosis patients, resulted in the formation of a biofilm – in the absence of bacteria. A major component of P. aeruginosa biofilms was found to be Pf bacteriophages. When purifed Pf bacteriophages were mixed with hyaluronan, biofilms formed. Similar biofilms also formed when the filamentous bacteriophage fd of E. coli was mixed with hyaluronan. Mixtures of Pf bacteriophages and various polymers (alginate, DNA, hyaluronan, polyethylene glycol) formed liquid crystals (matter in a state between a liquid and a solid crystal).

Pf phages were detected in sputum from patients with cystic fibrosis, but not in uninfected patients. Addition of Pf phage to sputum from patients infected with P. aeruginosa made the samples more birefringent, a property of liquid crystals. Compared with a strain of P. aeruginosa that does not produce Pf phage, colonies of virus-producing strains formed liquid crystals. These observations indicate that Pf phage help organize the bacteria into a biofilm matrix.

Some features of biofilms include their ability to adhere to surfaces, to protect bacteria from dessication, and to increase resistance to antibiotics. Addition of phage Pf increased biofilm adhesion and tolerance against dessication. Such addition also made the biofilm more resistant to aminoglycoside antibiotics, because these were sequestered in the biofilm. No phage-mediated increased resistance to ciprofloxacin was observed, probably because this antimicrobial does not interact with polyanions of the biofilm as do aminoglycosides.

These results show that presence of bacteriophage in a biofilm of P. aeruginosa helps organize the matrix while contributing to some of its fundamental properties. It seems likely that filamentous phages of other bacteria will play roles in biofilm formation, suggesting that targeting the phages in these matrices could be effectie strategies for treating biofilm infections.

TWiV 346: A double helical career

Episode #346 of the science show This Week in Virology was recorded at the 34th Annual Meeting of the American Society for Virology, where Vincent, Rich, and Kathy spoke with Joan Steitz, a tireless promoter of women in science and one of the greatest scientists of our generation.

You can find TWiV #346 at www.microbe.tv/twiv.

Your viral past

virusesDid you ever wonder what different virus infections you have had in your lifetime? Now you can find out with just a drop of your blood and about $25.

Immune defense systems of many hosts produce antibodies in response to virus infections. These large proteins, which are generally virus specific, can block or inhibit virus infection, and persist at low levels for many years after the initial infection. Hence it is possible to determine whether an individual has had a virus infection by looking for anti-viral antibodies in the blood. Up to now the process of identifying such antibodies has been slow and limited to one or a few viruses. A new assay called VirScan allows unbiased searches for all the virus antibodies in your blood, providing a picture of all your past infections.

To identify the human antivirome, DNAs were synthesized encoding proteins from all viruses known to infect humans – 206 species and over 1000 strains. These DNAs were inserted into the genome of a bacteriophage, so that upon infecting bacteria, the viral peptides are displayed on the phage capsid. These ‘display’ phages were then mixed with human serum, and those that were bound by antibodies were isolated. The DNA sequence of the phage genomes were then determined to identify the human virus bound by the antibodies.

This method was used to assay samples from 569 humans. The results show that each person had been exposed to an average of 10 viruses, with a range from a few to over 20 (two individuals had antibodies to 84 different virus species!). The most frequently identified viruses included herpesviruses, rhinoviruses, adenoviruses, influenza viruses, respiratory syncytial virus, and enteroviruses. The overall winner, found in 88% of samples, is Epstein-Barr virus.

These results are not unexpected: all of us are infected with at least a dozen viruses at any time, and the viruses identified in this study known to infect much of the human population. What was surprising is the absence of some common viruses, such as rotaviruses, and the ubiquitous polyomaviruses. According to serological surveys, the most common human viruses are the small, single-stranded DNA containing anelloviruses. Yet the related torque teno virus was only found in 1.7% of samples. These differences are likely due to a combination of technical and biological issues (e.g., failure of antibodies to certain viruses to persist in serum).

This new assay may one day become a routine diagnostic tool that is used along with complete blood counts and chemistries to know if a patient’s signs and symptoms might be attributable to a past virus infection. VirScan technology is not limited to virus infections – it can be used to provide a history of bouts with bacteria, fungi, and parasites.

VirScan might also allow us to determine which virus infections are beneficial, and which contribute to chronic diseases such as autoimmune or neurodevelopmental disorders or cancer. The assay can be used to conduct unbiased population-based studies of the prevalence of virus infections and their possible association with these diseases. Such connections were not previously possible with antibody assays that search for one virus at a time. This approach was not only inefficient, but required guessing the responsible virus.

Some other findings of this study are noteworthy. As expected, children had fewer virus infections than adults. HIV-positive individuals had antibodies to more viruses than HIV-negative individuals, also expected given the damage done by this virus to the immune system. Frequencies of anti-viral antibodies were higher outside of the United States, possible due to differences in genetics, sanitation, or population density. In most samples, there was a single dominant peptide per virus, although there were occasional differences among populations. This information might be useful for improving vaccines, or tailoring them to specific countries or regions.

Update: It would be very informative to use VirScan to search for antibodies against viruses that are not known to infect humans. Other animal viruses, plant viruses, insect viruses: to which do a significant fraction of humans respond? The information might identify other viruses that replicate in humans and which might constitute future threats (or present benefits).