TWiV 468: Zika by the slice

Amy Rosenfeld joins the TWiV team to talk about her career and her work on Zika virus neurotropism using embryonic mouse organotypic brain slice cultures.

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Lymphocytes after dark

B cell

When you are infected with a microbe, pieces of the pathogen are picked up by sentinel dendritic cells and brought to local lymph nodes. There the sentinels present their gifts to lymphocytes – B (pictured; image credit) and T cells – who then decide if they are foreign, in which case an immune response begins. These lymphocytes circulate throughout the body not continuously, but in a circadian manner – a 24 hour cycle.

In mice, lymphocyte numbers peak in lymph nodes about one hour after lights are turned off. During the day, these cells leave the lymph nodes. However, the same pattern was observed when mice were kept entirely in the dark, showing that the change in lymphocyte numbers is due to an endogenous circadian clock, not a response to a change in lighting.

The homing of lymphocytes into lymph nodes depends on a number of different proteins, including adhesion molecules and attractants. The levels of these proteins also peaked at night. When the receptor for one attractant, CCR7, was genetically ablated, no oscillations of lymphocyte numbers in lymph nodes was observed.

Dendritic cells, the major antigen presenting cells, are also more numerous in lymph nodes during night hours. This observation makes sense given that dendritic cells present antigen to lymphocytes in the lymph node.

Circadian rhythms are under the control of clock genes – the discovery of which was recently recognized by the Nobel Prize in Physiology or Medicine. Deletion of the gene encoding one such clock gene in B or T cells of mice caused loss of the rhythmicity of lymphocytes in lymph nodes. In these mice, the levels of CCR7 mRNAs also lost their rhythmic character. These observations show that circadian clocks regulate lymphocyte migration.

Earth has a 24 hour day and life has adapted this cycle by turning genes on and off only when needed. It seems reasonable that lymphocyte trafficking into lymph nodes would peak during the hours when animals are moving about and potentially encountering pathogens. But not all animals are diurnal – active during the day. Mice are nocturnal, a behavior that probably helps to avoid predators. It makes perfect sense that lymphocyte trafficking in mice peaks at the onset of night.

It seems likely that in humans, a diurnal species, lymphocyte numbers peak in lymph nodes during the day. Consistent with this idea, it has been shown that in humans, vaccination in the morning produces higher antibody titers compared with vaccination in the afternoon. The take away message is clear: do not get your vaccines at night!

TWiV 467: Jon and Ted’s Excellent Adventure

Jon and Teddy Yewdell join the TWiV team to talk about their careers, their research, and the problems with biomedical research.

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Zika virus blocks the neuron road

Written with Amy Rosenfeld, Ph.D.

By infecting organotypic brain slice cultures from embryonic mice, we have shown that Zika virus has always been neurotropic. The same culture system provides information on how Zika virus infection of the developing brain might lead to microcephaly.

The small heads observed in microcephalic children reflect a physically smaller brain – specifically, the neocortex is thinner than in a normal brain. The neocortex, only found in mammals, is the largest part of the cerebral cortex of the brain. It is composed of six distinct layers of neurons, which are established during embryonic development (illustrated below). First, glial cells originating from progenitor cells in the ventricular zone extend their processes throughout the cortex and anchor at the pia, the outer surface of the brain. These long fibers provide a scaffold on which neurons, produced from the same progenitor cells, migrate outwards to establish the six layers of the cortex. Movies have been made that show the migration of neurons on glial fibers, and they are amazing.

embryonic brain development

The glial fibers are visible as parallel tracks in our embryonic brain slice cultures stained with an antibody to vimentin, a protein component of the fibers (image below, left panel). When embryonic brain slice cultures were infected with Zika virus, the structure of the glial tracks was altered. Instead of parallel tracks, the fibers assumed a twisted morphology that would not allow neurons to travel from the ventricular zone to the developing neocortex (image below, right panel). Disruption of glial fibers was observed after infection with Zika viruses isolated from 1947 to 2016.

Zika fiber disruption

Image credit: Rosenfeld AB et al.

To determine if Zika virus-mediated disruption of glial fibers could impair neuronal migration, we isolated brains from embryonic mice as described above, but before virus infection, a plasmid encoding green fluorescent protein was injected into the ventricle. An electrical current was then applied to the brain to encourage uptake of the plasmid in neuronal progenitors lining the ventricle. The brains were then sliced, placed in culture, and infected with Zika virus.

Four days later, in the uninfected brain slices, green fluorescent neurons could be seen in the ventricular zone, and some of these had already migrated through the developing cortical plate to the pial surface. In brain slices infected with Zika virus, the migration of green neurons to the cortical plate was impaired, and the cells remained in the area where the plasmid was injected. This observation indicates that the disruption of glial fibers caused by Zika virus infection has caused fewer neurons to reach the cortical plate.

We think it is likely that Zika virus disruption of glial fibers during embryonic development contributes to microcephaly: if neurons cannot migrate to the pial surface, the neocortex will be thinner. Zika virus infection also inhibits the proliferation of progenitor cells that line the ventricular surface, which is likely a contributing factor to microcephaly. Other embryonic brain cells are infected with Zika virus, and these could play a role in microcephaly. Furthermore, there are other effects of Zika virus infection on the developing brain, including calcifications, hypoplasia (reduced cell density), lissencephaly (smooth brain), ventriculomegaly (enlarged ventricle), and brainstem dysfunction.

We are particularly interested in identifying the viral protein that disrupts the glial fibers in embryonic mouse brains. Once that protein is identified, it might be possible to understand the mechanisms by which the glial fibers are disrupted. Such information would not only lead to a better understanding of how Zika virus causes microcephaly, but should also provide a better understanding of how the brain develops.

TWiV 466: The Capsid Club

From Indiana University, Vincent and Kathy speak with Tuli Mukhopadhyay, John Patton, and Adam Zlotnick about their careers and their work on alphaviruses, hepatitis B virus, and rotaviruses.

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Zika virus has always been neurotropic

Third trimester embryonic mouse brains

Written with Amy Rosenfeld, Ph.D.

Zika virus has been infecting humans since at least the 1950s (and probably earlier), but epidemics of infection have only been observed in the past ten years and congenital Zika syndrome in the last two. Two hypotheses emerged to explain this new pattern of disease: evolution of the virus, or random introduction into large, immunologically naive populations. Results from our laboratory show that one component of these disease patterns – neurotropism, the ability to infect cells of the nervous system – has always been a feature of Zika virus.

If evolution has selected for Zika viruses that cause epidemics and congenital neurological disease, there are many steps in the infection pathway that could be affected. Let’s focus on the ability of Zika virus infection during pregnancy to cause microcephaly. Mutations that affect multiple stages of infection might be responsible. These could include any or all of the following:

  • Mutations that increase viremia in the human host, increasing the likelihood that virus will be captured by a mosquito taking a blood meal.
  • Mutations that increase viral replication in the mosquito vector.
  • Mutations that increase the ability of the virus to cross the placenta.
  • Mutations that allow efficient replication in the fetus.
  • Mutations that promote virus entry of the nervous system (neuroinvasion).
  • Mutations that enhance replication in neural cells (neurotropism).

This list is by no means exhaustive. The point is that no small animal model is likely to capture all of these steps. For example, no mouse model of Zika virus infection has so far lead to the development of microcephalic offspring. Therefore testing whether any of the the mutations observed in different Zika virus isolates are responsible for new disease patterns is likely impossible.

We have chosen to look at the question of how Zika virus disease has changed by looking at a very specific part of the replication cycle: growth of the virus in fetal brain, specifically in organtypic brain slice cultures. Here’s how it works: we remove the developing embryos from pregnant mice during the first, second or third trimesters of development (see photo). The fetal brain is removed, sliced (slices are about 300 nm thick), are placed into culture medium. The slices live up to 8 days, during which time brain development continues. The Vallee laboratory here at Columbia has used a similar system utilizing rats to study the genetic basis of microcephaly.

Next, we infect the embryonic brain slices with different isolates of Zika virus from 1947 to 2016, from Africa, Asia, South America, and Puerto Rico. All of the isolates replicated in brain slice cultures from the first and second trimesters of development. These observations show that Zika virus has been neurotropic since at least 1947. Similar observations have been made with the 1947 isolate using human neurospheres, organoids, and fetal organotypic brain slice cultures.

The incidence of microcephaly is greatly reduced when mothers are infected during the third trimester of development. Consistent with this observation, we found that organotypic brain slice cultures from the third trimester of mouse development support the replication of only two of seven Zika virus isolates examined – the original 1947 isolate from Uganda, and 2016 isolate from Honduras. Furthermore, these viruses replicate in different cells of the third trimester embryonic brain compared with second trimester brain. We are interesting in identifying the changes in the virus responsible for these differences.

Our approach asks only whether different Zika virus isolates can infect brain cells when the virus is placed directly on these cells. We cannot make any conclusions about the ability of the virus to invade the brain from the blood (neuroinvasion), or any of the other steps in infection listed above.

Our experimental system also reveals how Zika virus infection of the developing brain might lead to microcephaly, a topic that we’ll explore next week.

TWiV 465: Theodora the explorer

Theodora Hatziioannou joins the TWiV team to discuss a macaque model for AIDS, and how a cell protein that blocks HIV-1 infection interacts with double-stranded RNA.

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Good viruses visiting bad neighborhoods

Marco VignuzziWhat would happen to an RNA virus if its genome were placed in a bad neighborhood? The answer is that fitness plummets.

RNA virus populations are not composed of a single defined nucleic acid sequence, but are dynamic distributions of many nonidentical but related members. In the past I have referred to these populations as quasispecies but that is no longer the preferred term: mutant swarms or heterogeneous virus populations should be used instead.

The term for all possible combinations of a viral genome sequence is sequence space; for a 10,000 nucleotide genome this would be theoretically 410,000 different genomes – a huge number, more than the atoms in the universe. Any RNA virus population occupies only a fraction of this sequence space, in part because many mutations are deleterious. Studies have shown that viral genomes occupy specific parts of sequence space, called neighborhoods, and movement to different neighborhoods is important for viability. If the viral genome is placed in a bad neighborhood – one that is detrimental for virus fitness – the ability to explore sequence space is restricted.

An example of the effect of changing viral sequence space is shown by a study in which hundreds of synonymous mutations (they did not change the amino acid sequence) were introduced in the capsid region of poliovirus (link to paper). Such rewiring, which placed the virus in a different sequence space, reduced viral fitness and attenuated pathogenicity in a mouse model. In other words, the viral genome was placed in a bad neighborhood, from where it could not move to other neighborhoods needed for optimal replication and pathogenesis. While the genome rewiring did not affect the protein sequence, it might have had deleterious effects on RNA structures or codon or dinucleotide frequency. For example, introduction of codon pairs that are under-represented in the human genome can produce less fit viruses.

A recent study avoids these potential issues by introducing changes in the viral genome that do not affect protein coding, RNA structures or codon or dinucleotide frequency, yet place the viral genome in a different sequence space (link to paper). All 117 serine/leucine codons in the capsid region of Coxsackievirus B3 were changed so that a single nucleotide mutation would lead to a stop codon, terminating protein synthesis and virus replication (this virus is called 1-to-Stop). The serine codons were changed to UUA or UUG; one mutation changes these to the terminators UAA, UGA, or UAG. Another virus was made in which two mutations were needed to produce a stop codon (NoStop virus).

1-to-Stop viruses replicated normally, but when mutagenized, they had significantly lower fitness than wild type or NoStop viruses. Extensive passage of the virus in cells, which would be expected to cause accumulation of mutations, had the same effect on fitness. When a high fidelity RNA polymerase was introduced into 1-to-Stop virus, it replicated like wild type virus. Similar results were obtained with an influenza virus when one of its 8 genome segments was rewired to produce 1-to-Stop and NoStop counterparts.

The 1-to-Stop Coxsackieviruses were attenuated in a mouse model of infection. Furthermore, mice infected with 1-to-Stop virus were protected against replication and disease after challenge with wild type virus. These observations suggest that rewiring viral RNA genomes could be used to design vaccines.

These findings show that recoding a viral genome places it an different sequence space than wild type virus, in which single mutations can lead to inactivation of viral replication. This new neighborhood is unfavorable (‘bad’) because the virus cannot readily move to other neighborhoods to accommodate the effects of mutation.

For more discussion of viral sequence space and rewiring viral genomes, listen to the podcast This Week in Evolution #24: our guest is Marco Vignuzzi (pictured), senior author on the second paper discussed here.

TWiV 464: Boston baked viruses

At Tufts University Dental School in Boston, Vincent speaks with Katya Heldwein and Sean Whelan about their careers and their work on herpesvirus structure and replication of vesicular stomatitis virus.

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From cell proteins to viral capsids

Origin of virusesWe have previously discussed the idea that viruses originated from selfish genetic elements such as plasmids and transposons when these nucleic acids acquired structural proteins (see A plasmid on the road to becoming a virus). I want to explore in more detail the idea that the structural proteins of  viruses likely originated from cell proteins (link to paper).

Three ideas have emerged to explain the origin of viruses: 1. viruses evolved first on Earth, before cells, and when cells evolved, the viruses became their genetic parasites; 2. viruses are cells that lost many genes and became intracellular parasites; 3. viruses are collections of genes that escaped from cells. Missing from these hypothesis is how nucleic acids became virus particles – that is, how they acquired structural proteins. It seems likely that viral structural proteins originated from cellular genes.

An analysis of the sequence an structure of major virion proteins has identified likely ancestors in cellular proteins. Following are some examples to illustrate this conclusion.

A very common motif among viral capsid proteins is called the single jelly roll, made up of eight beta strands in two four-stranded sheets. Many cell proteins have jelly role motifs, and some form 60-subunit virus-like particles in cells. The extra sequences at the N-termini of viral jelly roll capsid proteins, involved in recognizing the viral genome, likely evolved after the capture of these proteins from cells.

The core proteins of alphaviruses (think Semliki Forest virus) has structural similarity with chymotrypsin-like serine proteases. The viral core protein retains protease activity, needed for cleavage from a protein precursor.

Retroviral structural proteins also appear to have originated from cell proteins, with clear homologies with matrix, capsid, and nucleocapsid proteins. The matrix Z proteins of arenaviruses are related to cellular RING domain proteins, and the matrix proteins of some negative strand RNA viruses are related to cellular cyclophilin. There are many more examples, providing support for the hypothesis that viruses evolved on multiple instances by recruiting different cell proteins.

Given this information on the origin of viral capsid proteins, we can modify the three hypotheses for the origin of viruses into one. Self-replicating, virus like nucleic acids emerged in the pre-cellular world and from the emerged the first cells. The replicating nucleic acids entered the cells, where they replicated and became genetic parasites. At some point these genetic elements acquired structural proteins from the cells and became bona fide virus particles. As cells evolved, new viruses emerged from them.

It is important to point out that the genes do not always flow from cells to viruses. We know that viral proteins can be returned to cells, where they serve useful functions. One example is syncytin, a retroviral protein used for the construction of the mammalian placenta.