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TWiV 937: Pediatric hepatitis with Emma Thomson

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From the European Society for Clinical Virology 2022 Conference in Manchester UK, Vincent speaks with Emma Thomson about the recent outbreak of pediatric hepatitis of unknown etiology and the finding that it is linked to infection by adenovirus-associated virus 2.

Hosts: Vincent Racaniello

Guest: Emma Thomson

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A Viral Risk Factor for Multiple Sclerosis

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by Gertrud U. Rey

Epstein-Barr virus (EBV) is a prevalent human herpesvirus that is most commonly transmitted through saliva and is well known for causing infectious mononucleosis (i.e., “mono”). Various recent lines of evidence suggest that EBV infection may also be a risk factor for the development of multiple sclerosis (MS), an autoimmune disease in which the protective covering of nerves is progressively damaged, leading to severe neurological problems.

In an effort to identify a possible causal link between EBV infection and MS, several research groups collaborated with the US military in an impressive epidemiological survey spanning a 20-year period. The study drew from a population of more than 10 million military personnel. The authors then investigated/documented 801 subjects who had developed MS during the course of service. Active duty members are routinely screened for HIV infection at the start of service and every two years thereafter, and residual serum samples are stored in an archive at the Department of Defense. The authors analyzed up to three of these residual samples from each person who had developed MS after entering service for the presence of antibodies against EBV. As a control, they also screened the samples for antibodies against cytomegalovirus, another common human herpesvirus that is also transmitted through saliva but that has no known causal link to MS. Remarkably, only one of the 801 MS subjects was EBV-negative at the time of the last sample – all others had tested positive for EBV at some time prior to onset of MS but had been EBV negative when they first joined the service. The presence of EBV antibodies in the serum by the time the third sample was drawn was associated with a 32-fold increased risk of developing MS, with a median time interval of 7.5 years from estimated EBV infection to onset of MS, suggesting that EBV infection was a causal factor for the development of MS. In contrast, there was no correlation between cytomegalovirus infection and development of MS. In fact, the risk of developing MS was lower for people who had been previously infected with cytomegalovirus.

Based in part on these results, the authors of a different study aimed to identify a molecular mechanism by which EBV ultimately leads to the development of MS. Because MS patients almost always have high levels of antibodies against the EBV protein Epstein-Barr nuclear antigen 1 (EBNA1), it is possible that these antibodies cross-react to self antigens with similar amino acid sequences in an autoimmune mechanism known as “molecular mimicry.” One such self antigen with sequence similarity to EBNA1 is GlialCAM, a protein produced by cells of the central nervous system, where it mediates the binding of cells to each other and their surrounding materials.  

As a first step, the authors collected serum and cerebrospinal fluid (CSF) samples from MS patients and control patients suffering from a non-MS neuroinflammatory condition. They then isolated single B cells from each of these samples and analyzed the cells for the presence of membrane-bound antibodies (i.e., B cell receptors) that are characteristic of MS. B cells isolated from the CSF of MS patients displayed a range of B cell receptors that were not present in the sera of these patients or the CSF or sera of non-MS patients, a disparity that is indicative of central nervous system inflammation and a hallmark of MS. When the genes encoding these B cell receptors were sequenced, they were found to be highly clonal, meaning that there were only a few different sequences present, with each sequence present in large numbers of copies or “clones.” In other words, the clonal antibodies were the offspring of only a few select precursor B cells that were repeatedly expanded within the CSF of MS patients. In contrast, the sequences of B cell receptor genes from the CSF of non-MS patients were mostly different from each other (i.e., non-clonal). This finding suggested that the antibody-encoding gene sequences in the B cells of MS patients were undergoing a process called somatic hypermutation – a rearrangement of the original germline sequences that leads to an optimized antibody response. And although somatic hypermutation typically occurs in the germinal centers of lymphoid tissues, in MS patients it seemed to be happening in the CSF.

Antibodies that were secreted from B cells in the CSF of MS patients were isolated and characterized by mass spectrometry, a technique that reveals the chemical identity of molecules and the amino acid composition of protein peptides. The amino acid sequences of these antibodies were then compared/matched to the previously sequenced B cell receptor genes to determine whether these antibodies originated from those B cell receptors. This comparison revealed that for 87% of the clonal B cell receptor sequences, the CSF of MS patients contained matching peptide sequences that were unique to the patient. In contrast, only 40% of the non-clonal sequences had matching peptides in the CSF. Antibodies that were highly abundant in the CSF matched to a few clonally expanded B cell receptors, confirming that a small fraction of B cells produced most of the antibodies in the CSF.

As a next step, the authors generated monoclonal antibodies from each of 148 individual B cell receptors isolated from the CSF of MS patients and tested their ability to bind EBNA1. Antibodies from six out of nine MS patients bound full EBNA1 protein, and antibodies from eight of those nine patients bound shorter peptides of EBNA1. One of these monoclonal antibodies, called MS39p2w174 (shortened here to “MS39”), bound EBNA1 in a region that was previously shown to induce stronger antibody responses in subjects with MS than in healthy individuals, and was thus used in all subsequent binding studies. Comparison of the amino acid sequence of MS39 to that of the known wild type version of this antibody (i.e., “germline MS39”) revealed that only two of the amino acids in MS39 that interact with EBNA1 were different from those found in germline MS39, suggesting that only these two amino acids resulted from gene rearrangement during somatic hypermutation. Because the remaining sequence of this “hypermutated MS39” was identical to the sequence of germline MS39, it is likely that naïve B cells producing germline MS39 can bind EBNA1.

MS39 also bound GlialCAM, although this binding was significantly reduced for germline MS39 compared to hypermutated MS39. In contrast, both germline and hypermutated MS39 bound EBNA1 with similar affinity. This observation suggested that somatic hypermutation of MS39 increased its binding affinity for GlialCAM.

A comparison of hypermutated MS39 binding affinity to whole EBNA1 protein, whole GlialCAM protein, and shorter peptide versions of EBNA1 and GlialCAM revealed that the antibody bound both versions of EBNA1 with equal affinity but bound whole GlialCAM much more strongly than its peptide version. In its native state, GlialCAM protein is heavily phosphorylated – a modification that is mediated by a group of enzymes called kinases, which attach phosphoryl groups to certain amino acids. When the authors phosphorylated two specific amino acids on GlialCAM, the binding affinity of hypermutated MS39 to GlialCAM increased by at least 50-fold. Considering that certain kinases are known risk factors for MS, it makes sense that phosphorylation of GlialCAM by these kinases would lead to an increased ability of hypermutated MS39 to cross-react with GlialCAM, thereby potentially leading to MS.

The authors also tested plasma samples from MS patients and healthy individuals for their reactivity to EBNA1 and GlialCAM. Plasma from all MS patients and most healthy individuals were reactive to EBNA1, suggesting that all of these samples contained antibodies to EBNA1 protein. However, only plasma from MS patients had any significant reactivity to GlialCAM, suggesting that only MS patients have antibodies that bind GlialCAM with high affinity. This confirms that there is likely a molecular mechanism that causes MS patients to develop antibodies that target and attack GlialCAM.

Considering that EBV infects about 95% of adults, it is currently unclear why most people never develop MS. However, because humans all have different cell surface molecules that present antigens to cells of the immune system, it is possible that different people present different portions of EBNA1 and/or GlialCAM on their cell surfaces, resulting in varied B and T cell responses that lead to different immune outcomes. Despite the low incidence of MS, it would be great to have a vaccine that prevents EBV infection, thereby possibly also preventing MS. 

[For a more detailed discussion of these two papers, please check out TWiV 869.]

A Viral Heist

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by Gertrud U. Rey

Viruses that require access to the host cell nucleus typically get there by exploiting cellular motor proteins for their own transportation purposes. For example, herpesviruses use two cellular proteins called dynein and kinesin to shuttle their cargo to and from the nucleus. However, the exact mechanism by which these viruses engage the motor proteins was unclear until recently.

Dynein and kinesin traffic around the cell on long cylindrical structures called microtubules, which not only serve as a “railway” system for a variety of motor proteins that transport cargo to support various cellular functions, but also provide a framework for the cytoskeleton. Microtubules have a well-defined polarity that is integral to their biological function: one end is designated the plus end, while the other end is the minus end. Dynein moves from the plus end of the microtubule towards the minus end in the direction of the nucleus, while kinesin moves in the opposite direction, typically leading to the cell periphery.

Herpes simplex virus type 1 (HSV-1) initially infects epithelial cells of the skin and mucous membranes. During this infection, viral particles fuse with the host cell membrane and release the protein shell that encompasses the viral genome (the “capsid”), into the cytoplasm. A virally encoded and capsid-bound protein called pUL36 then binds dynein, allowing the capsid to travel along a microtubule to the nucleus, where the viral genome is injected through a nuclear pore. When newly assembled capsids carrying newly replicated viral genomes exit from the nucleus at a later time, they engage kinesin to motor along the microtubules toward the cell membrane. Newly formed virions emerging from the cell membrane subsequently infect nearby sensory neurons, where they establish a lifelong, persistent infection.

Studies have shown that deletion of the portion of pUL36 that tethers it to the viral capsid results in its ability to move along microtubules in both directions, suggesting that in addition to binding dynein, pUL36 also binds kinesin. Subsequent follow-up studies exploring the kinetics of this presumed interaction revealed that pUL36 does indeed bind kinesin through a series of tryptophan-aspartate (WD) motifs. Two of these motifs – WD3 and WD4 – appear to be particularly important: WD3 improves pUL36-kinesin binding and WD4 is essential for the interaction.

In an effort to more clearly define the role of kinesin during herpesvirus infection, Caitlin Pegg, Sofia Zaichick and co-authors of a recent study, carried out a series of experiments using HSV-1 and pseudorabiesvirus (PRV), a herpesvirus in the same subfamily as HSV-1, which is often used as a model to study the basic processes of herpesvirus infection. To determine whether WD3 and WD4 are needed for PRV replication and viability, the authors introduced mutations in the DNA sequences encoding these motifs in PRV. Plaque assays done using cells infected with the respective modified viruses revealed that viruses with a mutation in WD3 produced smaller plaques, suggesting that this motif enhanced viral replication. Viruses with a mutation in WD4 produced almost no plaques, suggesting that this motif was essential for viral replication. These results complemented the data obtained from the pUL36-kinesin binding studies, implying that an interaction between pUL36 and kinesin is needed for viral replication.

The authors also determined that a mutation in the sequence encoding the acidic amino acids of the WD4 motif led to reduced kinesin binding but did not impair PRV replication. To study the transport mechanism of PRV, they generated a virus with reduced kinesin binding ability (“RKB”) by changing the amino acids in both WD3 and the acidic amino acids in WD4. They further modified the virus by inserting a gene encoding a fluorescent marker next to the capsid genes. The joint expression of the capsid and marker genes in cells would result in fluorescently tagged capsids, allowing one to visualize these particles using a fluorescence microscope. Microscopic analysis of sensory neurons infected with fluorescently tagged RKB viruses revealed that the capsids traveled through the cytoplasm toward the nucleus, but never actually reached the nucleus. This suggests that pUL36 binding to kinesin is important for delivery of viral particles into the neuronal nucleus during infection.

To further substantiate this finding, the authors performed infection experiments using retinal pigment epithelial (RPE) cells that do not express kinesin. Infection of these cells with fluorescently-tagged HSV-1 revealed that viral capsids only advanced to the centrosome, an organelle involved in cell division that is somewhat removed from the nucleus. In contrast, infection of wild type RPE cells having functional kinesin resulted in trafficking of viral capsids right up to the nucleus, confirming that kinesin is needed for transporting capsids from the centrosome to the nucleus.

To determine what happens when virions are produced in the absence of kinesin, the authors infected kinesin-deficient RPE cells with either wild type HSV-1 or wild type PRV, isolated the resulting viral particles and then used those viruses to infect wild type (i.e., kinesin-expressing) RPE cells. Visualization of capsid movement in the wild type RPE cells revealed that the capsids only moved as far as the centrosome, and that centrosome-to-nucleus trafficking was impaired, an effect that was particularly pronounced when the experiment was repeated using wild type sensory neurons. The fact that the virus particles isolated from kinesin-deficient cells were unable to traffic to the nucleus in neuronal cells in spite of the fact that these cells produced kinesin suggests that viruses that do make it to the neuronal nucleus must typically carry kinesin when they enter these cells, presumably inside the virion. However, analysis of wild type cell-originating HSV-1 extracellular virions using anti-kinesin antibodies did not reveal the presence of kinesin in these virions.

In a different approach aimed at identifying kinesin inside HSV-1 virions, the authors generated a DNA construct consisting of kinesin fused to the reporter enzyme β-lactamase and inserted this construct into kinesin-deficient RPE cells. They then infected the cells with HSV-1, extracted newly replicated virions from the cells and mixed the virions with nitrocefin, a molecule that reacts with β-lactamase to produce a color change that can be visually detected. A color change would indicate the presence of β-lactamase, which would mean that kinesin was present as well. This method revealed that the virions isolated from the kinesin/β-lactamase-expressing cells did contain β-lactamase, further suggesting that they also contained kinesin. These results suggested that HSV-1 virions capture kinesin from initially infected epithelial cells and ferry it to subsequently infected neuronal cells to deliver their genome into the nucleus of those cells.

To confirm whether this hypothesis was true, the authors prepared HSV-1 virions expressing kinesin fused to DmrB, a protein that can be induced to dimerize through addition of a drug. When the drug was added to primary sensory neurons infected with HSV-1 carrying the DmrB-kinesin fusion protein, capsid transport from the centrosome to the nucleus was reduced, presumably because DmrB formed a dimer which structurally impaired the motility of kinesin. These results confirmed that kinesin mediates the movement of capsids from the centrosome to the nucleus. And although kinesin-mediated transport of HSV-1 presumably leads the virions to the cell membrane instead of the nucleus, the microtubules radiating out from the centrosome toward the nucleus are oriented such that their minus end is anchored in the centrosome, while the plus end is at the nucleus, consistent with the directional movement of kinesin.

The fact that viruses can capture a cellular protein and incorporate it for later use is a remarkable discovery that the authors refer to as “assimilation.” As more research emerges in this field, it will be interesting to see whether herpesviruses steal other cellular proteins and whether members of other virus families also assimilate cellular proteins to suit their purposes.     

[For an in-depth discussion of this paper, please check out TWiV 833]

TWiV 833: Grand theft kinesin

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TWiV reviews Michael Worobey’s dissection of the early COVID-19 cases in Wuhan, and the discovery that herpesviruses assimilate cellular kinesin to produce motorized virus particles.

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Show notes at microbe.tv/twiv

CRISPR-ing herpes simplex virus

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herpesvirus latencyby Gertrud U. Rey

Herpes simplex viruses establish lifelong persistent infection in sensory neurons of infected individuals, a phenomenon called latency. Latent viral genomes are “dormant” but can sporadically reactivate and begin replicating in a phase called lytic replication, which is often accompanied by shedding of virus particles and the appearance of painful lesions. There is no vaccine to prevent infection with either herpes simplex virus type 1 or 2 (HSV-1 or -2), and currently available therapeutics do not clear latent viruses or prevent their reactivation.

The emergence of CRISPR genome editing tools has inspired renewed efforts for preventing the reactivation of latent viruses by targeting and cleaving their genomes. An exemplary CRISPR editing system consists of the bacterial nuclease Cas9 and a small “guide” RNA molecule. The RNA molecule, which is complementary to the target sequence, guides the nuclease to its destination, where the nuclease cleaves the target DNA. CRISPR/Cas9 complexes can be introduced into cells by various mechanisms. For example, viruses engineered to encode the nuclease and the guide RNA can be transferred into cells using a technique called transduction.

A team at Harvard Medical School recently determined that specifically designed guide RNAs not only inhibit lytic replication of HSV-1, but can also cleave and edit latent HSV-1 genomes, thereby inhibiting their reactivation.

The authors of the study screened 58 potential guide RNAs for their ability to direct the cleavage of HSV-1 target DNAs in vitro. In this assay, they incubated individual nuclease/guide RNA complexes together with different DNA substrates containing various target sequences and measured cleavage efficiency by gel electrophoresis. The guide RNAs that led to the best cleavage efficiency were then further tested for their efficacy in inhibiting HSV-1 lytic replication in human fibroblast cells. The authors transduced the cells with the various nuclease/guide RNA complexes, infected them with HSV-1, and measured viral (lytic) replication by plaque assay. Although several of the guide RNAs significantly reduced viral replication, the guide RNA targeting the UL30 region, which encodes the viral DNA polymerase, reduced viral levels by more than 10,000-fold.

To see whether this editing system could inhibit reactivation of latent HSV-1 genomes, the authors infected cells with a replication-defective HSV-1 strain, thus mimicking latency, and transduced the cells with Cas9 nuclease and various guide RNAs that had been effective in the in vitro cleavage screen. They then reactivated the latent virus by “superinfecting” the latently infected cells with wild type HSV-1 and measured the ability of the individual guide RNAs to inhibit this reactivation. The replication-defective strain encodes a green fluorescent protein, allowing the authors to distinguish between replication of the wild type input HSV-1 and the reactivated virus. When used individually, four of the guide RNAs reduced reactivation of latent viruses by about 100-fold. However, the authors were able to reduce reactivation by an additional 10-fold by targeting two genes simultaneously with two different guide RNAs, suggesting that one can achieve an increased effect by combining several guide RNAs.

Sequencing analyses also showed that some of the CRISPR/Cas9 complexes introduced detrimental mutations into the target sequence, and that the guide RNA targeting the UL30 gene led to mutations in about 40-80% of the latent viral genomes. Although these mutations did not reduce the actual number of latent genomes, they did reduce their ability to reactivate.

During latency, HSV-1 and HSV-2 exist as circular chromosomes wrapped around cellular chromatin components called nucleosomes. This temporary association with nucleosomes implies that portions of the latent viral DNA are tightly folded and inaccessible to guide RNAs. Because the UL30 target site was consistently cleaved so efficiently, the authors speculate that this site may be in an open portion of the viral DNA that is more accessible to guide RNAs than other sites in the viral genome. If this is true, future guide RNA design strategies could include sequencing latent genomes using methods that identify open or accessible DNA.

Previous attempts to eliminate and/or prevent the reactivation of latent HSV virus in infected cells have had limited success. This study provides the first evidence that CRISPR/Cas9 can efficiently edit latent HSV genomes. Other studies are underway to determine whether CRISPR/Cas9 can edit the HSV genome during latent infection in the resting sensory neuron host cell and other in vivo models. Although more work is needed to figure out how to deliver Cas9 and guide RNAs to latently infected sensory or other neurons in vivo, the therapeutic potential of CRISPR/Cas9 in the context of HSV latency is encouraging, particularly when considered in combination with other existing therapies.

TWiV 579: Reno viral

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Vincent speaks with virologists at the University of Nevada at Reno about their careers and their work on herpesviruses, arboviruses, and the development of diagnostics for infectious diseases.

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Show notes at microbe.tv/twiv