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An Appreciation for Viruses

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by Gertrud U. Rey

EV-D68

Most people associate viruses with illness and suffering. After all, the word “virus” is derived from the Latin word for “poison.” However, considering that the vast majority of viruses cause no illness and are actually beneficial to humans and the planet as a whole, this sentiment is largely misplaced. Let me explain.

The ability of viruses to enter cells by attaching to host cell receptors and releasing their genome into the cell can be exploited for various purposes. For example, viruses can be used as vectors for delivering vaccines, healthy copies of defective genes (i.e., for “gene therapy”), and therapeutic drugs to specific cells.

Several SARS-CoV-2 vaccines, including those made by AstraZeneca and Johnson & Johnson, consist of a “vector virus” (an adenovirus) that contains a gene for the SARS-CoV-2 spike protein. Upon injection into a vaccine recipient, the vector virus should enter cells and serve as a code for host proteins to synthesize the encoded spike protein. Genes that regulate replication of the vector virus are removed to ensure that the vector itself cannot cause an infection in human cells. Other genes not needed for purposes of vaccine delivery are also typically removed to create more room inside the vector for the inserted antigen gene. Adenoviruses are particularly suitable for delivering foreign genes into cells because they have a double-stranded DNA genome that can accommodate segments of foreign DNA and because they infect most cell types without integrating into the host genome. However, poxviruses, retroviruses, vesicular stomatitis virus, and other viruses can also be used for vaccine delivery. As of today, six viral-vectored vaccines have been authorized for use in humans: four SARS-CoV-2 vaccines (two of which were previously described here and here) and two Ebola virus vaccines.

Viruses may also serve as vectors for targeted gene therapy to treat genetic disorders caused by mutations in the sequence of a person’s DNA. By replacing the mutated, non-functional portion of DNA with its healthy counterpart, the function of the defective gene could potentially be restored. Some viruses, like retroviruses, already insert their genetic material into the host genome as part of their replication cycle, making them suitable for delivering such functional genes to target cells. Recent advances in technology may even allow for the delivery of CRISPR-mediated gene editing tools to edit the target genome in the cell by excising the defective gene and replacing it with a functional version. One such targeted therapy aimed at treating genetic muscle disease by specifically targeting muscle cells was recently discussed on TWiV 812. Another exemplary gene therapy method for potentially deleting integrated HIV-1 from the genomes of infected individuals using CRISPR technology was described in a previous post.

A similar vector approach can also be used for cell-specific delivery of therapeutic drugs. For example, replication-incompetent viruses (viruses that have been engineered so they can’t replicate) can be further modified. These modifications may allow the viruses to specifically target dividing tumor cells or cells that display surface proteins that are unique to cancer cells, and deliver chemotherapeutic drugs only to those cells. Alternatively, replication-competent viruses can be manipulated to directly target and kill cancer cells in a mechanism known as oncolytic virotherapy. An example of this mechanism described previously involves a herpes simplex virus engineered to target a receptor that is practically absent in healthy brain cells, but is specifically expressed on glioblastoma multiforme tumor cells. The engineered virus also encodes a gene for a cytokine that increases the effectiveness of oncolytic viruses by recruiting cytotoxic T lymphocytes, which cause the tumor cells to burst. An accumulating body of evidence suggests that the cancer-specific antigens that emerge from burst cancer cells may also trigger additional downstream immune responses, further enhancing the potency of oncolytic viruses.

Considering that we are on the brink of a major antibiotic resistance crisis, viruses may just come to our rescue in this regard as well. Bacteriophages (“phages” for short) are viruses that only infect bacteria, and as it turns out, they can be used to treat pathogenic bacterial infections. There are numerous potential advantages to phage therapy compared to traditional antibiotic therapy. Phages are equally effective against antibiotic-sensitive and antibiotic-resistant bacteria. They are also more specific than antibiotics, and this specificity leads to reduced impact on commensal bacteria, which are typically obliterated by conventional antibiotics. Unlike most antibiotics, phages are capable of disrupting bacterial biofilms, and their use would lead to reduced incidence of opportunistic infections and reduced toxic effects of bacterial infection. Although bacteria can become resistant to phages, phages can likewise evolve to overcome this resistance, making bacterial resistance to phages less of a challenge than their resistance to antibiotics. Furthermore, scientists have found that the efficacy of phage therapy can be improved by combining phages with an antibiotic treatment regimen, or by combining several phages in a “phage cocktail.” In a highly publicized phage therapy success story, infectious disease epidemiologist Steffanie Strathdee describes how she recruited the help of an international team of physicians to cure her husband of a life-threatening multi-drug-resistant Acinetobacter baumanii infection using an intravenous phage therapy cocktail.

Phages can also be used as an alternative energy source by powering the electrodes in batteries. As repeatedly demonstrated by materials scientist Angela Belcher at MIT, biological scaffolds composed of M13 phages that display the negatively charged peptide sequence glutamate-glutamate-alanine-glutamate (E-E-A-E) inevitably attract nickel phosphide molecules, and the resulting nanostructures can be used directly as freestanding negative electrodes in batteries. These “virus batteries” have multiple advantages over traditional batteries. They are more environmentally friendly because they’re made from non-toxic materials. Their synthesis requires relatively little equipment, so they are inexpensive to produce. They are lightweight and flexible and can thus be woven into fabrics, which makes them suitable for military clothing. They also have higher conductivity than conventional lithium-ion batteries, making them extremely useful for portable electronics, medical implants, and various aerospace applications. It is even possible that they could one day be used to power electric cars.

The examples described so far are ones in which people have capitalized on virus functions for the benefit of humans. However, viruses have other benefits that just relate to their natural functions. For instance, phages are also an essential component of our environment, where they help control pests and recycle nutrients. If phages didn’t exist, some bacterial populations would explode and outcompete other populations, causing them to disappear completely. This imbalance would be especially disastrous in the oceans, where microbes make up more than 70% of the total biomass. Phages kill a large portion of oceanic bacteria every day, allowing the organic molecules released from the dead bacterial cells to be recycled as nutrients for other organisms. Perhaps the most important organisms to benefit from these recycled nutrients are microscopic plants called phytoplankton, which produce oxygen by removing carbon dioxide from the atmosphere. In fact, phytoplankton are a crucial element of the global carbon cycle and one of the largest contributors to our atmospheric oxygen. This means that without viruses, we would not have air to breathe.

Viruses are deeply integrated in life on earth, and their functions in sustaining environmental equilibrium and our ongoing survival are too numerous to describe in a single blog post. Moreover, our current appreciation of what can be accomplished using viruses is cursory, at best. Future research will lead to a deeper understanding of how viruses can be utilized to do more good.

[This post was written in honor of Virus Appreciation Day, which occurs annually on October 3]

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.

Phi Kappa Zeta Builds a Panic Room

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Phikzlikevirus_virionby Gertrud U. Rey

Bacteria use at least a couple of pathways for protecting themselves against viral infection. In return, multiple viral strategies for inhibiting or evading these pathways have evolved.

[Read more…] about Phi Kappa Zeta Builds a Panic Room

TWiV 437: Kathy’s new spindle virus

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The TWiVsters reveal new giant viruses that argue against a fourth domain of life, and discovery of viruses in the oceanic basement.

You can find TWiV #437 at microbe.tv/twiv, or listen below.

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TWiV 433: Poops viruses and worms

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The lovely TWiV team explore evolution of our fecal virome, and the antiviral RNA interference response in the nematode C. elegans.

You can find TWiV #433 at microbe.tv/twiv, or listen below.

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Do giant viruses have a CRISPR-like immune system or a protein restriction factor?

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Zamilon virophageA battle is brewing between two research groups in Marseille, France that are involved in the discovery and study of giant viruses. Didier Raoul and colleagues believe that they have discovered a CRISPR-like, DNA based defense system in mimivirus that confers resistance to virophage (paper link). Claverie and Abergel disagree: they think that the defense system involves proteins, not nucleic acids (paper link).

Virophages are DNA viruses that can only replicate in cells infected by giant viruses like mimivirus. Their name, which means ‘virus eater’, comes from the observation that they inhibit mimivirus replication. A specific virophage called Zamilon was discovered that can inhibit the replication of lineage B and C mimivirus but not lineage A.

Examination of the DNA sequences of 60 different mimivirus strains revealed that the genomes of lineage A contained a 28 nucleotide sequence identical to Zamilon virophage. This sequence was not found in any lineage B mimivirus and in only one out of 19 lineage C mimiviruses. In addition, a 15 nucleotide subset of this sequence is repeated four times in the lineage B and C mimivirus genomes.

Near the 15 nucleotide Zamilon-derived repeated sequences in lineage B and C mimivirus genomes are genes encoding several proteins related to components of the bacterial CRISPR-Cas system. These include a nuclease, an RNAse, and an ATP-dependent DNA helicase.

The CRISPR system provides defense against invading DNA. When a foreign DNA, such as a bacteriophage genome, enters a bacterial cell, some is fragmented and integrated into the CRISPR locus as a ’spacer’ (sequences in the foreign DNA are called ‘protospacers’). Following transcription, CRISPR RNAs (crRNA) are processed by a multiprotein complex to produce ~60 nucleotide RNAs. When the spacer of a crRNA base pairs with a complementary sequence in an invading DNA molecule, CRISPR-associated endonucleases cleave the DNA. The integration of the sequences of the invading DNA into the host cell genome, from which they can be mobilized in the form of crRNAs, provides a form of “memory” and acquired immunity. It should be noted that there are six known types of CRISPR systems that differ in their components and mechanisms.

Because the CRISPR-Cas system is an adaptive immune system that protects bacteria and Archaea from virus infections and invasion of foreign DNA, the authors propose that they have discovered a new adaptive immune system that protects mimiviruses from virophage infection. They call this system mimivirus virophage resistance element, or MIMIVIRE.

The authors provide experimental support for their hypothesis by showing that silencing the genes encoding the endonuclease, the helicase, and the repeated insert using siRNA allows Zamilon replication in mimivirus-infected cells.

Claverie and Abergel think that Raoult and colleagues are wrong (paper link). They provide three reasons to dispute their findings, and ‘propose a simpler protein-based interaction model that explains the observed phenomena without having to extend the realm of adaptive immunity to the world of eukaryotic viruses, a revolutionary step that would require stronger experimental evidences.’

The first problem is that mimivirus and Zamilon virophage replicate in the same location in the infected cell, making a CRISPR-like defense system difficult to conceptualize. In contrast, CRISPR sequences reside in the bacterial genome, from which RNAs are produced that target the destruction of invading DNAs elsewhere in the cell.

The second problem is that the Zamilon sequences in the mimivirus genome are not regularly spaced or flanked by recognizable repeats, a hallmark of the CRISPR system (the name stands for ‘clustered regularly interspersed short palindromic repeats). However it should be noted that type VI CRISPR systems have no CRISPR locus and likely function via mechanisms that are different from other CRISPR systems.

Finally, Claverie and Abergel argue that there is no way for the proposed nucleic acid defense system to distinguish between the virophage and the virophage sequences in the mimivirus genome. In some CRISPR systems this discrimination is achieved by protospacer adjacent motifs (PAMs), short (2-5 nt) sequences next to the invader protospacer sequences that are recognized by the endonuclease complex guided by the crRNA. PAMs are not present in the bacterial genome, sparing it from endonucleolytic cleavage. Nevertheless, non PAM-based mechanisms of discriminating invader from host are known, for example, in the type III CRISPR system.

If Raoult and colleagues have not discovered a CRISPR-like mimivirus defense system, then why would silencing the genes encoding CRISPR-like proteins allow Zamilon replication? Claverie and Abergel think that it is not the 15 nucleotide Zamilon repeats that are important to mimivirus, but the encoded amino acids: Asp-Asn-Glu-Ser (DNES in one letter code). They believe that DNES is a motif present in proteins that block Zamilon replication by as yet unidentified mechanisms.

Many cellular proteins have been identified that interfere with virus replication, such as those encoded by interferon induced genes (ISGs). DNES containing proteins that inhibit Zamilon replication would be conceptually analogous, except that they are encoded by a virus, not the host.

Claverie and Abergele appear to have a strong case that mimivirus defense against Zamilon virophage is mediated by protein, not nucleic acid, but further experimentation is certainly needed to support their position. Nevertheless they recognize that the discovery by Raoult and colleagues “remains fascinating even if it falls short of demonstrating the existence of a CRISPR-Cas-like adaptive immune system”.