Covid Builds a Fortress Within

 Viral proteins build peculiar vesicles to hide the viral replication apparatus.

SARS-CoV is still with us, a brutal addition to the already extensive army of respiratory viruses infecting humanity. While most people clear it, we have a hard time doing so, a testament to a tough evolutionary arms race. A fair portion of our extremely complicated immune system is devoted to viruses, including basics like recognizing double-stranded RNA and viral replication structures. A trick that coronaviruses and allied species possess has gradually come to light, which is the formation of vesicular structures that appear to host their replication apparatus. 

Coronavirus-infected cells display a variety of vesicular structures, including "zippered" endoplasmic reticulum, convoluted membranes (CV), dense membrane spherules (DMS) and double-membrane vesicles (DMV). The endoplasmic reticulum (ER) is the cellular organelle where membrane proteins and secreted proteins are first made, before they are sorted out to various other membrane systems and the outside (and where the bulk of membrane lipid production happens, among much else). Coronaviruses appear to commandeer the ER and divert its membranes to the new structures. It is the DMV that turns out to have an important function- hosting viral replication. How do we know this? Researchers recently turned to a classic technique- radioactive labeling of new RNA production in infected cells, followed by electron microscopy combined with auto-radiography. The image below shows in stunning detail various organelles within an infected cell, and the black dots are film grains turned by the radioactive RNA to mark synthesis sites. They are quite closely aligned with the DMV structures.

Exquisite auto-radiograph and electron micrograph of a SARS-CoV-infected cell. The mitochondria (m) are most apparent, followed by the viral replication organelles (RO, aka DMV), followed by the endoplasmic reticulum (ER), lipid droplets (LD), nucleus (N), and virion-containing region (VCR). The black dots from photo-sensitive film exposed by radioactive RNA is clustered around the DMV structures.

This finding leads to several questions. How do these structures form? And, given the need for replication to both get inputs such as nucleotides and to export outputs like the virus's genomic RNAs, why use membranes that are impermeable to such molecules? Why use two membranes, when one suffices for most cellular organelles like the ER, lysosomes, peroxisomes, etc? This had puzzled the field for some time. Now, it turns out (in another recent paper) a couple of powerful viral proteins solve both questions at once. Coronavirus products nsp3 and nsp4 have long been known as important for viral success, but recent work puts them at the heart of DMV formation, into what is now called a replication organelle (RO), as well as a DMV. They are expressed in the ER and seem to play the leading role (along with several host proteins and lipids) in curving its membranes into the DMV shape. They also form dimeric pairs (nsp3 on one membrane, and nsp4 on a facing membrane) that seal two membranes together, as seen in the DMV structure. And thirdly, they, once fully assembled and mated, form a pore which keeps out pretty much everything big, but lets through single stranded RNA and small molecules.

Structure determination of the multimeric nsp3/4 pore structure from purified DMV vesicles, several views. Note the tight pore going through the center, and differential sizes of the inner and outer membrane rings. It is a protein complex that both bends the membrane and keeps only the most essential traffic going through it.

This structure is beautiful in a way. The central pore, at about 1.5 nm, is lined with positive charges like lysine and asparagine, the better to conduct negatively charged RNA. The inner membrane structure is tighter than that of the outer membrane, the better to curve those membranes into the observed spherical size. While it is a little hard to believe that such DMV vesicles, even studded with such a bespoke pore, can conduct the kind of traffic, both in and out, needed to sustain high rates of viral replication, that is quite evidently how it works. These researchers make a few mutations in the newly revealed key positively charged central pore amino acids to show that, if those charges are lost, replication of the virus was "abolished". This creates an obvious drug target as well- some chemical that plugs this pore or otherwise blocks the assembly of this ornate structure.

Additionally, the assembly of all this out of flat ER was also studied. The nsp3/4 proteins are originally connected end-to-end and do a delicate dance of pulling on each other (after cleavage) to dramatically curve the membrane between them, forming a tight loop from the (future) outside DMV membrane to the (future) inside one. On the other hand, another way they can assemble (right side in diagram below) is from separated (ER) membranes, leading to the "zippered" ER conformation that is also seen in infected cells. Whether the latter can be transformed into the former remains a question. 

Models for assembly of the linked nsp3-nsp4 proteins into the curved membranes of the DMV pore, with super-curvature at the pore junction between outside and inside membranes. TM stands for transmembrane domain, NTD for N-terminal domain (front), CTD for C-terminal domain (rear), and Ecto for the ecto-domains of each protein that are not within the membrane.

It is naturally implicit in this work that, if the pores of nsp3/4 allow through the absolute essentials of viral replication, they also block the various cellular sensors of viral presence, such as the RIG and TLR proteins, thus delaying the host response. Perhaps the RNAs allowed out are modified prior to exit to make them look more host-like. All those assumptions have yet to be nailed down explicitly. At any rate, viral assembly takes place elsewhere, so it is not entirely clear yet what exactly is being hidden here.

There were some technical innovations along the way to these results. These researchers tagged the nsp proteins in a way that allowed them to easily purify DMV vesicles out of whole cells, speeding their cry-electron microscopy work of getting these structures. Did they just use the Alpha fold program and do all this the easy way? Not at all. They did use Alpha fold to refine some of the structures, to extract more atomic detail. But they notably did not trust the AI to cook this kind of finding up from scratch. Some things still need to be done empirically, if you really want the truth.

• How much good do social programs do?
• Fire sale in Ukraine.
• Leave that soil be!

Making America Great: First Quarter Report Card

Are we great yet? I give some grades.

Enhancing the rule of law, and adhering to the constitution: F

This administration is characterized by contempt. A juvenile contempt for its enemies, and thorough contempt for the law, separation of powers, and the constitution. In asserting its royal prerogative to eviscerate legislatively created agencies, it is taking more power from congress, as if congress weren't sufficiently neutered and ineffective already. We are watching a replay of the transition of Rome from a republic to a monarchy, though in much more ham-fisted fashion, as its senate was sidelined. So far, the Republicans in congress do not see the danger, as they cheer on the mayhem. But it will come for them more directly in due time, maybe in Trump's third term, as he grooms Eric to be next in line.

Economic growth: F

The markets have given their verdict, which is thumbs down. The trade war this administration has started, in royal fashion, is bad for us and bad for everyone else. Even putting aside the short-term insanity, the long-term implications are lower living standards and lower growth. To take one example, what is going to happen if people in the US are effectively confined to buying US-built cars? We will be going back to the 60's and 70's, when cars were poorly built, and the captive market meant that US car makers did not have to innovate. We should focus on strategic industries, to preserve base-line capacity to build things, but otherwise let foreign trade work its economic magic.

Peace on earth: F

The new administration is siding with aggressors all over the world now, especially Russia and Israel. China is the only exception, though its support for Taiwan is quite a bit more tepid than that of the last administration. Siding with aggressors is a recipe for more war. More broadly, the US has lost its moral high ground, such as it was, and is losing friends at a rapid clip. I mean, how can one alienate Canada? That really takes some serious stupidity. Trump was angling for a Nobel Peace Prize, by ending the Ukraine war. But predictably, Putin plays him for the fool he is, and keeps on doing what he wants to. The instability and madness of the current administration is another factor all by itself, leading to international instability and higher risk for war, not to mention driving countries around the world into the arms of the truly stable genius... China.

Education and innovation: F

Of all the things that make our country great, it is education that has the greatest long-term implications. That is where the human capital comes from, and the technological innovation. We can grant that Republicans rely on less educated voters, so logic dictates that they make voters less educated. And that is what this administration has been doing with determination, eviscerating the department of education, cancelling and slashing funding for research, and ultimately promoting the destruction of public schools, through vouchers and other long-standing hobbyhorses of the right. This may make a country more amenable to royal rule, but is unlikely to make the US anything other than a diminished and declining power with lower living standards, less attractive to foreign students and foreign investment. China will shortly be the leading nation in high-level scholarly research.

Health and Safety, Pro-worker Policy: F

Here as well, the administration has spoken loudly through its actions and appointments. Putting an anti-science vaccine denier in charge of HHS, and slashing personnel throughout the health agencies, and OSHA, and immediately kneecapping the labor relations board. Medicaid is slated to lose a trillion dollars, in favor of tax cuts for the rich. It all says that business and the rich are the true constituency of this administration, not people, let alone workers. Workers, indeed, are the evident enemy. How different this is from the campaign rhetoric! But that is how grifters work. And they will be gone before the real costs sink in.

Safeguarding democracy: F

Honestly, is this even a subject?

Culture and style: F

White Potus. Also, the Zelensky meeting. 

Drain the swamp: F

The inauguration set the tone, as Trump introduced an eponymous meme-coin, which his friends and insiders stocked up on before the public offering, in a naked pump and dump, even if the dump part of the operation has been delayed. Much bigger, however, is the tariff-palooza, which has the world "kissing my ass". No possibility of corruption there! If there were a lower grade than F, it would be awarded here.

Clear and elevated rhetoric: F

Again, the Zelensky meeting. I recently watched a documentary series on John F. Kennedy, which demonstrated that one needn't go back to the 19th century to encounter well-written, coherent, and civil political discourse in America. While admitting that the Biden administration was hardly a high point of forceful communication, at least it was civil- domestically and to our friends and partners abroad. Trump and his toadies compete for juvenile putdowns, unthinking meanness, and large helpings of lies. When actual policy is needed, elliptical "the weave" expressions clear the field of coherent thought, to make room for more chaos and cons.

OK, other than in these areas, things are going great. If your metric is owning the libs, destroying the government, giving away the store to the rich and to Russia, and having people line up to kiss the president's ass, then everything is going very well. 

It is important to understand that, generally speaking, the government exists to protect people from each other, especially protect the little people. The rich can take care of themselves, at least until things get really bad. It is the little people who need the Bill of Rights, the consumer protection bureau, the SEC, the FDA, the VA, OSHA, and all the other regulatory agencies that keep the rapacious wolves of capitalism on their leashes. Everyone benefits from civil service protections, transparency, rules, and law. But the little people benefit the most, because they are beset the worst in the capitalist system. All men are created equal, but not really. The insanity of giving up our government to the people with the most money is truly astounding, and we are seeing the fruits daily.

• Gary Kasparov gives some advice.
• Law, schmaw.
• The three-toed sloth posits AI is not intelligent, but another cultural technology, maybe a regurgitation machine. Or a feral card catalog. But does it help us think better?
• The barriers to knowing thyself.
• Making China great again.
• Gosh, if RFK wanted to get to the bottom of autism, did he attend this talk downstairs?

The Genome Remains Murky

A brilliant case study identifying the molecular cause of certain neuro-developmental disorders shows how difficult genome-based diagnoses remain.

Molecular medicine is increasingly effective in assessing both hereditary syndromes and cancers. The sequencing approach generally comes in two flavors- whole genome sequencing, or exome sequencing, where only the most important (protein-coding) parts are sampled. In each case, the hunt is for mutations (more blandly called variants) that cause the syndrome being investigated, from among the large number of variants we all carry. This approach is becoming standard-of-care in oncology, due to tremendous influence and clinical significance of cancer-driving mutations, many of which now match directly to tailored treatments that address them (thus the "precision" in precision medicine).

But another arm of precision medicine is the hunt for causes of congenital problems. There are innumerable genetic disorders whose causal analysis can lead not only to an informative diagnosis, and sometimes to useful treatments, but also to fundamental understanding of human biology. Sufferers of these syndromes may spend a lifetime searching for a diagnosis, being shuffled from one doctor or center to another and subject to various forms of hypothetical medicine, before some deep sequencing pinpoints the cause of their disease and founds a new diagnostic category that provides, if not relief, at least understanding and a medical home. 

A recent paper from Britain provided a classic of this form, investigating the causes of neurodevelopmental (NDD) disorders, which encompass a huge range of problems from mild to severe. They comment that even after the most modern analysis and intensive sequencing, 60% of NDD cases still can not be assigned causes. A large part of the problem is that, despite knowing the full sequence of the human genome, its function is less well-understood. The protein-coding genes (20,000 of those, roughly) are delineated and studied pretty closely. But that only accounts for 1 to 2% of the genome. The rest ranges from genes for a blizzard of non-coding RNAs, some of which are critical, to large regulatory regions with smatterings of important sites, to junk of various kinds- pseudogenes, relic retroviruses, repetitive elements, etc. The importance of any of these elements (and individual DNA base positions within them) varies tremendously. This means specifically that exome sequencing is not going to cut it. Exome sequencing focuses on a very small part of the genome, which is fine if your syndrome (such as a common cancer) is well characterized and known to arise from the usual suspects. But for orphan syndromes, it does not cast a wide enough net. Secondly, even with full genome sequencing, so little is known about the remoter regions of the genome that assigning a function to variations found there is difficult to impossible. It takes statistical analysis of incidence of the variation vs the incidence of the syndrome.

These authors used a trove of data- the Genomics England 100,000 genomes project, focusing on the ~9,000 genomes in this collection from people with NDD syndromes. (Plus additional genomes collected elsewhere.) (We can note in passing that Britain's nationalized health system remains at the forefront of innovative research and care.) What they found was an unusually high incidence of a particular mutation in a non-protein-coding gene called RNU4-2. The product of this gene is an RNA called U4, which is an important part of the spliceosome, where it pairs RNA-to-RNA with another RNA, U6, in a key step of selecting the first (5-prime) side of an intron that is to be spliced out of mRNA messages. This gene would never have come up in exome analysis, being non-protein-coding. Yet it is critically important, as splicing happens to the vast majority of human genes. Additionally, differential splicing- the selection of alternative exons and splice sites in a regulated way- happens frequently in developmental programs and neurological cell types. There is a class of syndromes called spliceosomopathies that are caused by defects in mRNA splicing, and tend to appear as syndromes in these processes.

As shown in the images (all based on a large corpus of other work on spliceosomes), RNU4-2/U4 pairs intimately with the U6 spliceosomal RNA, and the mutation found by the current group (which is a single nucleotide insertion) causes a bulge in this pairing, as marked. Meanwhile, the U6 RNA pairs at the same time with the exon-intron junction of the target mRNA (bottom image), at a site that is very close to the U4 pairing region (top image). The upshot is that this single base insertion into U4 causes some portion of the target mRNAs to be mis-spliced, using non-natural 5 prime splice sites and thus altering their encoded proteins. This may cause minor problems in the protein, but more often will cause a shift in translation frame, a premature stop codon, and total loss of the functional protein. So this tiny mutation can have severe effects and is indeed genetically dominant- that is, one copy overrides a second wild-type copy to generate the NDD diseases that were studied.

The U4 RNA (teal) paired with the U6 RNA (gray), within an early spliceosome complex. The mutation studied here is pointed out in black (n.64_65insT - i.e. insertion of a T). Note how it would cause a bulge in the pairing. Importantly, the location in the U6 RNA that pairs with the mRNA (see below) is right next door, at the ACAGAGA (light gray). The authors use this structural work from others to suggest how the mutation they found can alter selected splicing sites and thus lead to disease. Other single nucleotide insertions that cause similar syndromes are marked with black arrows, while single nucleotide substitutions that cause less severe syndromes are marked with orange RNA segments.

The U6 RNA (pink) paired with its mRNA target to be spliced. It binds right at the intron (gray) exon (black) boundary, where the cut will eventually be made the remove the intron. The bump from the mis-paired mutant U4 RNA (see above) distorts this binding, sending U6 to select wrong locations for spicing.

The researchers went on to survey this and other spliceosomal RNA genes for similar mutations, and found few to none outside the region marked in the diagram above. For example, there is a highly similar gene called RNU4-1. But this gene is expressed about 100-fold less in brain and other tissues, making RNU4-2 the principal source of U4 RNA, and much more significant as a causal factor for NDD. It appears that other locations in RNU4-2 (and other spliceosomal RNA genes) are even more important than the one mutated location found here, thus are never found, being lethal and heavily selected against, in this highly conserved gene. 

They also noted that, while this RNU4-2 mutation is severe, and thus must happen spontaneously (i.e. not inherited from parents), it only occurrs on the maternal alleles, not paternal alleles in the affected children. They speculate that this may be due to effects this gene may have in male gametogenesis, killing affected sperm preferentially, but not affected oocytes. Lastly, this set of mutations (in the small region shown in the first figure above) appears to account for, in their estimation, about 0.4 % of all NDD seen in Britain. This is a remarkably high rate for such a particular mutation that is not heritable. They speculate that some mutation hotspot kind of process may be causing these events, above the general mutation rate. What this all says about so-called "intelligent design", one may be reluctant to explore too deeply. On the other hand, this still leaves plenty of room to hunt for additional variations that cause these syndromes.

In this research, we see that clinically critical variations can pop up in many places, not just among the "usual suspects", genetically and genomically speaking. While much of the human genome is junk, most of it is also expressed (as RNA) and all of it is fair game for clinically important (if tragic) effects. The NDD syndromes caused by the mutation studied here are very severe- for more so than the ADD or mild autism diagnoses that make up most of the NDD spectrum. Understanding the causal nexus between the genome and human biology and its pathologies, remains an ongoing and complicated scientific adventure.

• Playing the heel. Being the heel. 
• It sure is great to be the victim.
• Oh, right.. now we really know what is going on.
• More spiritual warfare.
• Another grift.

Psilocybin and the Normie Network

Psychedelic mushrooms desynchronize parts of the brain, especially the default mode network, disrupting our normal sense of reality.

Default mode network- sounds rather dull, doesn't it? But somewhere within that pattern of brain activity lies at least some of our consciousness. It is the hum of the brain while we are resting without focus. When we are intensely focused on something outside, in contrast, it is turned down, as we "lose ourselves" in the flow of other activities. This network remains active during light sleep and has altered patterns during REM sleep and dreaming, as one might expect. A recent paper tracked its behavior during exposure to the psychedelic drug psilocybin.

These researchers measured the level of active connectivity between brain regions (by functional MRI) on human subjects given a high dose of psilocybin, or Ritalin- which stands in as another psychoactive stimulant- or nothing. Compared with the control of no treatment, Ritalin caused a slight loss of connectivity (or desynchronization), (below, b), while psilocybin (a) caused huge loss of connectivity, clearly correlated with the subjective intensity of the trip. They also found that if, while on psilocybin, they gave their subjects some task to focus on, their connectivity increased again, again tracking directly with the subjective experiences of their subjects. 

The researchers show a metric of connectivity between distinct brain regions, under three conditions. FC stands for functional connectivity, where high numbers (and brighter colors) stand for more distance, i.e. less connectivity/synchrony. Methylphenidate is Ritalin. Synchrony is heavily degraded under psilocybin.

Of all the networks they analyzed, the default mode network (DNM) was most affected. This network runs between the prefrontal cortex, the posterior cingulate cortex, the hippocampus, the angular gyrus, and temporoparietal junction, among others. These are key areas for awareness, memory, time, social relations, and much else that is highly relevant to conscious awareness and personhood. There is a long thread of work in the same vein that shows that psychedelic drugs have these highly correlated effects on subjective experience and brain patterns. A recent review suggested that while subnetworks like the DNM are weakened, the brain as a whole experiences higher synchrony as well as higher receptivity to outside influence in an edgy process that increases its level of (to put it in terms of complexity theory) chaos or criticality. 

So that is what is happening! But that is not all. The effects of pychedelics, even from one dose, can be long-lasting, even life-changing. The current researchers note that the DMN desynchronization they see persists, at weaker levels, for weeks. This correlates with the subjective experience of changed senses that can result from a significant drug trip. And that trip, as noted above regarding receptivity and chaos, is a delicate thing, highly subject to the environment and mood the subject is experiencing at the time. 

But when a task is being done, the subjects come back down towards normalcy.

These researchers note that brain plasticity comes into play, evidently as a homeostatic response to the wildly novel patterns of brain activation that took the subject out of their rut of DMN self-hood. Synapses change, genes are activated, and the brain reshapes usually in a way that increases receptivity to novel experiences and lifts mood. For example, depression correlates with strong DMN coherence, while successful treatment correlates with less connectivity.

"We propose that psychedelics induce a mode of brain function that is more dynamically flexible, diverse, integrated, and tuned for information sharing, consistent with greater criticality."

So, consider the mind blown. Psychedelics appear to disrupt the usual day-to-day, which is apparently a strongly positive thing to do, both subjectively and clinically. That raises the question of why. Why do we settle into such durable and often negative images of the self and ways of thinking? And why does shaking things up have such positive effects? Jung had a deep conviction that our brains are healing machines, with deep wisdom and powers that are exposed during unusual events, like intense dreams. While there are bad trips, and people who go over the edge from excessive psychedelic use, with a modicum of care, it appears that positive trips, taken in a mood of physical and social safety, let the mind reset in important ways, back to its natural open-ness.

• Lying, as usual.
• PFAS in the water.
• Miracles? .. and the life of Jesus.