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I have heard that the Coronavirus family have a proofreading and editing function in their polymerase enzymes which can recognize and excise mutations. This is obviously disastrous for the population of infected individuals. Is this true, and if so, would it be possible to knock out the proofreading and editing subunit selectively so that the mutational burden would progressively increase in the viral population ? Could this then lead to something like Muller's Ratchet and dampen the virulence in the population or would there still be a mechanism to remove the mutated viruses from the population and retain the virulence over time?
Many in the scientific community have mobilized to understand the virus that is causing the global coronavirus disease 2019 (COVID-19) pandemic. Gao et al. focused on a complex that plays a key role in the replication and transcription cycle of the virus. They used cryo–electron microscopy to determine a 2.9-angstrom-resolution structure of the RNA-dependent RNA polymerase nsp12, which catalyzes the synthesis of viral RNA, in complex with two cofactors, nsp7 and nsp8. nsp12 is a target for nucleotide analog antiviral inhibitors such as remdesivir, and the structure may provide a basis for designing new antiviral therapeutics.
A novel coronavirus [severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2)] outbreak has caused a global coronavirus disease 2019 (COVID-19) pandemic, resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase [(RdRp), also named nsp12] is the central component of coronaviral replication and transcription machinery, and it appears to be a primary target for the antiviral drug remdesivir. We report the cryo–electron microscopy structure of COVID-19 virus full-length nsp12 in complex with cofactors nsp7 and nsp8 at 2.9-angstrom resolution. In addition to the conserved architecture of the polymerase core of the viral polymerase family, nsp12 possesses a newly identified β-hairpin domain at its N terminus. A comparative analysis model shows how remdesivir binds to this polymerase. The structure provides a basis for the design of new antiviral therapeutics that target viral RdRp.
Coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus [severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2)] that emerged in December 2019 (1–3) and has since become a global pandemic. COVID-19 virus is reported to be a new member of the betacoronavirus genus and is closely related to severe acute respiratory syndrome–coronavirus (SARS-CoV) and several bat coronaviruses (4). Compared with SARS-CoV and Middle East respiratory syndrome–coronavirus (MERS-CoV), COVID-19 virus exhibits faster human-to-human transmission, which lead the World Health Organization to declare a worldwide public health emergency (1, 2).
Coronaviruses (CoVs) employ a multisubunit machinery for replication and transcription. A set of nonstructural proteins (nsps) produced as cleavage products of the ORF1a and ORF1ab viral polyproteins (5) assembles to facilitate viral replication and transcription. A key component, the RNA-dependent RNA polymerase [(RdRp), also known as nsp12], catalyzes the synthesis of viral RNA and thus plays a central role in the replication and transcription cycle of COVID-19 virus, possibly with the assistance of nsp7 and nsp8 as cofactors (6). Therefore, nsp12 is considered a primary target for nucleotide analog antiviral inhibitors such as remdesivir, which shows potential for the treatment of COVID-19 viral infections (7, 8). To inform drug design, we determined the structure of nsp12, in complex with its cofactors nsp7 and nsp8, by cryo–electron microscopy (cryo-EM) using two different protocols: one in the absence of dithiothreitol (DTT) (dataset 1) and the other in the presence of DTT (dataset 2).
The bacterially expressed full-length COVID-19 virus nsp12 (residues S1 to Q932) was incubated with nsp7 (residues S1 to Q83) and nsp8 (residues A1 to Q198), and the complex was then purified (fig. S1). Cryo-EM grids were prepared using this complex, and preliminary screening revealed excellent particle density with good dispersion. After the collection and processing of 7994 micrograph movies, we obtained a 2.9-Å resolution three-dimensional reconstruction of an nsp12 monomer in complex with one nsp7-nsp8 pair and an nsp8 monomer, as was previously observed for SARS-CoV (9). In addition to the nsp12-nsp7-nsp8 complex, we also observed single-particle classes corresponding to the nsp12-nsp8 dimer, as well as individual nsp12 monomers, but these do not produce atomic-resolution reconstructions (fig. S2). However, the nsp12-nsp7-nsp8 complex reconstruction provides the structural information for complete structural analysis.
The structure of the COVID-19 virus nsp12 contains a right-hand RdRp domain (residues S367 to F920) and a nidovirus-specific N-terminal extension domain (residues D60 to R249) that adopts a nidovirus RdRp-associated nucleotidyltransferase (NiRAN) (10) architecture. The polymerase domain and NiRAN domain are connected by an interface domain (residues A250 to R365) (Fig. 1, A and B). An additional N-terminal β hairpin (residues D29 to K50), built with the guidance of an unambiguous cryo-EM map (fig. S3A), inserts into the groove clamped by the NiRAN domain and the palm subdomain in the RdRp domain (Fig. 2). The nsp7-nsp8 pair shows a conserved structure similar to that of the SARS-CoV nsp7-nsp8 pair (9, 11). The orientation of the N-terminal helix of the separate nsp8 monomer bound to nsp12 is shifted compared with that in the nsp7-nsp8 pair (fig. S4A). The 13 additional amino acid residues resolved at the N-terminal of nsp8 show that the long shaft of its well-known golf club shape is bent (fig. S4B).
(A) Domain organization of COVID-19 virus nsp12. The interdomain borders are labeled with residue numbers. The N-terminal portion with no cryo-EM map density and the C-terminal residues that cannot be observed in the map are not included in the assignment. The polymerase motifs are colored as follows: motif A, yellow motif B, red motif C, green motif D, violet motif E, cyan motif F, blue and motif G, light brown. (B) Ribbon diagram of COVID-19 virus nsp12 polypeptide chain in three perpendicular views. Domains are colored the same as in (A). The individual nsp8 (nsp8-1) bound to nsp12 and that in the nsp7-nsp8 pair (nsp8-2) are shown in gray the nsp7 is in pink. The bottom left panel shows an overview of the cryo-EM reconstruction of the nsp12-nsp7-nsp8 complex.
(A) Overall structure of the N-terminal NiRAN domain and β hairpin of COVID-19 virus nsp12. The N-terminal NiRAN domain and β hairpin of COVID-19 virus nsp12 are shown as yellow and cyan cartoons, respectively, whereas the other regions of COVID-19 virus nsp12 are shown as a molecular surface with the same color scheme used in Fig. 1. The NiRAN domain of SARS-CoV nsp12 is superimposed to its counterpart in COVID-19 virus nsp12 and is shown in purple. (B) Key interactions between the β hairpin and other domains. The β hairpin is shown as a cyan tube with its key residues in stick mode. These have the closest contacts with other domains of COVID-19 virus nsp12. The interacting residues in the palm and fingers subdomain of the RdRp domain and the NiRAN domain are identified by the labels. Single-letter abbreviations for the amino acid residues are as follows: A, Ala C, Cys D, Asp E, Glu F, Phe G, Gly H, His I, Ile K, Lys L, Leu M, Met N, Asn P, Pro Q, Gln R, Arg S, Ser T, Thr V, Val W, Trp and Y, Tyr.
The overall architecture of the COVID-19 virus nsp12-nsp7-nsp8 complex is similar to that of SARS-CoV with a root mean square deviation (RMSD) value of 0.82 for 1078 Cɑ atoms (fig. S4C). However, there are key features that distinguish the two. The cryo-EM map allowed us to build the complete structure of COVID-19 virus nsp12, including all residues except S1 to D3 and G897 to D910. In contrast, the first 116 residues were not resolved in SARS-CoV nsp12 (9). The portion of the NiRAN domain resolved in SARS-CoV (residues 117 to 249) is composed of six helices with a three-stranded β sheet at the N terminus (9) (Fig. 2A). In the COVID-19 virus structure, we additionally resolved residues A4 to R118. These constitute a structural block with five antiparallel β strands and two helices. Residues N215 to D218 form a β strand in COVID-19 virus nsp12, whereas these residues are less ordered in SARS-CoV nsp12. This region makes contact with the strand that includes residues V96 to A100, thus contributing to the stabilization of its conformation. As a result, these four strands form a compact semi–β barrel architecture. Therefore, we identify residues A4 to T28 and Y69 to R249 as the complete coronaviral NiRAN domain. With the resolution of N-terminal residues, we are also able to identify an N-terminal β hairpin (D29 to K50 Figs. 1A and 2A). This β hairpin inserts into the groove clamped by the NiRAN domain and the palm subdomain in the RdRp domain and forms a set of close contacts to stabilize the overall structure (Fig. 2B and fig. S5). We have also observed C301 to C306 and C487 to C645 form disulfide bonds in the absence of DTT (dataset 1). However, in the presence of DTT (dataset 2), chelated zinc ions are present in the same location as that observed in SARS-CoV (fig. S3B).
The polymerase domain adopts the conserved architecture of the viral polymerase family (12) and is composed of three subdomains: a fingers subdomain (residues L366 to A581 and K621 to G679), a palm subdomain (residues T582 to P620 and T680 to Q815), and a thumb subdomain (residues H816 to E920) (Fig. 1). The catalytic metal ions, which are observed in several structures of viral polymerases that synthesize RNA (13, 14), are not observed in this work in the absence of primer-template RNA and nucleoside triphosphates (NTPs).
The active site of the COVID-19 virus RdRp domain is formed by the conserved polymerase motifs A to G in the palm domain and configured like other RNA polymerases (Figs. 1A and 3A and fig. S6). Motif A, composed of residues 611 to 626 (TPHLMGWDYPKCDRAM), contains the classic divalent-cation–binding residue D618, which is conserved in most viral polymerases including hepatitis C virus (HCV) ns5b (residue D220) and poliovirus (PV) 3D pol (residue D233) (13, 14) (Fig. 3, B and C). Motif C [residues 753 to 767 (FSMMILSDDAVVCFN)] contains the catalytic residues [759 to 761 (SDD)] in the turn between two β strands. These catalytic residues are also conserved in most viral RdRps, e.g., 317 to 319 (GDD) in HCV ns5b and 327 to 329 (GDD) PV 3D pol , with the first residue being either serine or glycine.
(A to C) Structural comparison of COVID-19 virus nsp12 (A), HCV ns5b (PDB ID: 4WTG) (13) (B), and PV 3D pol (PDB ID: 3OLB) (14) (C). The three structures are displayed in the same orientation. The polymerase motifs (motifs A to G) have the same color scheme used in Fig. 1A. (D) The template entry, NTP entry, and product hybrid exit paths in COVID-19 virus nsp12 are labeled in slate, deep teal, and orange colors, respectively. Two catalytic manganese ions (black spheres), pp-sofosbuvir (dark green spheres for carbon atoms), and primer template (orange) from the structure of HCV ns5b in complex pp-sofosbuvir (PDB ID: 4WTG) (13) are superposed to COVID-19 virus nsp12 to indicate the catalytic site and nucleotide binding position.
In this structure, as in other RNA polymerases, the primer-template entry, NTP entry, and nascent strand exit paths are positively charged and solvent accessible, and they converge in a central cavity where the RdRp motifs mediate template-directed RNA synthesis (Fig. 3D). The configurations of the template-primer entry paths, the NTP entry channel, and the nascent strand exit path are similar to those described for SARS-CoV and for other RNA polymerases, such as HCV and PV polymerase (14) (Fig. 3, B and C). The NTP entry channel is formed by a set of hydrophilic residues, including K545, R553, and R555 in motif F. The RNA template is expected to enter the active site composed of motifs A and C through a groove clamped by motifs F and G. Motif E and the thumb subdomain support the primer strand. The product-template hybrid exits the active site through the RNA exit tunnel at the front side of the polymerase.
Remdesivir, the single Sp isomer of the 2-ethylbutyl L-alaninate phosphoramidate prodrug (15) (fig. S7), has been reported to inhibit COVID-19 virus proliferation and therefore to have clinical potential (7, 8). We will briefly discuss its possible binding and inhibition mechanism on the basis of the results of this study. The efficacy of chain-terminating nucleotide analogs requires viral RdRps to recognize and successfully incorporate the active form of the inhibitors into the growing RNA strand. Sofosbuvir (2′-F-2′-C-methyluridine monophosphate) is a prodrug that targets HCV ns5b and has been approved for the treatment of chronic HCV infection (16). It acts by binding to the catalytic site of HCV ns5b polymerase (12, 16). Given that remdesivir and sofosbuvir are both nucleotide analogs and given the structural conservation of the catalytic site between COVID-19 virus nsp12 and HCV ns5b polymerase (13, 16) (fig. S7), we modeled remdesivir diphosphate binding to COVID-19 virus nsp12 on the basis of superposition with sofosbuvir bound to HCV ns5b (Fig. 4A and fig. S4D). Overall, we found that the nsp12 of COVID-19 virus has the highest similarity with the apo state of ns5b. Given the conformational changes of ns5b in apo, elongation, and inhibited states, it appears that catalytic residues D760, D761, and the classic D618 will undergo a conformational change to coordinate the divalent cations (Fig. 4B). The latter will anchor the phosphate group of the incoming nucleotide or inhibitors together with the allosteric R555 in motif F (Fig. 4C). In the structures of the HCV ns5b elongation complex or its complex with diphosphate sofosbuvir (pp-sofosbuvir), a key feature is that the incorporated pp-sofosbuvir interacts with N291 (equivalent to N691 in COVID-19 virus). However, because of a fluorine substitution on its sugar moiety, pp-sofosbuvir is not capable of joining the hydrogen bonding network with S282 and D225 (Fig. 4D), which is necessary to stabilize the incoming natural nucleotide (13). However, remdesivir keeps an intact ribose group, so it may be able to use this hydrogen bond network like a native substrate. Additionally, T680 in COVID-19 virus nsp12 is also likely to form hydrogen bonds with the 2′ hydroxyl of remdesivir and, of course, with incoming natural NTP (Fig. 4D). Moreover, the hydrophobic side chain of V557 in motif F is likely to stack with and stabilize the +1 template RNA uridine base to base pair with the incoming triphosphate remdesivir (ppp-remdesivir) (Fig. 4E).
(A) The polymerase motifs are colored as in Fig. 3. Superposition of the structure of HCV ns5b in complex with pp-sofosbuvir (PDB ID: 4WTG) (13) with COVID-19 virus nsp12 shows the possible positions of the two catalytic ions (purple spheres), the priming nucleotide (U 0), template strand, and the incoming pp-remdesivir in nsp12. (B to E) Structure comparison of HCV apo ns5b or its complex with UDP and pp-sofosbuvir with the COVID-19 virus nsp12.
The rapid global spread of COVID-19 virus has emphasized the need for the development of new coronavirus vaccines and therapeutics. The viral polymerase nsp12 appears to be an excellent target for new therapeutics, especially given the fact that lead inhibitors already exist in the form of compounds such as remdesivir. Considering the structural similarity of nucleoside analogs, the binding mode and inhibition mechanism discussed here may also be applicable to other similar drugs or drug candidates such as favipiravir, which has proven effective in clinical trials (17). This target, in addition to other promising drug targets such as the main protease, could support the development of a cocktail of anti-coronavirus treatments that potentially can be used for the discovery of broad-spectrum antivirals.
The Coronavirus Is Mutating. But That May Not Be A Problem For Humans
A colorized image of cells from a patient infected with the coronavirus SARS-CoV-2. The virus particles are colored pink. The image was captured from a scanning electron micrograph. NIAID/Flickr hide caption
A colorized image of cells from a patient infected with the coronavirus SARS-CoV-2. The virus particles are colored pink. The image was captured from a scanning electron micrograph.
As the new coronavirus continues to spread around the globe, researchers say the virus is changing its genetic makeup slightly. But does that mean it is becoming more dangerous to humans? And what would the impact be on any future vaccines?
"In the literal sense of 'is it changing genetically,' the answer is absolutely yes," says Marc Lipsitch, an infectious disease epidemiologist at Harvard University. "What is in question is whether there's been any change that's important to the course of disease or the transmissibility or other things that we as humans care about."
So far, "there is no credible evidence of a change in the biology of the virus either for better or for worse," says Lipsitch.
Coronaviruses — like all viruses — change small parts of their genetic code all the time.
"Viruses mutate naturally as part of their life cycle," says Ewan Harrison, scientific project manager for the COVID-19 Genomics UK Consortium, a new project that tracks the virus in the United Kingdom.
Like flu and measles, the coronavirus is an RNA virus. It's a microscopic package of genetic instructions bundled in a protein shell. When a virus infects a person, the string of genetic instructions enables the virus to spread by telling it how to replicate once it enters a cell. The virus makes copies of itself and pushes them out to other cells in the body. Infectious doses of the virus can be coughed out in droplets and inhaled by others.
Inevitably, viruses "make mistakes in their genomes" as they copy themselves, says Harrison. Those changes can accumulate and carry over to future copies of the virus. Researchers are using these small, cumulative changes to trace the pathway of the virus through groups of people.
So far, researchers who are tracking the genetic changes in SARS-CoV-2 — the official name for the coronavirus — say it seems relatively stable. It acquires about two mutations a month during this process of spread, Harrison says — about one-third to one-half the rate of the flu.
Coronaviruses differ from flu viruses in another key way that reduces the number of mutations. They proofread their own genomes when they copy themselves, cutting out things that don't seem right. "They maintain this ability to keep their genome pretty much intact," says Vineet Menachery, a virologist at the University of Texas Medical Branch. "The mutations that they incorporate are relatively rare."
This added proofreading function means that coronaviruses are also one of the largest RNA viruses. They're about 30,000 nucleotides long — double the size of flu viruses. But at 125 nanometers wide, they're still microscopic 800 of them could fit in the width of a human hair.
Nonetheless, their relatively larger size means "they have a lot more tools in their tool belt" compared with other RNA viruses, says Menachery — in other words, more capability of fighting off a host's immune system and making copies of themselves.
Researchers are on alert for changes that might affect how the coronavirus behaves in humans. For instance, if the coronavirus developed ways to block parts of our immune system, it could hide out in our bodies and establish itself better. If it evolved to bind more strongly to human cells, it could enter them more efficiently and replicate more quickly.
But it's not as if the coronavirus needs to become more potent to survive and thrive. It's already replicating itself around the world very successfully, says Justin Bahl, an evolutionary biologist at the University of Georgia. "The viruses themselves are not actually under much pressure to change."
Selective pressures could come from introducing treatments and vaccines that are effective against a narrow group of coronavirus strains. If that happens, strains that aren't targeted by these measures would likely proliferate.
The small genetic changes that researchers have observed so far don't appear to be changing the function of the virus. "I don't think we're going to see major new traits, but I do think that we're going to see different variants emerge in the population," says Bahl.
And that slower rate of change is potentially good news for treatments and vaccines. Researchers think that once a person gains immunity against SARS-CoV-2, either by recovering from an infection or by getting a future vaccine, they will likely be protected against the strains in circulation for "years rather than months," predicts Trevor Bedford, an evolutionary biologist at the Fred Hutchinson Cancer Research Center, in an assessment shared on Twitter.
Projects such as the COVID-19 Genomics UK Consortium will use these genetic drifts to track the path of the virus and figure out if there are hospitals or community hubs that are hot spots for contagion, according to Harrison. This will give public health officials a sense of where and how the virus is being transmitted now.
Will the coronavirus surge when schools reopen? Will new strains emerge that develop resistance to drugs or vaccines that are introduced? To answer such questions, Harrison says, the long-term plan is to track the virus in real time — and see how it changes as it spreads.
Researchers are actively using these structures to search for compounds that block the action of the proteases, for use as antiviral drugs. The diversity of coronaviruses poses a great challenge with this effort: coronaviruses have been classified into four separate genera, and sequence and structural studies have shown that the proteases of these viruses can be very different, so drugs designed to fight one may not be effective against others. One possible way to address this challenge is to try to design a broad-spectrum inhibitor targeted against the progenitor bat coronavirus, such as the one shown here from PDB entry 4yoi, which may then provide a head-start for discovering inhibitors against newly emerging viruses. The active site cysteine and histidine are shown in the illustration, with an inhibitor in turquoise. To explore this structure in more detail, click on the image for an interactive JSmol.
Topics for Further Discussion
- An unusual octameric form of the main protease may be involved in its maturation. You can see it in PDB entry 3iwm.
- You can compare the folds of coronavirus main proteases and serine proteases using the “Structure Align” tool. Try using trypsinogen (PDB entry 1tgs), so that the whole enzyme is one chain for the alignment.
Related PDB-101 Resources
- Cui, J., Li, F., Shi, Z.L. (2019) Origin and evolution of pathogenic coronaviruses. Nat. Rev. Microbiol. 17, 181-192.
- 4yoi: St John, S.E., Tomar, S., Stauffer, S.R., Mesecar, A.D. (2015) Targeting zoonotic viruses: Structure-based inhibition of the 3C-like protease from bat coronavirus HKU4-The likely reservoir host to the human coronavirus that causes Middle East Respiratory Syndrome (MERS). Bioorg.Med.Chem. 23: 6036-6048
- 4ow0: Baez-Santos, Y.M., Barraza, S.J., Wilson, M.W., Agius, M.P., Mielech, A.M., Davis, N.M., Baker, S.C., Larsen, S.D., Mesecar, A.D. (2014) X-ray Structural and Biological Evaluation of a Series of Potent and Highly Selective Inhibitors of Human Coronavirus Papain-like Proteases. J.Med.Chem. 57: 2393-2412
- Hilgenfeld, R. (2014) From SARS to MERS: crystallographic studies on coronaviral proteases enable antiviral drug design. FEBS J. 281,4085-4096
- 1q2w: Pollack, A. (2003) Company says it mapped part of SARS virus. New York Times, July 30, section C, page 2.
February 2020, David Goodsell
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Why PDB-101? Researchers around the globe make these 3D structures freely available at the Protein Data Bank (PDB) archive. PDB-101 builds introductory materials to help beginners get started in the subject ("101", as in an entry level course) as well as resources for extended learning.
1: DNA Replication, Transcription and Translation
A. Function: DNA base sequence encodes information for amino acid sequence of proteins. Genetic code: 1 to 1 relationship between a codon (specific sequence of 3 bases) and 1 amino acid. Central Dogma of genetics/info flow in cells -Foundation Figure: Flow of Genetic Info p 1. DNA will be replicated and passed on to &ldquodaughter cells&rdquo
B. DNA Structure: figure 8.3 Double stranded (2 strands of DNA), helical &ldquodouble helix&rdquo, antiparallel
1. Two strands held together by hydrogen bonds between complementary bases inside helix
2. Strong outer &ldquosugar-phosphate&rdquo backbone covalent phosphodiester bonds link nucleotides
3. DNA strands: polymers of nucleotides
4. Nucleotides: 3 components. Sugar=deoxyribose, phosphate, nitrogenous base
5. Nitrogenous bases of DNA
a. purines (2 rings)= adenine (A) and guanine (G) pyrimidines (1 ring)= thymine (T) and cytosine (C)
b. Chagraff&rsquos rules: amount of A=T and amount of C=G this is because of complementary base-pairing rules
A=T form 2 hydrogen bonds
G=C form 3 hydrogen bonds
*c. complementary base pairing permits the precise replication of DNA
6. Deoxyribose: pentose 5 carbons. C1' covalently linked to nitrogenous base.
C3&rsquo= free OH (tail)
C5&rsquo linked to phosphate group (head)
7. Prokaryotic chromosomes see figure Most bacteria have a single circular chromosome. 1 copy of chromosomes=&ldquohaploid cells&rdquo (most human cells have 2 copies of linear chromosomes and are called &ldquodiploid cells&rdquo see &ldquoeukaryotic chromosomes).
8. Topoisomerases and Bacterial Gyrase
-Topoisomerases Enzymes which &ldquosupercoil&rdquo DNA or relieve supercoiling different types of toposiomerases in E. coli.
Type I/III&rdquo &ldquorelax&rdquo DNA supercoils
Type II= Bacterial Gyrase: introduces negative supercoils
&ldquoThrough the action of topoisomerases, the DNA molecule can be alternately coiled and relaxed. Because coiling is necessary for packing DNA into the confines of a cell and relaxing is necessary so DNA can be replicated (and transcribed), these two complementary processes ..play an important role in the behavior of DNA in the cell.&ldquo Brock Biology of Microorganisms 8th edition p 185 )
-bacterial gyrase is involved in supercoiling/relief of supercoiling of DNA
-antibiotics quinolones (e.g. nalidixic acid) and fluoroquinolones (such as ciprofloxacin) and novobiocin inhibit bacterial gyrase and interfere with DNA replication/transcription see p
C. DNA synthesis by DNA polymerases fig ___ Table _____
1. DNA polymerase requires template strand (guide), primer strand with free 3&rsquoOH group, activated substrates/precursors= nucleoside triphosphates
*2. DNA replicated in 5&rsquo to 3&rsquo direction (5&rsquo->3&rsquo). Incoming nucleotides can only be added to 3&rsquoOH tail of a growing DNA strand
3. Oxygen of 3&rsquoOH groups makes a nucleophilic attack on inner most phosphorus atom of incoming nucleoside triphosphate. Pyrophosphate split off and will be hydrolyzed by cellular phosphatases with the release of energy to drive synthesis. Nucleotide is linked to primer strand by phosphodiester bond (ester bond= bond between alcohol and acid)
4. If no 3&rsquoOH present , DNA strand cannot be lengthened=DNA chain termination. Use of dideoxynucleoside triphosphates as base analogues and in DNA sequencing reactions.
II. Replication of Bacterial Chromosome fig ____
A. Recall bacterial chromosome: singular, circular double stranded DNA in cytoplasm
B. DNA replication begins at specific site &ldquoori&rdquo = origin of replication
C. DNA replication proceeds bidirectionally from ori, with formation of replication bubble and 2 replication forks. Replication forks= regions where d.s. DNA unwound, form s.s. DNA templates, DNA polymerase makes complementary copy of parent ssDNA template.
D. DNA replication is semiconservative. 1 parent &ldquoold&rdquo DNA strand is used as template or guide for synthesis of 1 new daughter DNA strand.-result: 1 parent chromosome -> 2 daughter chromosomes. Each daughter chromosome is a copy of parent chromosome. Each daughter chromosome consists of 1 old parent DNA strand and 1 new daughter DNA strand. 1 parent strand is &ldquoconserved&rdquo in each new daughter chromosome
E. Enzymes/proteins involved in DNA synthesis. KNOW FOR EXAM. Fig 8___ Table ___
1.* Topoisomerases e.g., Bacterial Gyrase involved in DNA supercoiling/relief of
supercoiling (target of quinolones e.g., ciprofloxacin &ldquocipro&rdquo used to treat/prevent
1. Helicase: unwinds ds DNA, breaks H bonds between bases, forms ss DNA template
2. Single Strand Binding Proteins SSBP bind, stabilize and protect ssDNA
3. RNA Primase: an RNA polymerase which does not require a primer strand to start
primer synthesis. Synthesizes a short complementary RNA primer strand with free 3&rsquoOH
group using ss DNA as template. Creates RNA primer, permitting DNA polymerase to
start DNA synthesis. (RNA polymerase do not &ldquoproof read&rdquo and therefore can make
4-5. DNA polymerase: requires primer strand, template and activated nucleoside
triphosphates (dATP, dTTP,DCTP,dGTP). Must have DNA template. Synthesizes complementary DNA
strand using parent strand as template/guide. DNA polymerase have &ldquoproofreading abilities&rdquo, they &ldquocheck&rdquo
each nucleotide they add, remove if incorrect and add correct nucleotide. DNA polymerases have high
fidelity, they make very few mistakes. Original mistake rates 10-4 following proofreading, mistake rate=
10-9 ie one incorrect base in every 109 bases added E. coli: DNA polymerase III performs most of DNA synthesis
DNA polymerase I: will remove RNA primer and replace with DNA sequence
6. Ligase: links short sequences of DNA (called Okazaki fragments) together on &ldquolagging
strand&rdquo homework see inhibition of nucleic acid synthesis. What are nucleotide analogs? What are their uses?
Compare and contrast bacterial DNA polymerases and RNA polymerases
Note: ss=single strand ds=double strand P=phosphate
DNA polymerases synthesize complementary DNA using a DNA template/guide
e.g., ssDNA template base sequence: A T A G G C
Complementary DNA sequence T A T C C G dna
synthesized by DNA polymerase
RNA polymerases synthesize complementary RNA sequences using DNA as a template/guide
e.g., ssDNA template base sequence: A T A G G C
Complementary RNA sequence U A U C C G rna
synthesized by RNA polymerase
Synthesis of DNA and RNA require input of energy, both ATP and charged precursors
Compare and Contrast DNA Polymerase and cellular RNA Polymerase
DNA Polymerase RNA Polymerase
Template/guide ss DNA ssDNA
Synthesize complementary DNA complementary RNA
Charged precursors deoxyadenosine tri-P= dATP adenosine tri-P= ATP
deoxythymidine tri-P=dTTP uridine tri-P=UTP
deoxycytodine tri-P= dCTP cytodine tri-P=CTP
deoxyguanosine tri-P=dGTP guanosine tri-P=GTP
primer required? yes no
proofreading/editing? yes * no
*DNA polymerase proofreading/editting
Polymerases have a &rdquonormal&rdquo or &ldquointrinsic&rdquo mistake rate of approximately 10 -4 &ndash 10 -5 nucleotides (this means the polymerases introduce the incorrect nucleotide every 10,000 to 100, 000 nucleotides). DNA polymerases have the ability to &ldquoproofread
and edit&rdquo their mistakes. If they introduce the wrong nucleotide, they can remove or &ldquoexcise&rdquo the wrong nucleotide and try again to make a correct match. This reduces the mistake rate of DNA polymerases to approximately 10 -9 &ndash 10 -10 (or only one incorrect
nucleotide every 1,000,000,000 &ndash 10,000,000,000 nucleotides). RNA polymerase cannot proofread or edit so RNA polymerase make many mistakes (one reason many RNA viruses, for example HIV, mutate so rapidly. more later)
Review flow of information in cell
DNA--------> RNA --------->Protein
replication transcription translation
I. Genetic Code: one to one relationship between specific codon (specific 3 base sequence) and an amino acid
II. Bacterial Transcription: use of DNA as template/guide to synthesize complementary RNA.
DNA info is rewritten in RNA sequence. Fig ___
A. First step in gene expression
B. Products of transcription
1. messenger RNA=mRNA: will be translated into specific amino acid
sequence of a protein
2. transfer RNA=tRNA: actual &ldquotranslator&rdquo molecule, recognizes both a
specific codon and specific amino acid
3. ribosomal RNA=rRNA: combined with ribosomal proteins, will form
the ribosome, the &ldquoworkbench&rdquo at which mRNA is translated into a specific amino acid
4. additional RNA products
III. Promoters and Bacterial RNA polymerases
A. Promoters: specific DNA sequences which signal the &ldquostart&rdquo points for gene
transcription. Sigma factor/subunit of RNA polymerase binds to promoters to
B. Bacterial RNA polymerases: enzyme complex which recognizes DNA promoters, binds
to promoter and synthesizes complementary RNA copy using DNA as
E. coli RNA Polymerase: 2 subunits, sigma subunit and core
a. sigma subunit/factor= &ldquobrains&rdquo of RNA polymerase. Travels
along DNA until it reaches a promoter, binds promoter
b. core subunit: binds to sigma attached at promoter. &ldquoWorkhorse&rdquo
of RNA polymerase, carries out actual RNA synthesis. Requires
activated precursors and template strand, DOES NOT REQUIRE
PRIMER (compare to DNA Polymerase). Synthesizes RNA in 5&rsquo -
to->3&rsquo , similar to DNA polymerase. No proofreading ability
therefore will make more mistakes than DNA Polymerase
c. sigma subunit will drop off after the first few ribonucleotides
have been linked together, core continues alone. Note: core would
start transcription randomly of DNA without direction of sigma
subunit. Polycistronic mRNA (prok. only)
IV. Termination of transcription (over-simplified)
Terminators: DNA sequences which signal transcription stop signals. RNA
polymerase releases DNA when transcription terminator sequence encountered
Homework Describe antimicrobial drugs which bind to and inhibit function of bacterial RNA
polymerases (answer: rifampin _used to treat which pathogen?)
Bacterial Translation fig
Translation: RNA base sequence translated into amino acid sequence of protein. mRNA is template for
polypeptide synthesis. Second step in gene expression.
A.Translation of mRNA into a polypeptide chain is possible because of the genetic code:
1. genetic code: One to one relationship between a codon (specific sequence of 3 bases)
and a specific amino acid. Figure __ Genetic code table
Genetic code: Redundant (more than one codon for each amino acid) yet specific (each codon
encodes info for 1 amino acid only). Universal most cellular organisms use same genetic code
B. Translation requires tRNA, amino acids, ATP/GTP, ribosomes and mRNA
C. tRNA =transfer RNA. Adaptor/translator molecule. Only molecule which can "recognize" correct amino acid AND correct codon
1. structure: ss RNA, stem and loop
a. amino acid attachment site at one end
b. anticodon which "recognizes"(forms H bonds with) codon of mRNA
2. *45 different tRNA&rsquos for 20 different amino acids &ldquowobble&rdquo permits some tRNA&rsquos to bind to more than one codon (&ldquorelaxed&rdquo/improper base painring between 3 base of codon and anticodon)
D. amino acyl tRNA synthetases* : &ldquoload&rdquo proper amino acid on proper tRNA= amino acid activation. 20 different transferases for 20 different amino acids/tRNA&rsquos amino acid x+ ATP + tRNAx--> tRNAx:amino acid x + AMP + 2 P* &ldquocharged tRNA&rdquo or &ldquoactivated amino acid&rdquo
E. Ribosomes: 70S in prokaryotes. 2 subunits 30S (small subunit) + 50S (large subunit) S=Svedberg Unit, use to express sedimentation rates, ultracentrifugation
made of rRNA and ribosomal proteins. &ldquoWorkbench&rdquo at which mRNA will be translated into a polypeptide. 16s rRNA binds RBS (Ribosomal Binding Site on mRNA). 23s rRNA acts a ribozyme, forms peptide bonds between amino acids E, P and A sites.
F. Mechanics of translation: text. GTP is hydrolyzed during translation
Translation Initiation (note: tRNA-f met may first bind 30S subunit before 30S subunit binds RBS)
1. 30S subunit recognizes ribosomal binding site RBS/Shine-Dalgarno sequence. Complementary to 16s rRNA sequence of ribosome.
2. Translation begins at start codon AUG closest to ribosomal binding site
3. An initiator tRNA:methionine ( more precisely a formyl methionine in bacteria) enters the &ldquoP&rdquo or peptidyl binding site of the ribosome. A tRNA fits into the binding site when its anticodon base-pairs with the mRNA codon
4. The larger ribosomal 50S subunit then binds the complex
5. Additional proteins called initiation factors are required to bring all components together
Translation Elongation: amino acids are added one by one to first amino acid. Additional protein
elongation factors required
1. A second appropriately charged tRNA enters the &ldquoA&rdquo or aminoacyl binding site of the ribosome, bearing the next amino acid.
2. Peptide bond formation. 23s rRNA of large subunit catalyzes formation of peptide bond between amino acid at P site and amino acid at A site (rRNA acts as a &ldquoribozyme&rdquo, RNA catalyst) -amino acid of tRNA at P site is transferred to amino acid bond of tRNA at A site
3. Now ribosome moves &ldquodownstream&rdquo by one codon. tRNA carrying dipeptide is now in P site, A site is empty.
4. New appropriately amino acid charged tRNA enters A site
5. Ribosome catalyzes peptide bond formation between dipeptide and new incoming amino acid. Tripeptide is carried by tRNA at A site
7. Requires energy (GTP )
1. Ribosome reaches one of 3 nonsense codons/stop codons: UAA, UGA, UAG
2. Release factor binds A site, causes polypeptide and ribosome to be released from mRNA (by activation of ribozyme)
G. Polycistronic mRNA in prokaryotes permit coordinated gene expression in prokaryotes
mRNA encodes more than one gene so ribosomes can coordinately produce several
different proteins. For example 3 genes for proteins involved in lactose transport/metabolism in E. coli are
transcribed into a single mRNA molecule. Ribosomes translate all 3 into proteins at same time
H. Simultaneous transcription and translation in prokaryotes only. Ribosomes can bind mRNA and begin translation before transcription is finished. Very efficient. Fig ____
We are attempting to understand the processes required to accurately replicate the repetitive DNA sequences whose instability is associated with several human diseases. Here we test the hypothesis that the contribution of exonucleolytic proofreading to frameshift fidelity during replication of repetitive DNA sequences diminishes as the number of repeats in the sequence increases. The error rates of proofreading-proficient T7, T4, and Pyrococcus furiosis DNA polymerases are compared to their exonuclease-deficient derivatives, for +1 and −1 base errors in homopolymeric repeat sequences of three to eight base pairs. All three exonuclease-deficient polymerases produce frameshift errors during synthesis at rates that increase as a function of run length, suggesting the involvement of misaligned intermediates. Their wild-type counterparts are all much more accurate, suggesting that the majority of the intermediates are corrected by proofreading. However, the contribution of the exonuclease to fidelity decreases substantially as the length of the homopolymeric run increases. For example, the exonuclease enhances the frameshift fidelity of T7 DNA polymerase in a run of three A·T base pairs by 160-fold, similar to its contribution to base substitution fidelity. However, in a run of eight consecutive A·T base pairs, the exonuclease only enhances frameshift fidelity by 7-fold. A similar pattern was observed with T4 and Pfu DNA polymerases. Thus, both polymerase selectivity and exonucleolytic proofreading efficiency are diminished during replication of repetitive sequences. This may place an increased relative burden on post-replication repair processes to reduce rates of addition and deletion mutations in organisms whose genome contains abundant simple repeat DNA sequences.
To whom correspondence should be addressed. Phone: (919)-541-2644. Fax: (919)-541-7613. Email: [email protected]
From Bats to Human Lungs, the Evolution of a Coronavirus
For thousands of years, a parasite with no name lived happily among horseshoe bats in southern China. The bats had evolved to the point that they did not notice they went about their nightly flights unbothered. One day, the parasite—an ancestor of the coronavirus, SARS-CoV-2—had an opportunity to expand its realm. Perhaps it was a pangolin, the scaly anteater, an endangered species that is a victim of incessant wildlife trafficking and sold, often secretly, in live-animal markets throughout Southeast Asia and China. Or not. The genetic pathway remains unclear. But to survive in a new species, whatever it was, the virus had to mutate dramatically. It might even have taken a segment of a different coronavirus strain that already inhabited its new host, and morphed into a hybrid—a better, stronger version of itself, a pathogenic Everyman capable of thriving in diverse species. More recently, the coronavirus found a new species: ours. Perhaps a weary traveller rubbed his eyes, or scratched his nose, or was anxiously, unconsciously, biting his fingernails. One tiny, invisible blob of virus. One human face. And here we are, battling a global pandemic.
The world’s confirmed cases (those with a positive lab test for COVID-19, the disease caused by SARS-CoV-2) doubled in seven days, from nearly two hundred and thirteen thousand, on March 19th, to four hundred and sixty-seven thousand, on March 26th. Nearly twenty-one thousand people have died. The United States now has more confirmed cases than any country on earth, with more than eighty thousand on March 26th. These numbers are a fraction of the real, unknown total in this country and around the world, and the numbers will keep going up. Scientists behind a new study, published earlier this month in the journal Science, have found that for every confirmed case there are likely five to ten more people in the community with an undetected infection. This will likely remain the case. “The testing is not near adequate,” one of the study’s authors, Jeffrey Shaman, an environmental-health sciences professor at Columbia University, said. Comments from emergency-room doctors have been circulating on social media like S.O.S. flares. One, from Daniele Macchini, a doctor in Bergamo, north of Milan, described the situation as a “tsunami that has overwhelmed us.”
Scientists first discovered that coronaviruses originate among bats following the outbreak of Severe Acute Respiratory Syndrome (SARS) in 2003. Jonathan Epstein, an epidemiologist at the EcoHealth Alliance in New York who studies zoonotic viruses—those that can jump from animals to people—was part of a research team that went hunting for the source in China’s Guangdong Province, where simultaneous SARS outbreaks had occurred, suggesting multiple spillovers from animals to people. At first, health officials believed palm civets, a mongoose-like species commonly eaten in parts of China, were responsible, as they were widely sold at markets connected to the SARS outbreak, and tested positive for the virus. But civets bred elsewhere in Guangdong had no antibodies for the virus, indicating that the market animals were only an intermediary, highly infectious host. Epstein and others suspected that bats, which are ubiquitous in the area’s rural, agricultural hills, and were, at the time, also sold from cages at Guangdong’s wet markets, might be the coronavirus’s natural reservoir.
The researchers travelled through the countryside, setting up field labs inside limestone caverns and taking swabs from dozens of bats through the night. After months of investigation, Epstein’s team discovered four species of horseshoe bats that carried coronaviruses similar to SARS, one of which carried a coronavirus that was, genetically, a more than ninety per cent match. “They were found in all of the locations where SARS clusters were happening,” he said.
After years of further bat surveillance, researchers eventually found the direct coronavirus antecedent to SARS, as well as hundreds of other coronaviruses circulating among some of the fourteen hundred bats species that live on six continents. Coronaviruses, and other virus families, it turns out, have been co-evolving with bats for the entire span of human civilization, and possibly much longer. As the coronavirus family grows, different strains simultaneously co-infect individual bats, turning their little bodies into virus blenders, creating new strains of every sort, some more powerful than others. This process happens without making bats sick—a phenomenon that scientists have linked to bats’ singular ability, among mammals, to fly. The feat takes a severe toll, such that their immune systems have evolved a better way to repair cell damage and to fight off viruses without provoking further inflammation. But when these viruses leap into a new species—whether a pangolin or a civet or a human—the result can be severe, sometimes deadly, sickness.
In 2013, Epstein’s main collaborator in China, Shi Zheng-Li, sequenced a coronavirus found in bats, which, in January, she discovered shares ninety-six per cent of its genome with SARS-CoV-2. The two viruses have a common ancestor that dates back thirty to fifty years, but the absence of a perfect match suggests that further mutation took place in other bat colonies, and then in an intermediate host. When forty-one severe cases of pneumonia were first announced in Wuhan, in December, many of them were connected to a wet market with a notorious wildlife section. Animals are stacked in cages—rabbits on top of civets on top of ferret-badgers. “That’s just a gravitational exchange of fecal matter and viruses,” Epstein said. Chinese authorities reported that they tested animals at the market—all of which came back negative—but they have not specified which animals they tested, information that is crucial for Epstein’s detective work. Authorities later found the virus in samples taken from the market’s tables and gutters. But, because not all of the first patients were tied to the market, nor were they connected to one another, Epstein said, “it raised the question of, well, perhaps those forty-one weren’t the first cases.”
Analyses of the SARS-CoV-2 genome indicate a single spillover event, meaning the virus jumped only once from an animal to a person, which makes it likely that the virus was circulating among people before December. Unless more information about the animals at the Wuhan market is released, the transmission chain may never be clear. There are, however, numerous possibilities. A bat hunter or a wildlife trafficker might have brought the virus to the market. Pangolins happen to carry a coronavirus, which they might have picked up from bats years ago, and which is, in one crucial part of its genome, virtually identical to SARS-CoV-2. But no one has yet found evidence that pangolins were at the Wuhan market, or even that venders there trafficked pangolins. “We’ve created circumstances in our world somehow that allows for these viruses, which would otherwise not be known to cause any problems, to get into human populations,” Mark Denison, the director of pediatric infectious diseases at Vanderbilt University Medical Center’s Institute for Infection, Immunology, and Inflammation, told me. “And this one happened to say, ‘I really like it here.’ ”
The new coronavirus is an elusive killer. Since people have never seen this strain before, there is much about it that remains a mystery. But, in just the past few weeks, genetic sleuthing, atomic-level imaging, computer modelling, and prior research on other types of coronaviruses, including SARS and MERS (Middle East Respiratory Syndrome), have helped researchers to quickly learn an extraordinary amount—particularly what might treat or eradicate it, through social-distancing measures, antiviral drugs, and, eventually, a vaccine. Since January, nearly eight hundred papers about the virus have been posted on BIORxiv, a preprint server for studies that have not yet been peer-reviewed. More than a thousand coronavirus genome sequences, from different cases around the world, have been shared in public databases. “It’s insane,” Kristian Andersen, a professor in the Department of Immunology and Microbiology at Scripps Research, told me. “Almost the entire scientific field is focussed on this virus now. We’re talking about a warlike situation.”
There are endless viruses in our midst, made either of RNA or DNA. DNA viruses, which exist in much greater abundance around the planet, are capable of causing systemic diseases that are endemic, latent, and persistent—like the herpes viruses (which includes chicken pox), hepatitis B, and the papilloma viruses that cause cancer. “DNA viruses are the ones that live with us and stay with us,” Denison said. “They’re lifelong.” Retroviruses, like H.I.V., have RNA in their genomes but behave like DNA viruses in the host. RNA viruses, on the other hand, have simpler structures and mutate rapidly. “Viruses mutate quickly, and they can retain advantageous traits,” Epstein told me. “A virus that’s more promiscuous, more generalist, that can inhabit and propagate in lots of other hosts ultimately has a better chance of surviving.” They also tend to cause epidemics—such as measles, Ebola, Zika, and a raft of respiratory infections, including influenza and coronaviruses. Paul Turner, a Rachel Carson professor of ecology and evolutionary biology at Yale University, told me, “They’re the ones that surprise us the most and do the most damage.”
Scientists discovered the coronavirus family in the nineteen-fifties, while peering through early electron microscopes at samples taken from chickens suffering from infectious bronchitis. The coronavirus’s RNA, its genetic code, is swathed in three different kinds of proteins, one of which decorates the virus’s surface with mushroom-like spikes, giving the virus the eponymous appearance of a crown. Scientists found other coronaviruses that caused disease in pigs and cows, and then, in the mid-nineteen-sixties, two more that caused a common cold in people. (Later, widespread screening identified two more human coronaviruses, responsible for colds.) These four common-cold viruses might have come, long ago, from animals, but they are now entirely human viruses, responsible for fifteen to thirty per cent of the seasonal colds in a given year. We are their natural reservoir, just as bats are the natural reservoir for hundreds of other coronaviruses. But, since they did not seem to cause severe disease, they were mostly ignored. In 2003, a conference for nidovirales (the taxonomic order under which coronaviruses fall) was nearly cancelled, due to lack of interest. Then SARS emerged, leaping from bats to civets to people. The conference sold out.
SARS is closely related to the new virus we currently face. Whereas common-cold coronaviruses tend to infect only the upper respiratory tract (mainly the nose and throat), making them highly contagious, SARS primarily infects the lower respiratory system (the lungs), and therefore causes a much more lethal disease, with a fatality rate of approximately ten per cent. (MERS, which emerged in Saudi Arabia, in 2012, and was transmitted from bats to camels to people, also caused severe disease in the lower respiratory system, with a thirty-seven per cent fatality rate.) SARS-CoV-2 behaves like a monstrous mutant hybrid of all the human coronaviruses that came before it. It can infect and replicate throughout our airways. “That’s why it is so bad,” Stanley Perlman, a professor of microbiology and immunology who has been studying coronaviruses for more than three decades, told me. “It has the lower-respiratory severity of SARS and MERS coronaviruses, and the transmissibility of cold coronaviruses.”
One reason that SARS-CoV-2 may be so versatile, and therefore so successful, has to do with its particular talent for binding and fusing with lung cells. All coronaviruses use their spike proteins to gain entry to human cells, through a complex, multistep process. First, if one imagines the spike’s mushroom shape, the cap acts like a molecular key, fitting into our cells’ locks. Scientists call these locks receptors. In SARS-CoV-2, the cap binds perfectly to a receptor called the ACE-2, which can be found in various parts of the human body, including the lungs and kidney cells. Coronaviruses attack the respiratory system because their ACE-2 receptors are so accessible to the outside world. “The virus just hops in,” Perlman told me, “whereas it’s not easy to get to the kidney.”
While the first SARS virus attached to the ACE-2 receptor, as well, SARS-CoV-2 binds to it ten times more efficiently, Kizzmekia Corbett, the scientific lead of the coronavirus program at the National Institutes of Health Vaccine Research Center, told me. “The binding is tighter, which could potentially mean that the beginning of the infection process is just more efficient.” SARS-CoV-2 also seems to have a unique ability, which SARS and MERS did not have, to use enzymes from our human tissue—including one, widely available in our bodies, named furin—to sever the spike protein’s cap from its stem. Only then can the stem fuse the virus membrane and the human-cell membrane together, allowing the virus to spit its RNA into the cell. According to Lisa Gralinski, an assistant professor in the Department of Epidemiology at the University of North Carolina at Chapel Hill, this supercharged ability to bind to the ACE-2 receptor, and to use human enzymes to activate fusion, “could aid a lot in the transmissibility of this new virus and in seeding infections at a higher level.”
Once a coronavirus enters a person—lodging itself in the upper respiratory system and hijacking the cell’s hardware—it rapidly replicates. When most RNA viruses replicate themselves in a host, the process is quick and dirty, as they have no proofreading mechanism. This can lead to frequent and random mutations. “But the vast majority of those mutations just kill the virus immediately,” Andersen told me. Unlike other RNA viruses, however, coronaviruses do have some capacity to check for errors when they replicate. “They have an enzyme that actually corrects mistakes,” Denison told me.
It was Denison’s lab at Vanderbilt that first confirmed, in experiments on live viruses, the existence of this enzyme, which makes coronaviruses, in a sense, cunning mutators. The viruses can remain stable in a host when there is no selective pressure to change, but rapidly evolve when necessary. Each time they leap into a new species, for example, they are able to hastily transform in order to survive in the new environment, with its new physiology and a new immune system to battle. Once the virus is spreading easily within a species, though, its attitude is, “I’m happy, I’m good, no need to change,” Denison said. That seems to be playing out now in humans as SARS-CoV-2 circles the globe, there are slight variations among its strains, but none of them seem to affect the virus’s behavior. “This is not a virus that is rapidly adapting. It’s like the best car in the Indy 500. It’s out in front and there is no obstacle in its path. So there is no benefit to changing that car.”
A virus replicates in order to shed from its host—through mucus, snot, phlegm, and even our breath—as soon as possible, in great quantities, so that it can keep spreading. The coronavirus happens to be a brilliant shedder. A preprint study by German researchers, published earlier this month, and one of the first outside China to examine data from patients diagnosed with COVID-19, found clear evidence that infected people shed the coronavirus at significant rates before they develop symptoms. In effect—possibly due to that supercharged ability to bind and fuse to our cells—the virus wears an invisibility cloak. Scientists recently estimated that undocumented cases of COVID-19, or infected people with mild symptoms, are fifty-five per cent as contagious as severe cases. Another study found that in more severe cases (requiring hospitalization), patients shed the virus from their respiratory tracts for as long as thirty-seven days.
Outside a host, in parasitical purgatory, a virus is inert, not quite alive, but not dead, either. A hundred million coronavirus particles could fit on the head of a pin—typically, thousands or tens of thousands are necessary to infect an animal or a person—and they might remain viable for long stretches. Researchers at the Virus Ecology Unit of Rocky Mountain Laboratories, in Montana, a facility connected to the National Institute of Allergy and Infectious Diseases, have found that the virus can linger on copper for four hours, on a piece of cardboard for twenty-four hours, and on plastic or stainless steel for as long as three days. They also found that the virus can survive, for three hours, floating through the air, transmitted by the tiny respiratory droplets an infected person exhales, sneezes, or coughs out. (Other research suggests the virus might be able to exist as an aerosol, but only in very limited conditions.) Most virus particles, though, seem to lose their virulency fairly quickly. The infection window is highest in the first ten minutes. Still, the risk of infection has turned many of us, understandably, into germophobes.
All a virus wants is an endless chain of hosts. Contagion is the evolutionary end goal. Based on experiments so far, researchers estimate that COVID-19 is slightly more communicable than the common flu and less communicable than the most highly infectious viruses, like measles, with which a single sick person can infect around twelve other people. There are likely coronavirus super-spreaders—people who, for whatever reason, are almost entirely asymptomatic but transmit the disease to many other people. But pinning down an exact infection rate, at this point, is an impossible task. “We tend to focus on these absolute numbers as telling us how worried we should be,” Denison said. “Look, it’s like flooding. You know, is it up to my knees or is it up to my chin? It doesn’t matter. I need to do something to try to make sure I’m not gonna drive my car into the flood.”
In many places, we already have driven into the flood. As hundreds of people die each day, hospitals are running out of supplies, beds, and ventilators. In these severe COVID-19 cases, according to scientists’ current understanding, the disease may have more to do with a haywire immune response to the virus than anything else. Because the virus can gain a foothold in our lower respiratory system while still wearing that invisibility cloak, it “basically beats the immune system to the punch and starts replicating too rapidly,” Perlman said. When the immune system finally does register its presence, it might go into overdrive, and send everything in its arsenal to attack, since it has no specific antibodies to fight these strange new invaders. “It’s like pouring gas on the fire,” Denison told me. The lung tissue swells and fills with fluid. Breathing is restricted, as is the exchange of oxygen. “The host immune response just gets triggered to such an extreme level, and then builds on itself and builds on itself until ultimately the body kind of goes into shock,” Gralinski said. It is almost like an autoimmune disease the immune system is attacking parts of the body that it should not.
This type of response might be why the elderly are, on the whole, more vulnerable to COVID-19, just as they were to the SARS outbreak in 2003. (In that outbreak, there were almost no deaths among children under the age of thirteen, and, when kids did get sick, the disease was, on average, milder than what affected adults.) When studying SARS in mice models, Denison told me that he has observed a phenomenon known as “immune senescence,” in which older mice no longer had the capacity to respond in a balanced way to a new virus their immune systems’ overreaction then caused even more severe disease. This occurred in some of the worst cases during the first SARS outbreak, too, Denison said, and explains why antiviral drugs may be significantly more helpful at the onset of illness, before the immune system has had a chance to wreak havoc.
In the last decade, Denison’s lab and collaborators at the University of North Carolina have been researching antiviral treatments to try to find something that worked not just against SARS and MERS but for a new coronavirus which, they knew, would inevitably arrive. Together, they did much of the early research into the drug now known as Remdesivir, which is currently in development by Gilead and in studies on infected patients, and another antiviral drug compound, known as NHC. Both drugs, in animal models, were able to bypass, avoid, or block the coronavirus’s proofreading function, which helped stop the virus from replicating successfully in the body. “They worked very effectively against all the coronaviruses that we’ve tested,” Denison told me.
Coronaviruses likely have that proofreading enzyme because they are huge—one of the largest RNA viruses in existence—and they need a mechanism that maintains such a long genome’s structure. From our perspective, the benefit of such a big genome, Andersen told me, “is that the more genes and protein products a virus has, the more opportunities we have to design specific treatments against them.” For instance, the virus’s unique ability to use the human enzyme furin offers promise for antiviral drugs that act as furin inhibitors.
COVID-19, while still new to us hosts, will continue to be responsible for widespread infection and death. But, Epstein said, “Over time, as viruses evolve with their natural habitats, they tend to cause less severe disease. And that is good for both the host and the virus.” The more virulent strains might burn out (which, however, means many more awful deaths), while the remaining hosts might build up some immunity. More immediately, and urgently, the virus’s stability—how much it is thriving among us right now, and mutating only minimally—bodes well for the performance of antiviral drugs and, eventually, a vaccine. If the growing number of mitigation measures—this unprecedented national and international shutdown—are held in place for enough time, the speed at which the virus is spreading should slow, giving hospitals and health workers some relief. “The virus is our teacher,” Denison told me. It has spent thousands of years evolving to get where it is. We’re now just rushing to catch up.
The pace of evolution
Mutations may happen randomly, but the rate at which they occur depends on the virus. The enzymes that copy DNA viruses, called DNA polymerases, can proofread and fix errors in the resulting strings of genetic letters, leaving few mutations in each generation of copies.
But RNA viruses, like SARS-CoV-2, are the evolutionary gamblers of the microscopic world. The RNA polymerase that copies the virus’s genes generally lacks proofreading skills, which makes RNA viruses prone to high mutation rates—up to a million times greater than the DNA-containing cells of their hosts.
Coronaviruses have a slightly lower mutation rate than many other RNA viruses because they can do some light genetic proofreading. “But it’s not enough that it prevents these mutations from accumulating,” says virologist Louis Mansky, the director for the Institute for Molecular Virology at the University of Minnesota. So as the novel coronavirus ran amok around the world, it was inevitable that a range of variants would arise.
The true mutation rate of a virus is difficult to measure though. “Most of those mutations are going to be lethal to the virus, and you’ll never see them in the actively growing, evolving virus population,” Mansky says.
Instead, genetic surveys of sick people can help determine what’s known as the fixation rate, which is a measure of how often accumulated mutations become “fixed” within a viral population. Unlike mutation rate, this is measured over a period of time. So the more a virus spreads, the more opportunities it has to replicate, the higher its fixation rate will be, and the more the virus will evolve, Duffy says.
For SARS-CoV-2, scientists estimate that one mutation becomes established in the population every 11 days or so. But this process may not always happen at a steady pace.
In December 2020, the variant B.1.1.7 caught scientists’ attention when its 23 mutations seemed to suddenly crop up as the virus rampaged through Kent. Some scientists speculate that a chronically ill patient provided more opportunities for replication and mutation, and the use of therapies such as convalescent plasma may have pressured the virus to evolve. Not every change was necessarily useful to the virus, Duffy notes, yet some mutations that emerged allowed the variant to spread rapidly.
- DNA labeling by nick translation
- DNA end blunting of 5'- and 3'-overhangs
- cDNA synthesis from DNA or RNA template
One unit is defined as the amount of enzyme that catalyzes the incorporation of 10 nmol of total nucleotides into acid-insoluble product in 30 minutes at 37°C and pH 7.4, using poly d(A-T) as the template-primer.
Friedberg, E. C. The eureka enzyme: the discovery of DNA polymerase. Nat. Rev. Mol. Cell Biol. 7, 143&ndash7 (2006).
Lehman, I. R., Bessman, M. J., Simms, E. S. & Kornberg, A. Enzymatic synthesis of deoxyribonucleic acid. I. Preparation of substrates and partial purification of an enzyme from Escherichia coli. J. Biol. Chem. 233, 163&ndash70 (1958).
Okayama, H. & Berg, P. High-efficiency cloning of full-length cDNA. Mol. Cell. Biol. 2, 161&ndash70 (1982).
Ricchetti, M. & Buc, H. E. coli DNA polymerase I as a reverse transcriptase. EMBO J. 12, 387&ndash96 (1993).
Rigby, P. W., Dieckmann, M., Rhodes, C. & Berg, P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237&ndash51 (1977).
Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular cloning : a laboratory manual. (Cold Spring Harbor Laboratory, 1989).
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