Module 4 Flashcards
Briefly describe the nature of viruses.
Viruses are obligate intracellular parasites.
Can the extracellular forms of viruses repair themselves?
No. The extracellular forms can’t undergo repair because they are metabolically inert.
What makes viruses unlike cellular life forms?
Virus genomes can be made of either DNA or RNA, however no singular virus has both RNA genomic material and DNA genomic material in its capsid at the same time
The extracellular for is metabolically inert, only the intracellular form is metabolically active, and even then it’s situational
They cannot repair their extracellular forms
Where are you more likely to find an envelope virus: amongst animal viroids, or bacterial viroids?
Animal viroids
How do enveloped viruses mediate adhesion and membrane fusion to their target cells?
Their envelopes are usually studded with glycoproteins, which mediate adhesion and membrane fusion with the target cell.
Describe the double duty positive sense single stranded RNA genomes pull.
They are basically mRNA in addition to being the genome. They are used to make a negative sense copy of the genome in addition to coding for proteins.
What do all RNA viruses, aside from retroviruses, require in order to replicate?
Viral replicase, an RNA-dependent RNAP (RDRP)
How do RNA viruses make a positive sense copy upon entry to start replication if there’s no viral replicase to be found in the host?
They bring the viral replicase with them pre-formed.
What is the function of an interferon to the host cell?
It calls out to other cells to alert them that a viral infection is lurking.
True or false: Intramuscularly injected vaccines are great at stimulating circulating IgG and at producing mucosal antibodies.
False. They’re great at producing IgG antibodies, but not for mucosal antibodies.
How do viruses spread from one species to another?
The virus must undergo either antigenic shift, antigenic drift, or reassortment.
What is antigenic shift?
The rapid alteration in genotype and phenotype of a virus due to the acquisition of a different RNA segment. This is faster than antigenic drift and is the cause of most major influenza variants.
What is antigenic drift?
Mutations in the viral genome caused by RDRPs low fidelity rate (aka high viral mutation rate). This is slower than antigenic shift.
What is reassortment as it relates to viral genomes?
Simultaneous infection of two types of viruses in one host, leading to fragments of both viral genomes being combined into the capsids, creating a new virus. New HA and NA segments can cause especially severe pandemics because no one’s been exposed to the new proteins before, no immunity has been built up.
Why do viruses typically shut down the host translational machinery? What benefit do they gain from this?
Shutting down the host translational machinery shuts off host gene expression, which prevents the host from responding to the viral infection or from sending out an interferon to alert neighboring cells of the lurking virus.
Additionally, this global inhibition of host protein synthesis ensures maximal viral gene expression.
In addition to processing viral proteins, what other functions do viral proteases have?
They’re important for virulence as they stop the host from producing their own proteins, culling any response to the infection (like synthesis of an interferon) and allowing all of the host’s resources to go into viral replication.
Definition of the latent period:
No phages observed extracellularly
Definition of the eclipse period:
No phages seen either extracellularly or intracellularly
Definition of bust size:
A virus-specific measure of how many viruses can be produced from one bacterial cell.
How do T4 and T7 bacteriophages infect E. coli?
They randomly land and attack with their tail fibers (note that all 6 tail fibers must be engaged to initiate infection) to either the LPS core polysaccharide (the least variable portion of the LPS) or to certain strains of OmpC. Binding triggers a protein chain that drives the contractile sheath to push the genome into the cell. These bacteriophages also use lysozyme to penetrate/degrade the peptidoglycan.
What is a prophage?
A virus that incorporates its DNA into the bacterial genome.
What is a lysogen?
A bacterial strain with a prophage in its genome.
What’s the difference between a prophage and a lysogen?
A prophage refers to the viral DNA in a host bacterium’s genome. A lysogen refers to the bacterium that has the prophage in its genome.
True or false: Under most circumstances, most prophage genes are expressed in a lysogen.
False! Under most circumstances, most prophage genes are NOT expressed in a lysogen!
What causes cell death in a viral infection?
Viral gene expression (NOT presence or replication of a viral genome!)
How is a prophage maintained in a lysogen?
By using a viral repressor protein to minimize viral gene expression.
What happens if a lambda lysogen loses its ability to produce the C1 protein?
It will lose control of viral gene expression and the lytic phase will begin.
What is the signal that causes the lambda virus to be resurrected from the host genome and why?
The presence of LexA. It signals that the cell has activated its SOS response and things are going downhill, so it’s time to get out of the host genome and replicate before the host dies.
Lambda virus’s C1 repressor protein has structural and sequence similarity to what bacterial protein?
LexA
What is the effect of DNA damage on RecA?
It stimulates RecA to cleave both LexA and C1
What is the effect of C1 cleavage by RecA?
It releases transcriptional repression on all lambda genes, including Xis → excisionase
What is the function of excisionase in lambda’s viral infection?
Excisionase cuts the prophage out of the E. coli chromosome for circularization.
How is lambda’s prophage genome removed from the host genome?
Excisionase cuts it out
Why, under normal conditions, don’t infectious virions result in the lysis of other cells in a population of lysogens (this population is made up of lysogens of the same virus)?
Immunity. Lysogens are immune to superinfection by the same virus. This is due to the prophage-encoded repressor protein. C1 represses expression of other viral genes, maintaining lysogeny, and it also represses gene expression from the same incoming virus. A lysogen can be killed by other viruses, but not by the one already contained in its genome.
True or false: a lysogen can be killed during a superinfection with the same virus.
False
What mediates the “decision” to progress with lysogeny?
Poor nutrition, because without sufficient energy or resources, synthesis of new viruses is difficult.
Why do high Multiplicities of Infection (MOIs) favor lysogeny of lysis?
Because if a viral population greatly outnumbers the host cell population, the entire host pop. may be killed, forcing the virus to exist free in the environment where it’s vulnerable for longer.
What are restriction endonucleases?
Enzymes that bacteria have evolved to degrade foreign DNA. They cut DNA at specific sequences.
One famous example is TaqPolymerase, used in PCR
What is the Restriction-Modification system and what does it do?
For each restriction endonuclease, there is a cognate DNA methylase that recognizes the same palindrome and adds a methyl group to one nucleotide on each strand. This serves to protect the bacterium’s own DNA. By methylating its inverted repeats that its restriction endonucleases bind to, it prevents binding and subsequent destruction
What is the purpose of a toxin-antitoxin module?
To defend against viral infections and to protect the bacterial population.
How does a toxin-antitoxin module work?
During a viral infection, the virus stops normal host processes, like transcription and translation. In doing so, the host stops producing its toxin and antitoxin molecules. Because the antitoxin molecule is designed to have a shorter half life than the toxin and will subsequently degrade first, the toxin left behind kills the host, preventing the virus infecting it from replicating and releasing 100s of more baby viruses into the surrounding population. It’s almost a form of altruistic apoptosis.
What does CRISPR stand for?
Clustered regularly interspaced palindromic repeats.
What kind of genome do picornaviruses have?
Positive sense ssRNA
What are some examples of picornaviruses?
Hepatitis A, Hand+Foot+Mouth disease, polio, rhinoviruses.
What kind of genome does polio have?
Positive sense ssRNA
Polio can potentially be driven to extinction, although this hasn’t happened yet. What will allow us to one day make it extinct?
Polio has no animal reservoir, and thus nowhere else to hide except for in humans. This is pretty common amongst viruses, as most are specific for only certain tissues in certain species because of the presence or absence of appropriate host receptor molecules.
What host receptor protein does polio use and where are these receptors found?
Uses CD155 (aka necl5), found only on human enterocytes, pharyngeal cells, and the anterior horn of the spinal cord.
Why is polio technically not a central nervous system virus?
It’s not a central nervous system virus because it evolved for our intestinal tract. Entering the CNS is a dead end for them, it’s an accidental infection (GI infections are easy to spread from human to human, CNS infections not so much).
What’s the difference between the virus in the Salk vaccine vs the virus in the Sabin vaccine?
Salk → inactivated virus. Induced predominantly IgG antibodies, gave humoral immunity. Protected from disease, not infection.
Sabin → attenuated virus. Induced serum IgG and mucosal IgA antibodies, gave total immunity from infection, not just the disease
Why did poliomyelitis only become an epidemic disease in the 20th century, and why did it, a fecal contaminating virus, only proliferate in countries with high hygienic standards?
In areas where infants are more likely to be infected soon after birth, polio epidemics didn’t occur because most of the children are protected due to having passive immunity (from their moms) at the time of infection. The moms had antibodies for polio infections, and their passive immunity is still present in their infants very early in their life.
Why is polio an important virus to understand?
Because it represents a large subgroup of the picornaviruses, which are themselves a subgroup of enteroviruses. Also, viruses that infect the enteric (intestinal) tract occasionally can get out and infect other parts of the body (secondary viremia tissue)
What is humoral immunity?
Immunity mediated by antibodies circulating in bodily fluids
Which kind of antibodies did the Salk vaccine induce? What is the significance of this?
Predominantly IgG antibodies. This gave protection from the poliomyelitis disease, but not from poliomyelitis infection. It gave humoral immunity, not intestinal/mucosal immunity.
Which kind of antibodies did the Sabin vaccine induce? What is the significance of this?
Induces serum IgG and mucosal IgA antibodies.
Prevents poliomyelitis infection, not just poliomyelitis disease.
Why would a patient develop paralytic poliomyelitis after taking the Sabin vaccine?
The attenuated virus in the vaccine lost the mutation and became lethal again. The virus reverted to neurovirulence.
Describe the structure of the polio virus
Naked virus, no envelope. Capsid is geometric, isohedral with 20 faces, and is made of 3 proteins (VP1, VP2, VP3). VP4 lines the inner portion of the capsid. Inside, its ssRNA genome is capped by VPg and has a Poly A tail.
How do polio viruses enter host cells?
Their viral proteins (especially VP1) interact with host receptor molecule CD155/necl5, which triggers host receptor mediated endocytosis.
What is the significance of the host’s response of acidifying the endosome after uptaking poliovirus through receptor mediated endocytosis?
In acidic conditions, CD155/necl5 if attached to VP1-3 will undergo a structure change and create a pore in the capsid, allowing the viral genomic contents to be dumped out.
What is the function of polio’s Internal Ribosome Entry Site?
Polio’s IRES tricks our host initiation factors into binding and jumpstarting translation.
True or false: a hallmark of poliovirus is that it can survive the acid in our stomach.
True
How does poliovirus ensure that its proteins appear at temporally appropriate times?
Polio’s proteases have limited autoproteolytic activity, but their activity increases and the polyprotein is broken down. This makes polio’s proteins appear in waves, as the massive polyprotein is sequentially degraded into the individual proteins.
True or false: the poliovirus genome is polycistronic.
False. It’s monocistronic
Does VPg have anything to do with translation?
No. It’s a nuclear protein that functions as a primer for transcription and replication once inside the host.
Where does poliovirus replicate?
In the cytoplasm.
True or false: poliovirus uses the host’s nucleus during replication.
False. It uses only the cytoplasm
How does poliovirus stunt the host’s ability to recognize its infection, how does poliovirus redirect host translation machinery to viral translation?
PV protease 2A cleaves host cellular translation inhibition factor eIF-4G, which renders the host’s ribosomes unable to recognize 5’ capped mRNA.
PV protease 2A also cleaves a protein in the nuclear pore complex, resulting in no macromolecules entering the nucleus and preventing the host from mounting an antiviral response.
True or false: influenza uses only the nucleus when replicating.
False. It uses both the nucleus and the cytoplasm.
What kind of genome does influenza have? What is the significance of this kind of genome upon entry into a host?
Negative sense ssRNA. It means the virus must bring pre-formed RDRP with it as it can’t make its own proteins as it comes in.
Describe the structure of the extracellular form of influenza virus.
Enveloped virus. The envelope is from the host but is studded with exclusively viral proteins. In Influenza A, the genome is broken into 8 different segments, each covered with a nucleocapsid protein and capped with an RDRP on one end.
Why did the 1918 Spanish flu have a pronounced affinity for killing 20-45 y. olds?
The original spanish flu strain was so inflammatory that it caused a cytokine storm. Because that symptoms, damage, and subsequent death come from our immune response and not the virus itself, those with more robust immune systems reacted more to the infection, resulting in stronger cytokine storms and more deaths in the 20-45 age range.
How does influenza accomplish attachment, the first step in their pathogenesis?
Their hemagglutinin molecules studded throughout their envelope attach to host sialic acid receptor molecules, which are widespread on eukaryotic cells.
What is the function of neuraminidase to the influenza virus?
NA cleaves sialic acid. This allows a baby virus, should it get tangled up in host sialic acids as it tries to exit, to be cut free.
You have a patient who you suspect is sick with influenza. To test them, you take a sample that would contain influenza viroids (if they were indeed sick with influenza) and place it in a test tube with a solution of red blood cells. Describe what a positive result would look like.
The RBCs would remain spread out due to hemagglutination caused by the influenza’s HA molecules. The hemagglutination would create bridges between the RBCs, spreading them out. This is different from a negative test, where the RBCs would all clump together in a small dot at the bottom of the test tube.
What is the significance of the host cell’s acidification response to influenza pathogenesis?
The acidification of the contents of the endosome an influenza virus has been taken up in causes a conformational change of the HA molecules, eliciting the fusion of the viral lipid bilayer with the endosomal membrane, dumping the viral contents into the host cytoplasm.
What is the mechanism of action of amantadine/rimantadine antiviral drugs?
They target the cation channel and plug the M2 channel protein, which prevents/hinders endosome acidification.
Why is it difficult for viruses to develop resistance to tamiflu/oseltamivir?
Those antiviral drugs inhibit neuraminidase. Any mutations to NA would kill the virioid’s fitness, as baby viruses are much more likely to get and stay tangled in the host membrane on exit.
Compare and contrast the transcription and replication processes of influenza pathogenesis.
Both create RNA copies of the RNA genome
Transcription:
Uses 5’ methylguanosine cap snatching/decapitation, doesn’t create full length copies of the genome
Replication:
Creates full length copies of the genome, no cap snatching
How does influenza’s polyadenylation strategy differ from our own?
In Influenza, a signal in the negative sense copy of their genome causes the mRNA to be synthesized with a polyA tail.
In humans, a separate polyadenylation enzyme polyadenylates our mRNA.
In general, RNA viruses replicate in the cytoplasm, and DNa viruses replicate in the nucleus. What is an exception to this rule?
Influenza It uses both the nucleus and the cytoplasm to replicate.
Why does Influenza need to use the nucleus during its replication?
Because it infects eukaryotic hosts, and requires two of its genome segments to be alternatively spliced. Since alternative splicing in eukaryotes happens only in the nucleus, Influenza must use the nucleus to maintain pathogenicity.
True or false: Influenza genome segments 7 and 8 are polycistronic.
False. Despite both segments encoding two proteins each, they are monocistronic.
What proteins does Influenza genome segment 7 code for?
Matrix protein 1 (M1) and matrix protein 2 (M2)
What proteins does Influenza genome segment 8 code for?
Nonstructural protein 1 (NS1) and nonstructural protein 2 (NS2)
What is the role of NS1 in influenza pathogenesis?
NS1 binds to and inactivates host nucleoporin transport proteins, preventing host mRNA from exiting the nucleus. This prevents the host from producing interferons.
Which influenza viral proteins are cotranslationally synthesized in the endoplasmic reticulum?
HA, NA, and M2, because afterwards they are all membrane bound and trafficked through the golgi and sent to the host cell membrane, waiting to become capsid envelopes for the future babies.
What signals to the influenza virus that it’s time to switch from transcription to replication?
A critical concentration of the NP, NS1, NS2, and MS1 proteins inside the nucleus. These proteins are synthesized in the cytoplasm, then trafficked back into the nucleus. At the critical concentration the viral RDRP will stop cap snatching 5’ methylguanosine caps and instead start primer-independent replication, resting full length negative sense RNA copies of the RNA genome. Also at this critical concentration RDRP now ignores the Poly A signal and doesn’t synthesize a Poly A tail.
What is HA dependent on for influenza virus to be infectious?
Proteolytic processing by the host. The host must enzymatically cleave the HA into two proteins (though technically they’re still held together by a disulfide linkage)
What would happen to an influenza if its HA wasn’t proteolytically processed?
It would lose its virulence. The proteolytic processing (cleavage) allows for freedom of movement and allows HA to undergo the conformation change in acidic conditions that pulls the viral envelope close to the endosome membrane, allowing them to fuse. Without the proteolytic processing the membranes wouldn’t fuse and the viral contents wouldn’t be dumped into the cell, culling the infection.
Why haven’t we had a major avian flu outbreak yet?
Because only HAs 1, 2, and 3 can infect humans. All others infect some animal, and there hasn’t been a major crossover yet.
Another reason may be receptor locations. It’s been seen in humans infected with bird flu that the HA5 receptors are much deeper in the respiratory tract (making the infection more akin to pneumonia than a typical upper respiratory pneumonia). However, these haven’t caught on as lower respiratory infections are harder to spread to new hosts.
What kind of genome does HIV have?
A diploid positive sense ssRNA retrovirus genome.
Describe the structure of the extracellular form of the HIV virus.
It has an envelope that is studded with viral proteins.
What does HIV do immediately upon entry into a host cell?
It makes a dsDNA copy of the ssRNA genome and embeds it into the host genome using integrase and site-specific recombination.
True or false: HIV enters the host genome using homologous recombination.
False. It uses site-specific recombination.
What does a cell need to be infected by HIV1?
It needs to be permissive, meaning that it has receptor CD4 and either Ccr5 or Cxcr4
Where is CD4 usually found?
On lymphatic cells
Where is Ccr5 usually found?
Predominantly on macrophages
Where is Cxcr4 found?
Predominantly on T cells
Describe the mechanism by which HIV1 binds to a macrophage.
HIV1’s viral binding receptor gp120 undergoes a conformational change when bound to macrophage host receptors CD4 and Ccr5. This conformation change makes gp120 fall off, allowing the gp41 receptor beneath it to undergo a conformational change that exposes its hydrophobic amino acids, which pulls the viral envelope close to the membrane, allowing the two lipid bilayers to fuse, dumping HIV1’s viral contents into the macrophage.
Which influenza proteins are similar to gp120 and gp41 seen in HIV1?
HA1 and HA2
Which HIV strain is seen during the initial infection period and what type of cells does it predominantly infect?
HIV(R5), macrophage trophic strain. Binds to CD4 and Ccr5
Which HIV strain is seen during late state infection and is associated with AIDS? What type of cells does it predominantly infect?
HIV(X4), T-cell trophic strain. Binds to CD4 and Cxcr4
What larger family of viruses does HIV belong to?
Lenti retroviruses (remember that lent means slow and these viruses develop slowly)
True or false: HIV requires a primer to make its dsDNA copy of its genome.
True. It uses reverse transcriptase to synthesize the dsDNA copy, and uses tDNA (specifically tryptophan-charged tDNA) as a primer.
Describe the enzymatic activities of HIV1’s reverse transcriptase.
RDRP (RNA-dependent RNA polymerase, aka reverse transcriptase for RNA synthesis using RNA as a template)
DDRP (DNA-dependent RNA polymerase, aka reverse transcriptase for RNA synthesis using DNA as a template)
RNase H (degrades RNA when it the RNA is base paired with DNA)
How does HIV1 prevent superinfection?
Its nef gene. It codes for HIV1’s transcription repression factor, the protein that causes the infected host to downregulate its CD4 expression. This prevents superinfection, as other HIV1 bugs can no longer bind.
How does HIV1 ensure that GAG, a protein that is encoded on unspliced viral mRNA, is expressed?
Rev.
Rev mediates the transport of unspliced transcripts from the nucleus into the cytoplasm. Humans have very robust mechanisms to ensure mRNA doesn’t leave the nucleus, so this protein is necessary to circumvent these host mechanisms.
What is the role of HIV1’s Vpu gene?
The protein it codes for functions as NA does for influenza. It dissociates the virus from the cell should its gp120 get tangled in the host’s receptors on the way out.
Which of HIV1’s viral proteins is similar to influenza’s NA proteins?
Vpu
Describe the temporal aspect of HIV1’s gene expression.
The first of its mRNA transcripts to leave the nucleus are fully spliced, as the host’s blockade mechanisms preventing unspliced mRNA from entering the cytoplasm are still in place. Rev is encoded on one of these fully spliced mRNAs, and once synthesized re-enters the nucleus and binds to the Rev-Responsive Element, a tertiary RNA structure found on singly-spliced and unspliced HIV1 mRNAs. Once bound, it allows the minimally processed transcripts to circumvent the host’s blockade.
The use of Rev ensures that HIV1 gene expression happens in waves and that the proteins encoded on less-processed mRNAs aren’t expressed until the proteins on fully processed mRNA are.
What 3 genes are conserved across retroviruses?
GAG, POL, and ENV. There are some retroviruses with no more than these three genes. They are the bare minimum.
What is GAG, what does it code for, is it translated from processed or unprocessed mRNA?
Group antigens. These are the proteins that make up the capsid and envelope proteins. They are translated from unspliced mRNA
What is POL, what does it code for, is it translated from processed or unprocessed mRNA?
Polymerase. RNA-dependent-DNA-polymerase (RDDP), aka reverse transcriptase. Translated from unspliced mRNA
What is ENV, what does it code for, is it translated from processed or unprocessed mRNA?
Envelope proteins. Translated from spliced mRNA.
Even though the retroviral genome is basically mRNA (single strand positive sense), it can’t be directly translated immediately upon entry into a host cell. Why?
Splicing. The retroviral mRNA needs to be spliced, which only happens in the nucleus. This especially applies to HIV1, as it has a bunch of accessory proteins and its mRNA needs a lot of heterogeneous splicing (it has hella reading frames).
What type of genome does corona have?
Positive sense ssRNA (translated immediately upon entry)
Describe the structure of corona’s extracellular form.
Its capsid is enveloped with a lipid bilayer populated with spike proteins, the most common of which is Spike glycoprotein.
True or false: SARS-CoV2’s RDRP has proof-reading activity.
True. Its genome is enormous and it benefits from a higher fidelity RDRP
When is the most likely time frame during which coronaviruses first entered into humans?
In the 1890s, during the Russian flu pandemic.
Prediction technology has dated corona’s entry into humans to around the 1880s/1890s, and at this time there was a known outbreak of a corona respiratory virus in cattle. “Coincidentally”, the Russian flu pandemic of 1889-1891 happened at this time, the symptoms of which are remarkably similar to those of all other coronavirus infections.
Why are bats so frequently indicated in spillover events?
Their immune systems are hyperactive, allowing constant viral control.
Compared with terrestrial (non-flying) mammals, bats have a longer lifespan and a greater capacity to co-exist with a variety of viruses. Bats are a great reservoir because they are flying mammals, as flight has a tremendous metabolic demand (remember from 204 that birds by necessity have shrunk their genomes and hollowed their bones so they can fly).
How does CoV2/Covid-19 enter a host cell to begin infection?
Its viral spike proteins drive infection by binding to ACE2 (antigen-converting enzyme 2), where it is then proteolytically processed by protease cleavage by another host receptor protein.
The ACE2 receptor is widespread in humans, allowing for a multisystem infection.
Once the receptors and spike proteins bind, the host cell and viral envelope membranes fuse, allowing the viral contents to be dumped in.
What feature of SARS-CoV2 allows it to infect cells from multiple species?
Its spike protein has a polybasic cleavage site. This allows it to bind and be proteolytically processed (protease cleavage) by multiple different receptor molecules.
Corona needs to produce multiple proteins from the same piece of mRNA, but it infects only eukaryotic cells and eukaryotic ribosomes can’t bind internally to mRNA. How does it solve this problem?
Its mRNA genome is translated as one large polyprotein all at once, then its pp1ab protein cleaves the polyprotein into the 16 individual proteins using its autoproteolytic activity. This autoproteolytic cleavage mostly likely happens in waves, again leading to temporal regulation of viral gene expression.
True or false: The ribosome sometimes stops at the pp1ab/ORF1b site and sometimes goes on through.
False. This site is a hard stop, as it is the site just before the genes for the structural and accessory proteins (these genes require some gymnastics to translate)
When mRNA is synthesized in a lab and injected into a subject, it causes a massive inflammatory response. Why does this happen? Why does inflammation occur in response to foreign mRNA but not our own?
The nucleosides in the foreign mRNA aren’t modified, likely causing the inflammation. Modified nucleoside bases are seen in our own mRNA and tRNA, likely preventing the inflammatory response.
How is the expression of Gag and Pol proteins regulated?
In all retroviruses, the gag gene is located in the first reading frame on the genome, upstream of pol. Gag is translated/synthesized until the ribosome encounters a stop codon. The ribosome then uses a frameshift mechanism to pause, shift backwards by one nucleotide, then continue on (this happens just before the Gag gene’s stop codon, so the ribosome simply glides on through to synthesize Gag-Pol)
What feature of SARS-CoV2 allows it to infect cells from multiple species?
Its spike protein has a polybasic cleavage site. This allows it to bind and be proteolytically processed (protease cleavage) by multiple different receptor molecules.
Corona needs to produce multiple proteins from the same piece of mRNA, but it infects only eukaryotic cells and eukaryotic ribosomes can’t bind internally to mRNA. How does it solve this problem?
Its mRNA genome is translated as one large polyprotein all at once, then its pp1ab protein cleaves the polyprotein into the 16 individual proteins using its autoproteolytic activity. This autoproteolytic cleavage mostly likely happens in waves, again leading to temporal regulation of viral gene expression.
What happens once corona enters the host?
Immediately upon entry into the host, the ORF1a and ORF1b genes on the mRNA are translated.
The translation of ORF1a and ORF1b from the genomic RNA produces two polyproteins, pp1a and pp1ab, respectively. The latter results from a programmed –1 ribosomal frameshift at the short overlap of ORF1a and ORF1b.
Sixteen non-structural proteins are co-translationally and post-translationally released from pp1a (nsp1–11) and pp1ab (nsp1–10, nsp12–16) upon proteolytic cleavage by two cysteine proteases.
True or false: The ribosome sometimes stops at the pp1ab/ORF1b site and sometimes goes on through.
False. This site is a hard stop, as it is the site just before the genes for the structural and accessory proteins (these genes require some gymnastics to translate)
Describe all the bs with nested RNA and corona’s replication process.
A hallmark of coronaviruses is the discontinuous viral transcription process that produces a set of nested subgenomic RNAs (sgRNAs).
During negative-strand RNA synthesis, the RTC interrupts transcription following the encounter of transcription regulatory sequences (TRSs), located upstream to most ORFs. At these TRS elements, also called TRS ‘body’, the synthesis of the negative-strand RNA stops and is re-initiated at the TRS adjacent to a leader sequence (TRS-L).
This discontinuous step of coronavirus RNA synthesis involves the interaction between complementary TRSs of the nascent negative strand RNA (negative-sense TRS body) and the positive strand genomic RNA (positive-sense TRS-L).
Upon re-initiation of RNA synthesis at the TRS-L region, a negative strand copy of the leader sequence is added to the nascent RNA to complete the synthesis of negative-strand sgRNAs. The discontinuous step of negative strand RNA synthesis results in the production of a set of negative-strand sgRNAs that are then used as templates to synthesize a characteristic nested set of positive-sense sg mRNAs that are translated into structural and accessory proteins.
What function does the ExoN protein of coronaviruses serve?
Enhances proofreading activity during genome replication, part of the RTC
Why is Influenza considered a “mixing bowl” in pigs?
Pigs can be co-infected with human and avian Influenza strains, leading to reassortment.
What is the role of the TMPRSS2 protease in SARS-CoV-2 infection?
Cleaves the Spike protein to facilitate membrane fusion.
What distinguishes M-tropic and T-tropic HIV strains?
Their ability to use Ccr5 vs Cxcr4 coreceptors and their ability to infect either macrophages or T-cells
What is the role of nsp1 in SARS-CoV-2 infection?
Binds to ribosomes, prevents host mRNA from being translated (also degrades host mRNA?)
(In reference to the toxin-antitoxin antiviral bacterial system) How have bacteriophages evolved a way around this system?
The T4 bacteriophage has evolved a protein that mimics the antidote. The protein has enough structural similarity that it binds to the toxin and neutralizes it.
Latent period
Eclipse period
Rise period
Latent period → no extracellular bacteriophages
Eclipse period → no extra or intracellular bacteriophages (happens during the latent period)
Rise period → extracellular phage count increases
What is T6SS?
The Type IV secretion system
Why is amantadine no longer effective (what resistance strategy was developed against it)?
Influenza B was already naturally resistant.
Influenza A eventually evolved a single amino acid change in the transmembrane portion of the M2 protein.
Why are PB1-F2 strains of Influenza much more virulent?
PB1-F2 strains are generated from an alternate reading frame of PB1. PB1-F2 strains blocks a host’s mitochondrial apoptosis response, causing these strains to be more virulent and deadly to host organisms.
PB1-F2 can bind to Mitochondrial Antiviral Signaling protein (MAVS), the protein that turns on programmed cell apoptosis. It also inserts itself into the mitochondrial inner membrane to diminish the membrane’s charge differential, depleting the host’s PMF. These two actions prevent MAVS from inducing an interferon and apoptosis.
Explain why the H5N1 strain of Influenza can cause very severe viral pneumonia in the lungs, but cannot be easily transmitted by coughing or sneezing, as the H1N1 strain can.
It’s a lower respiratory infection, which isn’t as easily sneezed out like an upper respiratory infection (H1N1) is.
Why is it so important for HA to be proteolytically cleaved before the viral particle infects/enters another cell? How does this relate to the virus possibly being unable to be spread from person-to-person?
The proteolytic cleaving gives the HA the freedom of movement to undergo the acid-induced conformational change necessary to pull the endosomal membrane close to the viral envelope. Without this processing the viral contents would never leave their capsid.
Does HIV need to bring more than just the RdRP with it pre-formed?
Yes
It brings RdRP (reverse transcriptase), integrase, and some tRNA molecules (primers)
How is polio virus able to “trick” host ribosomes and use them for its advantage? How, specifically, are polio virus mRNAs transcribed and translated
Upon entry into the cytoplasm, poliovirus uses its IRESs (Internal Ribosome Entry Site) to mediate translation of its polyprotein. This polyprotein has autoproteolytic activity, and is sequentially degraded in waves. The positive sense ssRNA genome is then translated into negative sense ssRNA antigenome templates, using VPg as a primer (polio brought in pre-formed in its capsid)
What is the function of the Primer Binding Site (PBS) in the retroviral genome?
It’s the site (downstream of the 5’ UTR) where tRNA binds to initiate reverse transcription. It is also the attachment site where integrase cuts and pastes the 3’ long terminal repeat into the host genome.
Explain the function of some drugs targeting reverse transcription in retroviruses. Why is Reverse Transcriptase a major Achilles heel of HIV and retrovirus replication?
Reverse-transcriptase inhibitors are a class of antiretroviral drugs used to treat HIV and sometimes Hep B. REverse transcriptase is a good target here because they are the only enzyme required for the synthesis of DNA (ss and ds) from viral RNA. Knocking out the reverse transcriptase will effectively shut down the retrovirus.
What is a Multinucleate Syncytium? How does this occur?
A multinucleate syncytium is when several cells fuse together into one large cell or when several different viruses infect one cell. Multinucleate syncytiums caused by viruses happens when a cell is particularly permissive to multiple different viral pathogens.
Under what conditions is the AB toxin produced by the bacterium causing Diphtheria?
In the absence of iron.
Iron regulates the AB toxin’s synthesis. The region upstream of the AB toxin gene contains a sequence that is bound by an iron-binding repressor. This repressor protein dissociates only in the absence/low concentrations of iron.
Describe the receptor-mediated endocytic event with clathrin-coated pits when the AB toxin is secreted. What is the difference, chemically, between an inactive and active diphtheria toxin?
The B portion of the toxin binds to a specific receptor on a toxin-susceptible cell membrane, causing host-mediated endocytosis. Acidification of the endosome induces a conformational change in the B portion of the toxin, exposing hydrophobic regions of the T domain, which then interacts with the endosomal membrane to allow the now (barely) denatured A toxin fragment into the cytoplasm.
Active diphtheria toxin’s B portion is denatured, allowing the A portion to catalyze the transfer of an ADP-ribosyl moiety from NAD to EF-2 (elongation factor 2, it’s a GTP-binding protein involved in protein synthesis in eukaryotic cells). The now ADP-ribosylated EF-2 can’t mediate polypeptide elongation, shutting down host protein synthesis.
What is ADP-ribosyl transferase and what is its function?
The enzyme that catalyzed ADP-ribosylation (the addition of one or more ADP-ribose moieties to a protein, a reversible post-translational modification found in many cellular processes)
The Type III Secretion System is fairly widespread among pathogenic bacteria, for the purpose of secreting toxins directly into the host cells. What other bacterial structure is this system similar to?
It’s similar to the Type IV secretion system that conjugative plasmids use to infect other bacteria?
What is a characteristic of pathogenicity islands (makeup of the DNA)?
Pathogenicity islands are a class of genomic islands acquired by horizontal gene transfer (conjugation, transformation, transduction). Their characteristics include virulence genes, functional mobility elements, and areas of homology to tRNA genes and direct repeats. Pathogenicity islands enable a host to induce disease and can spread antibiotic resistance.
They are gene clusters present in pathogenic organisms but absent in nonpathogenic organisms of the same or closely related species.
All islands have GC content that differs from the surrounding DNA sequence, a connection with tRNA genes, flanking repeat sequences, and the capacity to recombine (meaning they usually contain integrase)
What gives Salmonella an advantage over other bacteria in the intestinal lumen?
It has a Type III secretion system and can secrete effector proteins into other nearby bacterial species?
It has at least five distinct pathogenicity islands…
It can use EMP glycolysis, pyruvate oxidation, and the TCA cycle? Some bugs can’t do all three…
Polio, influenza, HIV, corona: Genome type
+ssRNA, -ssRNA, diploid +ssRNA, +ssRNA
Polio, influenza, HIV, corona: Envelope
No, yes, yes, yes
Polio, influenza, HIV, corona: temporal aspect
Autoproteolytic activity, splicing, splicing, autoproteolytic activity
Polio, influenza, HIV, corona: entry
Endosome+acid, endosome+acid, wall+env fusion, wall+env fusion
Polio, influenza, HIV, corona: blocking host processes
PV protease 2A, NS1 (sometimes PB1-F2), Nef, NSp1
Polio, influenza, HIV, corona: replication location
Cytplasm, nucleus+cytoplasm, nucleus, replication organelles
Polio, influenza, HIV, corona: frameshifts
No, yes (PB1 vs PB1-F2), yes(gag vs gag-pro-pol), yes (ORF1a vs ORF1b)
Polio, influenza, HIV, corona: proofreading
No, no, no, ExoN/NSp14 built into RTC
Polio, influenza, HIV, corona: viral receptor
VP1-3, HA1+HA2, gp120+gp41, spike glycoprotein
Polio, influenza, HIV, corona: host receptor
CD155/necl5, sialic acid, CD4+Ccr5+Cxcr4, ACE2
