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Do they just pass through the membrane? Is there some specific transporter or mechanism? Does it vary?
I've seen pictures if retroviruses outside the cell but no details
Retroviruses, like many other enveloped viruses, exit the cell by a process called budding. The virus uses the host machinery for producing transmembrane proteins to enrich areas of the host cell membrane in viral transmembrane proteins, and co-opts a host process for releasing vesicles. You can see budding illustrated in the context of the life cycle of HIV in this figure from Murray Medical Microbiology.
The process has been studied in some detail in HIV and other retroviruses.
Retroviruses are unable to cross the cell membrane
The diversity of LTR retrotransposons
Eukaryotic genomes are full of long terminal repeat (LTR) retrotransposons. Although most LTR retrotransposons have common structural features and encode similar genes, there is nonetheless considerable diversity in their genomic organization, reflecting the different strategies they use to proliferate within the genomes of their hosts.
Transposons are mobile genetic elements that can multiply in the genome using a variety of mechanisms. Retrotransposons replicate through reverse transcription of their RNA and integration of the resulting cDNA into another locus. This mechanism of replication is shared with retroviruses, with the difference that retrotransposons do not form infectious particles that leave the cell to infect other cells. The long terminal repeat (LTR) retrotransposons, one of the main groups of retroelements (which include both LTR and non-LTR retrotransposons as well as retroviruses), are among the most abundant constituents of eukaryotic genomes. The LTRs are the direct sequence repeats that flank the internal coding region, which - in all autonomous (functional) LTR retrotransposons - includes genes encoding both structural and enzymatic proteins. The gag gene encodes structural proteins that form the virus-like particle (VLP), inside which reverse transcription takes place. The pol gene encodes several enzymatic functions, including a protease that cleaves the Pol polyprotein, a reverse transcriptase (RT) that copies the retrotransposon's RNA into cDNA, and an integrase that integrates the cDNA into the genome.
Much of what we know about the mechanisms of LTR retrotransposition (Figure 1) comes from work on yeast retrotransposons [1, 2], but it is generally assumed that the mechanism is very similar among LTR retrotransposons from divergent hosts. First, a retrotransposon's RNA is transcribed by the cellularly encoded RNA polymerase II from a promoter located within the 5' LTR. The RNA is then translated in the cytoplasm to give the proteins that form the VLP and carry out the reverse transcription and integration steps. Typically, two RNA molecules are packaged into one virus-like particle, and the RNA is subsequently made into a full-length DNA copy through a reverse transcription reaction that is first primed from a tRNA that pairs to a sequence near the 5' LTR (the primer-binding site). The resulting partial cDNA (called 'strong stop' DNA) is transferred from the 5' LTR to the 3' LTR, where reverse transcription proceeds. A second priming event initiates at a polypurine tract near the 3' LTR. The cDNA primed from the polypurine tract undergoes an additional strand transfer, ultimately giving rise to a double-stranded cDNA molecule. Finally, the cDNA is integrated back into the host DNA, adding another copy of the retrotransposon to the genome.
The life cycle of LTR retrotransposons. IN, integrase PR, protease RT, reverse transcriptase VLP, virus-like particle. Black triangles represent the LTRs.
The development and survival of a multicellular organism relies on cells proliferating by passing through a series of events known as the cell cycle. During the first stage of the cycle (G1), each cell makes an important decision: does it progress to the second stage (S-phase) and replicate its genome in preparation for cell division, or does it exit the cycle and become quiescent (Pardee, 1974 Johnson and Skotheim, 2013)?
Cells usually exit the cell cycle in order to differentiate into the distinct cell types of an organism, such as neurons, muscle or fat (Sun and Buttitta, 2017). If the decision to stop proliferating is somehow disrupted, this can affect the normal development or function of tissues and organs and lead to diseases, such as cancer. However, understanding the signals that control this decision can be challenging because a G1 cell preparing to enter S-phase is essentially indistinguishable from a G1 cell that will become quiescent.
One way to overcome this challenge is to visually monitor proteins that are only active in certain phases of the cell cycle (Sakaue-Sawano et al., 2008 Zielke et al., 2014 Grant et al., 2018). For example, a group of proteins called cyclin-dependent kinases (or CDKs for short), which drive cells into S-phase, are active in G1 but are permanently turned off when cells enter a state of quiescence.
In 2013, a group of researchers used this property of CDKs to distinguish cultured mammalian cells in G1 that were preparing to proliferate from those entering quiescence (Spencer et al., 2013). To do this they engineered a fluorescent reporter protein which sits in the nucleus when CDKs are inactive and moves into the cytoplasm when modified by active CDKs (Figure 1A). The nucleus and cytoplasm are easily distinguishable via time-lapse microscopy, making it possible to determine when CDKs are active in individual cells during G1.
Monitoring cell cycle progression using a fluorescent biosensor.
(A) As cells grow and prepare to divide they pass through four different phases of the cell cycle: G1, S, G2, M. During the course of this cycle, the activity of cyclin-dependent kinases (CDKs) changes. These fluctuations in activity can be monitored using a fluorescent reporter protein that contains a portion of human DNA helicase B (DHB), which moves from the nucleus to the cytoplasm when phosphorylated by active CDKs. Thus, changes in the levels of DHB in the nucleus and cytoplasm (depicted in shades of orange/red) can be used to determine a cell’s CDK activity. At the start of G1, CDK activity is low and DHB remains in the nucleus (shown in red). Some G1 cells maintain this low level of activity (CDK low ) and exit the cell cycle to become quiescent, while cells with increasing levels of CDK activity (CDK inc ) commit to another cell cycle and enter S-phase. (B) Adikes et al. showed that the DHB reporter could monitor cell cycle progression of individual cells in live animals, such as the embryo of a zebrafish. It can also identify which cells have exited the cell cycle and which are preparing for division.
Image credit: Joy H Meserve.
Now, in eLife, David Matus from Stony Brook University and co-workers – including Rebecca Adikes, Abraham Kohrman, and Michael Martinez as joint first authors – report how they used this technique to visualize when individual cells decide to stop dividing in living animals (Adikes et al., 2020). To adapt the CDK reporter to animals, the team (who are based at Stony Brook, the University of Colorado, Stanford University, Imperial College and University of Virginia) turned to two well studied experimental organisms: the nematode worm C. elegans and the zebrafish D. rerio.
Previous work using the CDK reporter in cultured mammalian cells showed that not all G1 cells behave the same after cell division: some never activate CDKs and enter quiescence (CDK low cells), while others begin increasing CDK activity during G1 (CDK inc cells) and ultimately commit to another cell cycle (Figure 1A Spencer et al., 2013). Adikes et al. found that this bifurcation in G1 cells could also be detected in the tissues of living C. elegans and D. rerio (Figure 1B). They found that the CDK low and CDK inc phenotypes of G1 cells could be used to predict whether a cell would enter quiescence or would re-commit to another cycle. Further experiments revealed that if a cell had high levels of a protein called p21, which inhibits the activity of CDKs, its daughter cells were more likely to become quiescent following division. This suggests that the decision to proliferate or exit the cell cycle may depend on how p21 levels are regulated in proliferating cells (Overton et al., 2014 Hsu et al., 2019).
The CDK reporter has a number of applications. It could make it easier to study how quiescence is regulated in tissues that are typically difficult to image for long periods of time. It might reveal early steps in tissue regeneration when cells are ramping up to re-enter the cell cycle. It could also be used to sort and recover populations of CDK low and CDK inc cells for further experiments to identify the pathways regulating entry into quiescence.
The CDK reporter will allow us to tackle many interesting questions in developmental biology. For instance, how might the organization of a tissue influence the decision to stop dividing and enter quiescence? Is it possible to identify cells very early in the differentiation process before genes that demark differentiation turn on? Whatever the application or research question, Adikes et al. demonstrate once again that important new insights into the complexities of biology arise when new tools are developed to visualize living organisms.
“Know your enemy,” Sun Tzu, the great sage of war, wrote some 2,500 years ago. Today, as COVID-19 spreads around the globe, the greatest army of medical scientists ever assembled is bent on learning all it can, as fast as it can, about SARS-CoV-2, the virus behind the pandemic.
Here’s a primer on viruses in general and SARS-CoV-2 in particular. As researchers learn more and more about the novel coronavirus that causes COVID-19, this knowledge — gathered through unmatched levels of scientific cooperation — is being turned against the virus in real time.
Not that this will be a simple pursuit. Compared with a lab dish, living people are complicated. The cells in that dish aren’t the same as the cells in living tissues affected by SARS-CoV-2. Plus, the environment surrounding, say, a lung cell in a person’s body is different from the one in a culture dish. And then there’s this thing called “side effects.” You don’t see those in a dish. But you may in a COVID-19 patient.
Illustration by Jeffrey Decoster
What, exactly, is a virus, anyway?
Viruses are easily the most abundant life form on Earth, if you accept the proposition that they’re alive. Try multiplying a billion by a billion, then multiplying that by 10 trillion. That — 10 to the 31st power — is the mind-numbing estimate of how many individual viral particles populate the planet.
Is a virus a living thing? Maybe. Sometimes. It depends on location. “Outside of a cell, a viral particle is inert,” virologist Jan Carette, PhD, associate professor of microbiology and immunology, told me. On its own, it can’t reproduce itself or, for that matter, produce anything at all. It’s the ultimate parasite.
Or, you could say more charitably, it’s very efficient. Viruses travel light, packing only the baggage they absolutely need to hack into a cell, commandeer its molecular machinery, multiply and make an escape.
There are exceptions to nearly every rule, but viruses do have things in common, said Carette.
A virus’s travel kit always includes its genome — its collection of genes, that is — and a surrounding protein shell, or capsid, which keeps the viral genome safe, helps the virus latch onto cells and climb inside, and, on occasion, abets a getaway by its offspring. The capsid consists of identical protein subunits whose shapes and properties determine the capsid’s structure and function.
Some viruses also wear greasy overcoats, called envelopes, made from stolen shreds of the membranes of the last cell they infected. Coronaviruses have envelopes, as do influenza and hepatitis C viruses, herpesviruses and HIV. Rhinoviruses, which are responsible for most common colds, and polioviruses don’t.
Enveloped viruses particularly despise soap because it disrupts greasy membranes. Soap and water are to these viruses what exhaling garlic is to a vampire, which is why washing your hands works wonders.
How do viruses enter cells, replicate and head for the exits?
For a virus to spread, it must first find a way into a cell. But, said Carette, “penetrating a cell’s perimeter isn’t easy.” The outer membranes of cells are normally tough to get into without some kind of special pass. Viruses have ways of tricking cells into letting them in, though.
Typically, a portion of the virus’s cloak will have a strong affinity to bind with one or another protein that dots the surfaces of one or another cell type. The binding of the virus with that cell-surface protein serves as an admission ticket, easing the virus’s invasion of the cell.
The viral genome, like ours, is an instruction kit for the production of proteins the organism needs. This genome can be made up of either DNA, as is the case with all creatures except for certain viruses, or DNA’s close chemical relative RNA, which is much more flexible and somewhat less stable. SARS-CoV-2’s genome is made of RNA, as are the genomes of most mammal-infecting viruses.
In addition to the gene coding for its capsid protein, every virus needs another gene for its own version of an enzyme known as a polymerase. Inside the cell, viral polymerases generate numerous copies of the invader’s genes, from whose instructions the cell’s obedient molecular assembly line produces capsid subunits and other viral proteins.
Among these can be proteins capable of co-opting the cellular machinery to help viruses replicate and escape, or of tweaking the virus’s own genome — or ours. Depending on the type of virus, the genome can contain as few as two genes — one for the protein from which the capsid is built, the other for the polymerase — or as many as hundreds.
Capsids self-assemble from their subunits, often with help from proteins originally made by the cell for other purposes, but co-opted by the virus. Fresh copies of the viral genome are packaged inside newly made capsids for export.
Often, the virus’s plentiful progeny punish the good deed of the cell that produced them by lysing it — punching holes in its outer membrane, busting out of it and destroying the cell in the process.
But enveloped viruses can escape by an alternative process called budding, whereby they wrap themselves in a piece of membrane from the infected cell and diffuse through the cell’s outer membrane without structurally damaging it. Even then, the cell, having birthed myriad baby viruses, is often left fatally weakened.
Illustration by Jeffrey Decoster
Introducing the coronavirus, and how it latches on
Now we know how your average virus — an essentially inert particle on its own — manages to enter cells, hijack their molecular machinery, make copies of itself and move on out to infect again.
That just scratches the surface. Of the millions of different viral species identified so far, only about 5,000 have been characterized in detail. Viruses come in many shapes and sizes — although they’re all small — and infect everything, including plants and bacteria. None of them works in precisely the same way.
So what about coronaviruses?
Enveloped viruses tend to be less hardy when they’re outside of cells because their envelopes are vulnerable to degradation by heat, humidity and the ultraviolet component of sunlight.
This should be good news for us when it comes to coronaviruses. However, the bad news is that the coronavirus can be quite stable outside of cells because its spikes, protruding like needles from a pincushion, shield it from direct contact, enabling it to survive on surfaces for relatively long periods. (Still, soap or alcohol-based hand sanitizers do a good job of disabling it.)
As mentioned earlier, viruses use proteins that are sitting on cells’ surfaces as docking stations. Coronaviruses’ attachment-enabling counterpart proteins are those same spikes.
But not all coronavirus spikes are alike. Relatively benign coronavirus variants, which at their worst might cause a scratchy throat and sniffles, attach to cells in the upper respiratory tract — the nasal cavities and throat. The viral variant that’s driving today’s pandemic is dangerous because its spike proteins can latch onto cells in the lower respiratory tract — the lung and bronchial cells — as well as cells in the heart, kidney, liver, brain, gut lining, stomach or blood vessels.
Antibody treatments could block binding
In a successful response to SARS-CoV-2 infection, the immune system manufactures a potpourri of specialized proteins called antibodies that glom on to the virus in various places, sometimes blocking its attachment to the cell-surface protein it’s trying to hook onto.
Stanford is participating in a clinical trial, sponsored by the National Institutes of Health, to see if antibody-rich plasma (the cell-free part of blood) from recovered COVID-19 patients (who no longer need these antibodies) can mitigate symptoms in patients with mild illness and prevent its progression from mild to severe.
Monoclonal antibodies are to the antibodies in convalescent plasma what a laser is to an incandescent light bulb. Biotechnologists have learned how to identify antibody variants that excel at clinging to specific spots on SARS-CoV-2’s spike protein, thus thwarting the binding of the virus to our cells — and they can produce just those variants in bulk. Stanford is conducting a clinical trial of a monoclonal antibody for treating COVID-19 patients.
A worry: Viral mutation rates are much higher than bacterial rates, which dwarf those of our sperm and egg cells. RNA viruses, including the coronavirus, mutate even more easily than DNA viruses do: Their polymerases (those genome-copying enzymes mentioned earlier) are typically less precise than those of DNA viruses, and RNA itself is inherently less stable than DNA. So viruses, and particularly RNA viruses, easily develop resistance to our immune system’s attempts to find and foil them.
The Stanford studies may help reveal whether the precision-targeted “laser” or kitchen-sink “lightbulb” approach works best.
The virus breaks into a cell
Assistant professor of chemical engineering and subcellular-compartment spelunker Monther Abu-Remaileh, PhD, described two key ways the coronavirus breaks into a cell and seeks comfort there, and how it might be possible to bar one of those entry routes with the right kind of drug.
Here’s one way: Once the coronavirus locks on to a cell, its greasy envelope comes into contact with the cell’s equally greasy outer membrane. Grease loves grease. The viral envelope and cell membrane fuse, and the viral contents dump into the cell.
The other way is more complicated. The viral attachment can set off a process in which the area on the cell’s outer membrane nearest the spot where the contact has been made caves in — with the virus (happily) trapped inside — until it gets completely pinched off, forming an inbound, membrane-coated, liquid-centered capsule called an endosome inside the cell. (To visualize this, imagine yourself with a wad of bubble gum in your mouth, blowing an internal bubble by inhaling, and then swallowing it. In this analogy, you’re the cell and all your skin, beginning with your lips, constitutes the cell’s outer membrane.)
Enclosed in this endosome is the viral particle that set the process in motion. The little devil has just hooked itself a ride into the cell’s inner sanctum. At this point, the viral particle consists of its envelope, its capsid and its enclosed genome — a blueprint for the more than two dozen proteins the virus needs and the invaded cell doesn’t provide.
But the endosome doesn’t remain an endosome indefinitely, Abu-Remaileh told me. Its mission is to become another entity called a lysosome, or to fuse with an existing lysosome.
Lysosomes serve as cells’ recycling factories, breaking down large biomolecules into their constituent building blocks for reuse. For this, they need an acidic environment, generated by protein pumps on their surface membranes that force protons into these vesicles.
The building internal acidity activates enzymes that chew up the cloistered coronavirus’s spike proteins. That brings the virus’s envelope in contact with the vesicle membrane and enables their fusion.
The viral genome gets squirted out into the greater expanse of the cell. There, the viral genome will find and commandeer the raw materials and molecular machinery required to carry out its genetic instructions. That machinery will furiously crank out viral proteins — including the customized polymerase SARS-CoV-2 needs to replicate its own genome. Copies of the genome and the virus’s capsid proteins will be brought together and repackaged into viral progeny.
A pair of closely related drugs, chloroquine and hydroxychloroquine, have gotten tons of press but, so far, mostly disappointing results in clinical trials for treating COVID-19. Some researchers advocate using hydroxychloroquine, with the caveat that use should be early in the course of the disease.
In a lab dish, these drugs diffuse into cells, where they diminish acidity in endosomes and prevent it from building up in lysosomes. Without that requisite acidity, the viral-membrane spike proteins can’t be chewed up and the viral envelope can’t make contact with the membrane of an endosome or lysosome. The virus remains locked in a prison of its own device.
That’s what happens in a dish, anyway. But only further clinical trials will tell how much that matters.
How the coronavirus reproduces
SARS-CoV-2 has entered the cell, either by fusion or by riding in like a Lilliputian aquanaut, stealthily stowed inside an endosome. If things go right for the virus, it fuses with the endosome’s membrane and spills its genome out into the (relatively) vast surrounding cellular ocean.
That lonely single strand of RNA that is the virus’s genome has a big job to do — two, in fact, Judith Frydman, PhD, professor of biology and genetics, told me — in order to bootstrap itself into parenting a pack of progeny. It must replicate itself in entirety and in bulk, with each copy constituting the potential seed of a new viral particle. And it must generate multiple partial copies of itself — sawed-off sections that serve as instructions, telling the cell’s protein-making machines, called ribosomes, how to manufacture the virus’s more than two dozen proteins.
To do both things, the virus needs a special kind of polymerase. Every living cell, including each of ours, uses polymerases to copy its DNA-based genome and to transcribe its contents (the genes) into RNA-based instructions that ribosomes can read.
The SARS-CoV-2 genome, unlike ours, is made of RNA, so it’s already ribosome-friendly, but replicating itself means making RNA copies of RNA. Our cells never need to do this, and they lack polymerases that can.
SARS-CoV-2’s genome, though, does carry a gene coding for an RNA-to-RNA polymerase. If that lone RNA strand can find and insert itself into a ribosome, the latter can translate the viral polymerase’s genetic blueprint into a working protein. Fortunately for the virus, there can be as many as 10 million ribosomes in a single cell.
Once made, the viral polymerase cranks out not only multiple copies of the full-length viral genome — replication — but also individual viral genes or groups of them. These snippets can clamber aboard ribosomes and command them to produce the entire repertoire of all the proteins needed to assemble numerous new viral offspring.
These newly created proteins include, notably, more polymerase molecules. Each copy of the SARS-CoV-2 genome can be fed repeatedly through prolific polymerase molecules, generating myriad faithful reproductions of the initial strand.
Well, mostly faithful. We all make mistakes, and the viral polymerase is no exception actually it’s pretty sloppy as polymerases go — much more so than our own cells’ polymerases, Carette and Frydman told me. So the copies of the initial strand — and their copies — are at risk of being riddled with copying errors, aka mutations.
However, coronavirus polymerases, including SARS-CoV-2’s, come uniquely equipped with a sidekick “proofreader protein” that catches most of those errors. It chops out the wrongly inserted chemical component and gives the polymerase another, generally successful, stab at inserting the proper chemical unit into the growing RNA sequence.
Coronavirus birth control
The experimental drug remdesivir, approved for emergency use among hospitalized COVID-19 patients, directly targets RNA viruses’ polymerases.
Stanford participated in clinical trials leading to this injectable drug’s approval. Initially developed for treating Ebola virus infection, it belongs to a class of drugs that work by posing as legitimate chemical building blocks of a DNA or RNA sequence. These poseurs get themselves stitched into the nascent strand and gum things up so badly that the polymerase stalls out or produces a defective product.
“Now, with the drug, the virus starts making a lot of rotten genomes that poison the viral replication process,” said Frydman.
Remdesivir has the virtue of not messing up our cells’ own polymerases, said Robert Shafer, MD, professor of infectious disease, who maintains a continuously updated database of results from trials of drugs targeting SARS-CoV-2.
But while remdesivir’s pretty good at faking out the viral polymerase’s companion proofreader protein, it’s far from perfect, Shafer said. Some intact viral genome copies still manage to be made, escape from the cell, and infect other cells — mission accomplished.
Using remdesivir in combination with some still-sought, as yet undiscovered drug that could block the proofreader might be a more surefire strategy than using remdesivir alone, Shafer said.
The final round in the cellular boxing ring
In addition to replicating its full-length genome, the virus has to make lots of proteins. And it knows just how. Those RNA snippets spun off by the viral polymerase are tailored to play by the cell’s protein-making rules — well, up to a point. They fit into ribosomes exactly as do the cell’s own strands of “messenger RNA” copied from the cell’s genes by its own DNA-reading polymerases. So-called mRNAs are instructions for making proteins.
But there’s a hitch: Among the proteins the virus forces ribosomes to manufacture are some that, once produced, bite the hand that fed them. Certain newly made viral proteins home to ribosomes in the act of reading one or another of the cell’s mRNA strands, hook themselves onto the strand and stick stubbornly, stalling out the ribosome until the cell’s mRNA strand falls apart.
The genomic RNA strands the virus generates, though, all have little blockades on their front ends that protect them from being snagged on the cell’s ribosomes by the viral wrecking crew. The result: The cell’s protein-making assembly line is overwhelmingly diverted to the production of viral proteins. That’s a two-fer: It both increases viral-component production and stifles the infected cell’s natural first line of defense.
Interferons as a potential treatment
Among the cell’s stillborn proteins are molecules called interferons, which the cell ordinarily makes when it senses it’s been infected by a virus. Interferons have ways of monkeying with the viral polymerase’s operations and squelching viral replication. In addition, when secreted from infected cells, interferons act as “call in the troops” distress signals that alert the body’s immune system to the presence and location of the infected cell.
Instead, silence. Advantage: virus.
There are several different kinds of interferons. A clinical trial is underway at Stanford to determine whether a single injection of one of them, called interferon-lambda, can keep just-diagnosed, mildly symptomatic COVID-19 patients out of the hospital, speed recovery and reduce transmission.
If you don’t hate and respect viruses by now, maybe you haven’t been paying attention. But there’s more.
Viruses don’t always kill the cells they take hostage. Some sew their genes into the genome of the cells they’ve invaded, and those insertions add up. Viral DNA sequences make up 8% of our genome — in contrast with the mere 1% that codes for the proteins of which we’re largely made and that do most of the making.
“Our genome has been ‘invaded’ by previous encounters with retroviruses after infection of sperm or egg cells,” Carette told me. “Through evolution, these retroviruses’ genes have become inactive.”
But, as always, there’s an exception. As Carette said: “An ancient viral gene has been repurposed to play an essential role in embryogenesis,” the process by which an embryo forms and develops.
The protein this gene encodes enables the fusion of two kinds of cells in the developing fetus’s placenta, allowing nutrient and waste exchange between the developing embryo and the maternal blood supply.
'Secret weapon' of retroviruses that cause cancer
Oncogenic retroviruses are a particular family of viruses that can cause some types of cancer. Thierry Heidmann and his colleagues in the CNRS-Institut Gustave Roussy-Université Paris Sud 11 "Rétrovirus endogènes et éléments rétroïdes des eucaryotes supérieurs" Laboratory have studied these viruses. They have identified a "virulence factor" that inhibits the host immune response and allows the virus to spread throughout the body. This factor is a sequence of amino acids that is located in the envelope protein of the virus.
These scientists have also shown that once mutated to lose its immunosuppressive capability, this envelope protein could serve as a basis for the development of vaccines.
These findings have been published online in the Proceedings of the National Academy of Sciences USA.
Retroviruses are viruses whose genome is made up of RNA. These viruses are unique in possessing an enzyme that enables synthesis from this RNA of a DNA molecule capable of integrating into the DNA of a host cell. The retrovirus then utilizes the cell machinery to replicate. HIV is one of the best-known retroviruses. Oncogenic retroviruses (or oncoretroviruses) are cancer-causing viruses. Numerous oncoretroviruses are associated with animal diseases. In humans, two retroviruses, called HTLV and XMRV, have been associated with a type of leukemia and with prostate cancer.
Researchers in the Rétrovirus Endogènes et Eléments Rétroïdes des Eucaryotes Supérieurs Laboratory (1), headed by Thierry Heidmann, CNRS Senior Researcher at Institut Gustave Roussy, have been working on the ability of retroviruses to propagate and persist in their hosts by escaping the immune system. They have studied the molecular basis of this process, and have shown that it is driven by the envelope protein of these viruses. First of all, this protein has an essential "mechanical" role, as it induces the fusion of viral particles with the target cell membrane, thus allowing them to penetrate into the cell. Using a mouse model of infection with a murine leukemia virus, the researchers showed that this envelope protein also has a second role that is equally essential to viral propagation in the body: it is immunosuppressive, or in other words it inhibits the host immune response in a radical manner, affecting both the "innate" and "adaptive" immune responses.
The researchers succeeded in locating the domain responsible for this property within the amino acid sequence of the envelope protein. This domain, an authentic virulence factor, is a crucial element in the arsenal that enables retroviruses to invade their host and produce their pathogenic effect. It thus becomes a target of choice for the design of novel antiretroviral therapeutic strategies, including vaccines. The results obtained by these scientists mean it will be possible to follow this path.
hey were able to introduce targeted point mutations into the envelope protein that could suppress its ability to inhibit the immune system which, as expected, reacted much more effectively than with the non-mutated protein, producing a high level of antibodies and inducing antiviral cellular immunity. By working on this mutated protein, it should be possible to develop vaccines for the future. Indeed, after the mouse model, the researchers were able to show that the HTLV and XMRV retroviruses associated with human diseases were both endowed with an immunosuppressive domain in their envelope protein.
▪ Abstract Human immunodeficiency virus (HIV) and other retroviruses acquire their envelopes and spread infection by budding through the limiting membranes of producer cells. To facilitate budding, retroviruses usurp a cellular pathway that is normally used to create vesicles that bud into late endosomal compartments called multivesicular bodies (MVB). Research on yeast and human MVB biogenesis has led to the identification of ∼25 human proteins that are required for vesicle formation and for HIV-1 budding, and has produced a working model for sequential recruitment of these proteins during MVB vesicle formation. Retroviruses can redirect this machinery to the plasma membrane and leave the cell in a single step or, alternatively, can bud directly into MVB compartments and then exit cells via the exosome pathway. Remarkably, virus release from both the plasma membrane and MVB compartments can occur directionally into specialized sites of cell-to-cell contact called virological synapses. Thus retroviruses have evolved elaborate mechanisms for escaping the cell and maximizing their chances of infecting a new host.
In February 1997 it was reported that pig cells contain a retrovirus capable of infecting human cells (at least, in vitro). This is troublesome because of the efforts that are being made to transplant pig tissue into humans (e.g., fetal pig cells into the brains of patients with Parkinson's disease). Transplant recipients must have their immune systems suppressed if the transplant is to avoid rejection. Could immunosuppressed patients be at risk from the retroviruses present in the transplanted cells? The probability that the original hosts for HIV-1 and HIV-2 were some other primate suggests that retroviruses can move from one species to another.
- an outer envelope which was derived from the plasma membrane of its host
- many copies of an envelope protein embedded in the lipid bilayer of its envelope
- a capsid a protein shell containing
- two molecules of RNA and
- molecules of the enzyme reverse transcriptase
Reverse transcriptase is a DNA polymerase that uses RNA as its template. Thus it is able to make genetic information flow in the reverse (RNA ->DNA) of its normal direction
(DNA -> RNA).
- CD4 molecules. It is this property that enables the virus to invade CD4 + T cells (and certain other cells that express CD4).
- CCR5 (CC chemokine Receptor 5) &mdash found on Th1 cells and macrophages.
All the proteins in the virus particle are encoded by its own genes.
When a retrovirus infects a cell
- its molecules of reverse transcriptase are carried into the cell attached to the viral RNA molecules.
- The reverse transcriptase synthesizes DNA copies of the RNA.
- These enter the nucleus and are
- inserted into the DNA of the host.
- These inserts are transcribed by the host's enzymes into fresh RNA molecules which re-enter the cytosol where
- some are translated by host ribosomes
- the gag gene is translated into molecules of the capsid protein
- the pol gene is transcribed into molecules of reverse transcriptase
- the env gene is translated into molecules of the envelope protein
The genome of retroviruses
- enable the DNA copy of the genome to be inserted into the DNA of the host and
- act as enhancers, causing the host nucleus to transcribe the DNA copies of the retroviral genome at a rapid rate.
The retroviral genome also contains a packaging signal sequence ("P") which is needed for the newly-synthesized RNA molecules to be incorporated in fresh virus particles [Example].
Most retroviruses also contain one or more additional genes. Some of these represent RNA copies of genes that earlier were picked up from their eukaryotic host. Several cancers in animals are caused by retroviruses that have, at some earlier time, picked up a proto-oncogene from their mammalian host and converted it into an oncogene.
How do retroviruses exit the cell - Biology
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A retrovirus is a single-stranded RNA virus that binds to specific cell surface receptors on a targeted host cell's outer membrane, fuses, and enters via endocytosis to replicate its genetic material in a unique way.
After enter the host's cell, the capsid is uncoated and the enzyme reverse transcriptase or RT binds to the viral RNA, synthesizing complementary DNA and with time, double-stranded DNA the reverse of the usual pattern.
Inside the nucleus of the host cell, the viral DNA is integrated into the host DNA forming a provirus. Thus the viral DNA is actively transcribed whenever the host DNA is transcribed, forming messenger RNA.
This mRNA exits the nucleus, enters the cytoplasm, and is translated to form new viral proteins, which can then assemble into new retroviruses that butt out of the cell and are ready to infect other cells.
16.5: Retrovirus Life Cycles
Retroviruses have a single-stranded RNA genome that undergoes a special form of replication. Once the retrovirus has entered the host cell, an enzyme called reverse transcriptase synthesizes double-stranded DNA from the retroviral RNA genome. This DNA copy of the genome is then integrated into the host&rsquos genome inside the nucleus via an enzyme called integrase. Consequently, the retroviral genome is transcribed into RNA whenever the host&rsquos genome is transcribed, allowing the retrovirus to replicate. New retroviral RNA is transported to the cytoplasm, where it is translated into proteins that assemble new retroviruses.
Antiretroviral Drugs Target Different Stages of the HIV Life Cycle
Particular drugs have been developed to fight retroviral infections. These drugs target specific aspects of the life cycle. One class of antiretroviral drugs, fusion inhibitors, prevents the entry of the retrovirus into the host cell by inhibiting the fusion of the retrovirus with the host cell membrane. Another class of antiretrovirals, reverse transcriptase inhibitors, inhibits the reverse transcriptase enzymes that make DNA copies of the retroviral RNA genome. Reverse transcriptase inhibitors are competitive inhibitors during the process of reverse transcription, the drug molecules are incorporated into the growing DNA strand instead of the usual DNA bases. Once incorporated, the drug molecules block further progress by the reverse transcriptase enzyme. The third class of drugs, integrase inhibitors, prevents the integrase enzymes from integrating the retroviral genome into the host genome. Finally, protease inhibitors interfere with the enzymatic reactions that are necessary for producing fully functioning retroviral particles.
Combinations (or &ldquococktails&rdquo) of antiretrovirals are used to fight Human Immunodeficiency Virus (HIV). If left untreated, this retrovirus causes AIDS. Cocktails of antiretrovirals are necessary to fight HIV infections because the retrovirus can quickly evolve resistance to any one drug. This capacity for rapid evolution stems from the single-stranded RNA genome of HIV, which accumulates mutations more rapidly than DNA or double-stranded genomes. Some of these mutations confer drug-resistance.
However, by combining drugs that target events at the beginning, middle, and end of the retroviral life cycle, antiretroviral cocktails (called highly active antiretroviral therapy, or HAART) dramatically reduce the HIV population in a patient. The likelihood of multiple mutations that confer resistance to various drugs in the HIV genome is much lower than that of a single resistant mutation, making the HAART strategy much more effective than single-drug therapies. This cocktail strategy has been enormously successful in treating HIV, such that it is now uncommon for treated individuals to develop AIDS.
Greenwood, Alex D., Yasuko Ishida, Sean P. O&rsquoBrien, Alfred L. Roca, and Maribeth V. Eiden. &ldquoTransmission, Evolution, and Endogenization: Lessons Learned from Recent Retroviral Invasions.&rdquo Microbiol. Mol. Biol. Rev. 82, no. 1 (March 1, 2018): e00044-17. [Source]
Atta, Mohamed G., Sophie De Seigneux, and Gregory M. Lucas. &ldquoClinical Pharmacology in HIV Therapy.&rdquo Clinical Journal of the American Society of Nephrology 14, no. 3 (March 7, 2019): 435&ndash44. [Source]
Retroviruses are a unique class of single-stranded ribonucleic acid (RNA) containing viruses, which replicate their genome through a double-stranded viral deoxyribonucleic acid (DNA) intermediate in the nucleus of the host cell. This is in contrast to all other RNA-containing viruses that replicate their genomes through double-stranded RNA intermediates almost always in the cytoplasm of host cells. Most retroviruses contain an RNA genome of 9 to 10 kilobases in length, which encodes a minimum of three genes required for replication. These are referred to as gag (structural proteins of the virus), pol (enzymes involved in replication), and env (envelope glycoproteins required for the virus to attach to a receptor of a new host cell). Human immunodeficiency virus (HIV), which causes acquired immunodeficiency syndrome (AIDS), belongs to a subclass of retroviruses, the lentiviruses, which encode additional viral genes that permit the virus to grow in nondividing cells, such as white blood cells.
The remarkable replication pathway of retroviruses requires that once the virus enters the host cell, a viral pol gene𠄾ncoded enzyme called reverse transcriptase (RT), which is packaged in virus particles, reverse transcribes the single-stranded RNA genome into a double-stranded DNA. This DNA intermediate migrates to the nucleus of the cell where it is integrated into the host cell genome. This process is catalyzed by another viral enzyme
Transcription of the viral sequence from the integrated DNA to make messenger RNA (mRNA) requires cellular enzymes. Full-length viral mRNA is transported to the cytoplasm where it is either packaged into progeny virus or translated on non-membrane-bound (free) ribosomes to yield viral Gag and Gag-Pol polyproteins (assemblies of many similar proteins). These polyproteins in turn migrate to the cell membrane where they assemble into virus particles, containing RNA, which bud from the cell surface. Concomitantly, viral glycoproteins are translated as polyproteins from a smaller-sized, spliced viral mRNA on membrane-bound ribosomes. These polyproteins are processed in the endoplasmic reticulum , where they also go through an additional modification known as glycosylation, in which sugar groups are added to the protein. When virus particles bud from the cell, they pinch off a portion of the cell membrane, containing the viral glycoproteins. This membrane becomes an outer coating of the virus particle.
The Gag and Gag-Pol polyproteins are cleaved into the mature-sized proteins during or immediately after the budding process by a third viralencoded enzyme called protease (PR). Once the protein-cleaving proteolytic processing is complete, an infectious virus results, which can infect new cells.
During an active infection process, approximately 1 percent of a cell's resources are diverted to synthesis of virus genomes and proteins. Infected cells are therefore not killed. Most retroviruses activate expression of a cancer-causing gene, called an "oncogene," which transforms host cells so that they become immortalized, providing a long-term home for the retrovirus. Lentiviruses, including HIV, do not transform cells. Instead they cause cell death in some of the cell types in which they replicate. When these cells are important components of the immune system, an infected person loses the ability to mount an effective immune response, resulting in AIDS. This leaves the person susceptible to almost any opportunistic infection. Patients with HIV infection are treated with drugs that inhibit either RT or PR to slow the spread of virus. As of May 2001, the treatment of choice for HIV patients included two RT inhibitors and one PR inhibitor, and is known therefore as "triple therapy." These drugs do not cure AIDS because the viral genome is integrated into the host chromosome. Also, virus-containing drug-resistant enzymes can be rapidly selected in a treated patient, necessitating the need for multidrug clinical strategies. Thus the only sure defense against AIDS is not to become infected by the virus.
Tips for keeping safe
If you are going to be working with retroviruses:
- Get trained to work with viruses, viral particles and biological material
- Wear the correct PPE (Personal protection equipment)
- Be informed as to emergency response and spill procedures
- Be extra careful with sharps and bio hazardous materials
- Know the right protocols for waste disposal and management
- Follow exact experimental protocols and safety procedures
- Report accidents, spills and unusual incidents
- Mosier D E (2004). Introduction for “Safety Considerations for Retroviral Vectors: A Short Review”. Applied Biosafety.9(2):68-75.
- Temin (1990). Safety considerations in somatic gene therapy of human disease with retrovirus vectors. Human Gene Therapy.1:111-123.
- Donahue et al (1992). Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer. J. Exp. Med.176:1125-1135.
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I don’t understand the paragraph about retroviruses being replication deficient. You say “Genes required for viral infection but unnecessary for packaging and transduction are deleted”. First, what is the difference between infection and transduction? Then you go on to say “Viral structural genes that are incorporated into the plasmid do not contain a ? (psi) sequence (which essentiallyhelps to incorporate the RNA into viral particles)”. The first sentence says that packaging is not affected, but the second sentence says that structural genes lack the Psi sequence which is required for packaging. How is replication prevented, and how does that affect the use of retrovirus as a tool for gene expression/knock-down?
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- some are translated by host ribosomes