VIRUSES & EXOSOMES
How They Are Different
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{This is long, so I’m showing the TABLE of CONTENTS at the beginning. I don’t know enough yet how to make links from Contents to sections of the post, so you’ll have to navigate yourself. I used my Bing A.I. to generate the first 6 sections. The last two are online articles that Bing A.I. recommended. I haven’t read everything yet, but I skimmed all of it. Bing A.I. is good for learning. It produces info very quickly. You just have to be careful to be skeptical of everything it says, since it’s got built-in mainstream biases.
Section 7 on Origins of Viruses says Flu vaccines are needed each year, because the virus mutates so fast, but that’s exactly one reason why such vaccines are useless. I’ve also read that vaxes suppress the immune system, instead of supporting it, so that’s another reason not to take them. And there are numerous toxic ingredients in them, including various viruses. So the author of that section is obviously not well-informed, or is lying to uphold mainstream disinfo. However, I expect that 90% or more of the info herein is accurate. I just skimmed the 2nd article and the 4th subsection mentions that exosomes could be used to deliver vaxes or drugs into cells. I don’t trust that at all. Do you?}
.1. THE DIFFERENCE BETWEEN VIRUSES & EXOSOMES
.2. HOW VIRUSES ENTER THE BODY
.3. HOW VIRUSES HIJACK HOST CELLS & REPLICATE
.4. SCIENTIFIC STUDIES THAT PROVED THAT SOME VIRUSES CAN INFECT CELLS AND REPLICATE THERE
.5. DIFFERENCES IN COMPOSITION BETWEEN VIRUSES AND EXOSOMES
.6. WHAT A PARENT CELL'S PHYSIOLOGICAL AND PATHOLOGICAL STATE CAUSE ITS EXOSOMES TO DO
.7. ORIGINS OF VIRUSES
.8. RELATIONSHIP BETWEEN EXOSOMES AND VIRUSES
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.1. THE DIFFERENCE BETWEEN VIRUSES & EXOSOMES
(Bing AI:) Viruses and exosomes are both small membrane-bound particles that can carry genetic material and proteins. However, they have some important differences. Viruses are infectious agents that can replicate only inside living cells. They have a genome that encodes their own proteins and can hijack the host cell's machinery to produce more viruses. Exosomes are extracellular vesicles that are released by many types of cells as a way of communication and waste disposal. They do not have a genome of their own, but they can carry RNA and proteins from the donor cell to the recipient cell. Exosomes are not infectious and do not replicate.
Some researchers have proposed that some viruses, such as HIV, are actually exosomes in disguise. They argue that these viruses use the same pathways and mechanisms as exosomes to bud from the cell membrane, fuse with target cells, and evade immune recognition. However, this hypothesis is controversial and has not been widely accepted by the scientific community. There are still many differences between viruses and exosomes, such as their origin, composition, function, and diversity.
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.2. HOW VIRUSES ENTER THE BODY
(Bing AI:) Viruses are microscopic agents that can cause various diseases in humans and other living organisms. But how do they enter the body and infect the cells? And which tissues are more susceptible to viral infection? In this blog post, we will explore these questions and learn more about the mechanisms of viral infection.
One of the main ways that viruses enter the body is through the respiratory tract, which includes the nose, mouth, throat and lungs. When we breathe, we inhale air that may contain viruses from the environment or from other people who are sick. Some of these viruses can attach to the cells that line the respiratory tract and invade them. For example, influenza virus, which causes the flu, and SARS-CoV-2, which causes COVID-19, are both respiratory viruses that enter the body this way.
Another way that viruses can enter the body is through the digestive system, which includes the stomach and intestines. When we eat or drink, we may ingest food or water that is contaminated with viruses from animals or humans. Some of these viruses can survive the acidic environment of the stomach and reach the intestines, where they can infect the cells that line the intestinal wall. For example, norovirus, rotavirus and astrovirus are all viruses that can cause gastroenteritis, sometimes called "stomach flu".
A third way that viruses can enter the body is through breaks in the skin or mucous membranes, which are thin layers of tissue that cover various organs and cavities. When we have a wound, a cut, a bite or an injection, we may expose our blood or tissues to viruses from external sources or from our own body fluids. Some of these viruses can enter the bloodstream or lymphatic system and spread throughout the body. For example, HIV, which causes AIDS, and hepatitis B and C viruses, which cause liver disease, are all blood-borne viruses that can enter the body this way.
Once inside the body, viruses need to find a way to enter the cells and use them as factories to make more copies of themselves. Viruses do this by bringing their genetic material (DNA or RNA) and some proteins to the cell surface and tricking the cell into letting them in. The cell then follows the instructions of the virus and uses its own tools and materials to make all the necessary parts for new viruses. These new viruses can then be assembled inside the cell or released outside to infect other cells.
However, not all cells are equally vulnerable to viral infection. Some cells have receptors on their surface that match specific proteins on certain viruses. These receptors act like locks that only certain keys (viruses) can open. For example, influenza virus can only infect cells that have sialic acid receptors on their surface. Other cells have defense mechanisms that can detect and destroy viral invaders before they cause damage. For example, macrophages are immune cells that can engulf and digest viruses.
Therefore, viral infection depends on many factors, such as how the virus enters the body, how it interacts with the cell surface, how it evades or overcomes the cell's defenses, and how it replicates and spreads. Understanding these mechanisms can help us develop better ways to prevent and treat viral diseases.
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.3. HOW VIRUSES HIJACK HOST CELLS & REPLICATE
(Bing AI:) Viruses are microscopic parasites that cannot survive or reproduce on their own. They need to infect a living cell and use its machinery to make more copies of themselves. But how do they do that? And what are the consequences for the host cell?
In this blog post, we will explore how viruses hijack the host cell's machinery to produce more viruses, and how this process affects the cell's function and fate.
The first step in viral infection is attachment. The virus recognizes and binds to a specific receptor on the surface of the host cell. This receptor is usually a protein that has a normal function for the cell, such as signaling or transport. The virus exploits this protein to gain entry into the cell.
The second step is penetration. Depending on the type of virus, there are different ways of penetrating the host cell. Some viruses fuse their membrane with the cell membrane and release their genetic material into the cytoplasm. Others are engulfed by the cell through a process called endocytosis and then escape from the endosome into the cytoplasm.
The third step is uncoating. This is when the virus releases its genetic material from its protective coat or capsid. The genetic material can be either DNA or RNA, depending on the virus. The genetic material carries the instructions for making more viruses.
The fourth step is replication. This is when the virus uses the host cell's machinery to make more copies of its genetic material and proteins. The virus may use different strategies to achieve this, depending on its type and complexity. Some viruses use the host cell's nucleus and enzymes to replicate their DNA or RNA. Others bring their own enzymes and replicate their RNA in the cytoplasm. Some viruses also integrate their DNA into the host cell's genome, becoming part of its DNA.
The fifth step is assembly. This is when the newly synthesized viral components are assembled into new virus particles. The assembly may take place in different locations within the cell, such as the nucleus, the cytoplasm, or the endoplasmic reticulum.
The sixth and final step is release. This is when the new virus particles exit the host cell and infect other cells. The release may occur by different mechanisms, depending on the virus. Some viruses cause the host cell to burst or lyse, releasing all the virus particles at once. Others bud off from the cell membrane, taking a piece of it with them as an envelope.
The viral infection has a profound impact on the host cell's function and fate. The virus may interfere with the cell's normal processes, such as metabolism, gene expression, signaling, and immunity. The virus may also trigger an immune response from the host organism, which may lead to inflammation, tissue damage, and disease symptoms.
Viruses are fascinating and formidable foes that challenge our understanding of life and health. By learning how they hijack our cells, we may find new ways to prevent and treat viral diseases.
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.4. SCIENTIFIC STUDIES THAT PROVED THAT SOME VIRUSES CAN INFECT CELLS AND REPLICATE THERE
(Bing AI:) Viruses are microscopic parasites that can infect living cells and cause diseases. But how do viruses enter cells and replicate inside them? In this blog post, we will explore some of the scientific studies that have revealed the mechanisms of viral infection and replication, focusing on coronaviruses, a group of viruses that includes the causative agent of COVID-19.
Coronaviruses are named after the crown-like spikes that protrude from their surface. These spikes are made of a protein called S (for spike), which is essential for the virus to attach to and enter the host cell. The S protein binds to a specific receptor on the cell membrane, which varies depending on the type of coronavirus. For example, SARS-CoV-2, the virus that causes COVID-19, uses a receptor called ACE2 (angiotensin-converting enzyme 2), which is found on many types of cells in the human body, including lung cells.
Once the S protein binds to the receptor, the virus can fuse with the cell membrane and release its genetic material (RNA) into the cell. The RNA then hijacks the cell's machinery to make copies of itself and produce more viral proteins. Some of these proteins form new spikes and others assemble into a shell that encloses the RNA, forming new viral particles. These particles then bud off from the cell membrane and infect other cells, spreading the infection.
The process of viral infection and replication is complex and involves many steps and factors. Scientists have used various methods and tools to study these mechanisms, such as cell culture, animal models, microscopy, molecular biology, biochemistry, and genetics. By understanding how viruses infect and replicate in cells, we can develop better ways to prevent and treat viral diseases.
{That didn't answer my question, so I switched to Chat mode, because Chat often provides references. Next is the answer it gave in Chat.}
There have been many scientific studies that have proven that viruses can infect cells and replicate within them. For example, a study published in Nature discusses the initial steps of coronavirus infection, which involve the specific binding of the coronavirus spike (S) protein to cellular entry receptors 1.
{I wanted more than one study, so I asked another question.}
What was the first scientific study that proved that viruses can infect cells and replicate there?
I’m sorry, but I couldn’t find information about the first scientific study that proved that viruses can infect cells and replicate within them. However, there have been many studies on this topic over the years. For example, Koonin and Martin (2005) postulated that viruses existed in a precellular world as self-replicating units.1
1. https://www.nature.com/scitable/topicpage/the-origins-of-viruses-14398218/
{That doesn't answer the question either, so maybe I should rephrase it. But, first I wanted the following info.}
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.5. DIFFERENCES IN COMPOSITION BETWEEN VIRUSES AND EXOSOMES
Viruses and exosomes are both small particles that can transfer genetic material and proteins from one cell to another. However, they have different origins, structures and functions. In this blog post, we will explore some of the main differences in composition between viruses and exosomes.
Viruses are infectious agents that consist of a nucleic acid genome (either DNA or RNA) enclosed by a protein coat called a capsid. Some viruses also have a lipid envelope derived from the host cell membrane. Viruses can only replicate inside living cells by hijacking their machinery and resources. Viruses can cause diseases in humans, animals and plants by disrupting the normal functions of the infected cells.
Exosomes are extracellular vesicles that are released by many types of cells under physiological and pathological conditions. Exosomes are formed by the inward budding of the endosomal membrane, resulting in multivesicular bodies (MVBs) that contain multiple exosomes. MVBs can then fuse with the plasma membrane and release exosomes into the extracellular space. Exosomes contain a variety of molecules, such as lipids, proteins, RNA and DNA, that reflect the origin and state of the parent cell. Exosomes can act as intercellular messengers by transferring these molecules to recipient cells and modulating their behavior.
One of the main differences in composition between viruses and exosomes is the presence or absence of a viral genome. Viruses carry their own genetic information that encodes for viral proteins and enzymes required for their replication and assembly. Exosomes do not have a specific genome, but they can contain fragments of cellular RNA and DNA that may have functional or regulatory roles in the recipient cells.
Another difference in composition between viruses and exosomes is the nature and diversity of their protein content. Viral proteins are mostly derived from the viral genome and are specific to each virus type. They include structural proteins that form the capsid and envelope, as well as non-structural proteins that mediate viral entry, replication, assembly and release. Exosomal proteins are mostly derived from the parent cell and are influenced by its physiological and pathological state. They include membrane proteins that facilitate exosome recognition and uptake by recipient cells, as well as cytosolic proteins that may have enzymatic or signaling activities.
A third difference in composition between viruses and exosomes is the lipid composition of their membranes. Viral envelopes are composed of lipids derived from the host cell membrane, but they may also incorporate viral proteins that modify their properties. For example, some viral envelope proteins can mediate fusion with target cell membranes or evade immune recognition. Exosomal membranes are composed of lipids derived from the endosomal membrane, but they may also reflect the lipid profile of the parent cell. For example, some exosomal lipids can modulate inflammation or angiogenesis in recipient cells.
In summary, viruses and exosomes are both small particles that can transfer genetic material and proteins from one cell to another. However, they have different origins, structures and functions. Viruses are infectious agents that carry their own genome and specific proteins that enable their replication and pathogenesis. Exosomes are extracellular vesicles that contain fragments of cellular RNA and DNA and diverse proteins that reflect the origin and state of the parent cell. Exosomes can act as intercellular messengers by transferring these molecules to recipient cells and modulating their behavior.
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.6. WHAT A PARENT CELL'S PHYSIOLOGICAL AND PATHOLOGICAL STATE CAUSE ITS EXOSOMES TO DO
Exosomes are small membrane-bound vesicles that are released by cells and carry various biomolecules, such as proteins, lipids, and nucleic acids. Exosomes play important roles in intercellular communication, as they can transfer their cargo to recipient cells and modulate their functions. However, exosomes are not just passive messengers; they are also influenced by the physiological and pathological state of the parent cell that produces them.
In this blog post, we will explore how the parent cell's condition affects the biogenesis, composition, and function of exosomes. We will also discuss some of the potential applications and challenges of using exosomes as biomarkers and therapeutics for various diseases.
Biogenesis of exosomes
Exosomes are derived from the endosomal system of the cell. They originate from the inward budding of the membrane of multivesicular bodies (MVBs), which are large endosomes that contain multiple intraluminal vesicles (ILVs). The MVBs can either fuse with lysosomes for degradation or with the plasma membrane for secretion. The ILVs that are released into the extracellular space are called exosomes.
The biogenesis of exosomes is regulated by several factors, such as the lipid composition of the membrane, the sorting machinery of MVBs, and the cytoskeleton dynamics. However, these factors can also be modulated by the physiological and pathological state of the parent cell. For example, hypoxia, oxidative stress, inflammation, infection, and cancer can alter the expression and activity of enzymes and proteins involved in exosome formation and release. These changes can affect the size, number, and morphology of exosomes.
Composition of exosomes
Exosomes contain a variety of biomolecules that reflect the origin and status of the parent cell. These include proteins, such as membrane receptors, signaling molecules, enzymes, cytoskeletal proteins, and heat shock proteins; lipids, such as cholesterol, sphingolipids, phospholipids, and ceramides; and nucleic acids, such as mRNA, miRNA, lncRNA, DNA, and circular RNA.
The composition of exosomes can also be influenced by the physiological and pathological state of the parent cell. For instance, hypoxia can induce the expression of hypoxia-inducible factor 1-alpha (HIF-1α) in exosomes, which can promote angiogenesis and tumor growth in recipient cells. Oxidative stress can increase the levels of reactive oxygen species (ROS) in exosomes, which can induce oxidative damage and inflammation in target cells. Infection can trigger the incorporation of viral or bacterial components into exosomes, which can modulate immune responses and pathogenesis. Cancer can alter the expression of oncogenes, tumor suppressors, and immune checkpoint molecules in exosomes, which can affect tumor progression and metastasis.
Function of exosomes
Exosomes can mediate various biological processes by transferring their cargo to recipient cells. Depending on the context and target cell type, exosomes can have different effects on cellular functions. Some examples are:
- Exosomes from immune cells can modulate immune responses by activating or suppressing immune cells, presenting antigens, or delivering cytokines and chemokines.
- Exosomes from stem cells can promote tissue regeneration and repair by delivering growth factors, anti-apoptotic molecules, or differentiation signals.
- Exosomes from neurons can modulate synaptic transmission and plasticity by delivering neurotransmitters, receptors, or synaptic proteins.
- Exosomes from endothelial cells can regulate vascular homeostasis and angiogenesis by delivering nitric oxide synthase (NOS), vascular endothelial growth factor (VEGF), or integrins.
- Exosomes from cancer cells can influence tumor microenvironment and metastasis by delivering oncogenic signals, extracellular matrix (ECM) components, or pro-angiogenic factors.
Applications and challenges of exosomes
Exosomes have emerged as promising biomarkers and therapeutics for various diseases. As biomarkers, exosomes can provide valuable information about the diagnosis, prognosis, and response to therapy of diseases. For example,
- Exosomes from blood or urine can be used to detect cancer biomarkers such as epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), or prostate-specific antigen (PSA).
- Exosomes from cerebrospinal fluid (CSF) can be used to diagnose neurode
{For an unknown reason my A.I. stopped writing at that point, so I asked the following question.}
Exosomes from cerebrospinal fluid (CSF) can be used to diagnose what?
Exosomes from cerebrospinal fluid (CSF) can be used to diagnose various neurological disorders, such as Parkinson's disease (PD) and Alzheimer's disease (AD). Exosomes are small vesicles that carry molecular information between cells. They can reflect the pathological changes in the brain and provide potential biomarkers for diagnosis and prognosis. For example, a recent study found that the levels of certain microRNAs in the exosomes from CSF were different between PD and AD patients. These microRNAs could be used as diagnostic indicators for these diseases.
{That's the short answer. Before giving me that answer, it gave me the following long answer, which might be more than I want to know right now.}
Exosomes are small vesicles that are released by cells and carry various molecules, such as proteins and nucleic acids. They play important roles in intercellular communication and can influence various biological processes. Exosomes can also reflect the pathological state of the cells that produce them, making them potential biomarkers for various diseases.
One of the sources of exosomes is the cerebrospinal fluid (CSF), which is a clear fluid that surrounds the brain and spinal cord. CSF exosomes can carry information about the health of the central nervous system (CNS) and may be useful for diagnosing neurological disorders. In this blog post, we will review some of the recent studies that have explored the use of CSF exosomes for diagnosing different CNS diseases.
One of the most common and devastating CNS diseases is Alzheimer's disease (AD), which is characterized by progressive cognitive decline and memory loss. AD is associated with the accumulation of amyloid-beta (Aβ) plaques and neurofibrillary tangles in the brain, as well as neuroinflammation and oxidative stress. CSF exosomes may reflect these pathological changes and provide clues for early diagnosis of AD.
For example, a study by Fiandaca et al. [1] found that CSF exosomes from AD patients had higher levels of Aβ42, tau, and phosphorylated tau than those from healthy controls or patients with mild cognitive impairment (MCI), which is a prodromal stage of AD. These exosomal biomarkers correlated well with the clinical diagnosis and could distinguish AD from MCI with high accuracy. Another study by Goetzl et al. [2] found that CSF exosomes from AD patients had lower levels of synaptic proteins, such as synaptophysin and synaptotagmin-1, than those from healthy controls or patients with frontotemporal dementia (FTD), another form of dementia. These exosomal biomarkers could also differentiate AD from FTD with high specificity and sensitivity.
Another CNS disease that may benefit from CSF exosome analysis is Parkinson's disease (PD), which is characterized by motor impairment, tremor, rigidity, and bradykinesia. PD is caused by the loss of dopaminergic neurons in the substantia nigra and the accumulation of alpha-synuclein aggregates in the brain. CSF exosomes may reflect these pathological features and help diagnose PD.
For instance, a study by Gui et al. [3] performed microRNA analysis on exosome miRNAs in the CSF of PD and AD patients. They found that PD patients had lower levels of miR-19b-3p, miR-153, miR-409-3p, and miR-10a-5p than AD patients or healthy controls. These miRNAs are involved in regulating neuronal development, survival, and function, and may be related to PD pathogenesis. The authors suggested that these exosomal miRNAs could be used as biomarkers for PD diagnosis.
Other CNS diseases that have been investigated using CSF exosome analysis include multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury (TBI), and glioblastoma multiforme (GBM). CSF exosomes may provide information about the inflammatory, oxidative, or tumorigenic state of the CNS in these diseases and help monitor their progression or response to treatment.
In summary, CSF exosomes are a promising source of biomarkers for diagnosing various CNS diseases. They can reflect the molecular changes that occur in the brain and spinal cord and provide insights into the pathophysiology of these diseases. However, there are still many challenges and limitations that need to be overcome before CSF exosome analysis can be widely applied in clinical practice. These include standardizing the methods for isolating, quantifying, and characterizing exosomes; validating the specificity and sensitivity of exosomal biomarkers; and establishing reference ranges and cut-off values for different diseases. Further research is needed to address these issues and to explore the potential therapeutic applications of CSF exosomes.
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.7. ORIGINS OF VIRUSES
{Finally, I asked this.}
What Nature article explains virus theory?
One article from Nature that explains virus theory is “The Origins of Viruses” 1. This article discusses various theories about the origin of viruses, including the idea that viruses existed in a precellular world as self-replicating units 1.
1. https://www.nature.com/scitable/topicpage/the-origins-of-viruses-14398218/
{This below is a copy of the article.}
The Origins of Viruses
By: David R. Wessner, Ph.D. (Dept. of Biology, Davidson College) © 2010 Nature Education
Citation: Wessner, D. R. (2010) The Origins of Viruses. Nature Education 3(9):37
How did viruses evolve? Are they a streamlined form of something that existed long ago, or an ultimate culmination of smaller genetic elements joined together?
The evolutionary history of viruses represents a fascinating, albeit murky, topic for virologists and cell biologists. Because of the great diversity among viruses, biologists have struggled with how to classify these entities and how to relate them to the conventional tree of life. They may represent genetic elements that gained the ability to move between cells. They may represent previously free-living organisms that became parasites. They may be the precursors of life as we know it.
The Basics of Viruses
We know that viruses are quite diverse. Unlike all other biological entities, some viruses, like poliovirus, have RNA genomes and some, like herpesvirus, have DNA genomes. Further, some viruses (like influenza virus) have single-stranded genomes, while others (like smallpox) have double-stranded genomes. Their structures and replication strategies are equally diverse. Viruses, do, however, share a few features: First, they generally are quite small, with a diameter of less than 200 nanometers (nm). Second, they can replicate only within a host cell. Third, no known virus contains ribosomes, a necessary component of a cell's protein-making translational machinery.
Are Viruses Alive?
A schematic diagram shows two groups of organisms: capsid-encoding organisms and ribosome-encoding organisms. The two groups are shown on a sphere that is divided in half, and lines radiating from where the two halves meet represent different taxa within the two groups. The top half of the sphere represents the capsid-encoding organisms, which are viruses, and includes viruses of Archaea, viruses of Bacteria, and viruses of Eukarya. The bottom half of the sphere represents ribosome-encoding organisms and includes Bacteria, Archaea, and Eukarya.
To consider this question, we need to have a good understanding of what we mean by "life." Although specific definitions may vary, biologists generally agree that all living organisms exhibit several key properties: They can grow, reproduce, maintain an internal homeostasis, respond to stimuli, and carry out various metabolic processes. In addition, populations of living organisms evolve over time.
Do viruses conform to these criteria? Yes and no. We probably all realize that viruses reproduce in some way. We can become infected with a small number of virus particles — by inhaling particles expelled when another person coughs, for instance — and then become sick several days later as the viruses replicate within our bodies. Likewise we probably all realize that viruses evolve over time. We need to get a flu vaccine every year primarily because the influenza virus changes, or evolves, from one year to the next (Nelson & Holmes 2007).
Viruses do not, however, carry out metabolic processes. Most notably, viruses differ from living organisms in that they cannot generate ATP. Viruses also do not possess the necessary machinery for translation, as mentioned above. They do not possess ribosomes and cannot independently form proteins from molecules of messenger RNA. Because of these limitations, viruses can replicate only within a living host cell. Therefore, viruses are obligate intracellular parasites. According to a stringent definition of life, they are nonliving. Not everyone, though, necessarily agrees with this conclusion. Perhaps viruses represent a different type of organism on the tree of life — the capsid-encoding organisms, or CEOs (Figure 1; Raoult & Forterre 2008).
Where Did Viruses Come From?
There is much debate among virologists about this question. Three main hypotheses have been articulated: 1. The progressive, or escape, hypothesis states that viruses arose from genetic elements that gained the ability to move between cells; 2. the regressive, or reduction, hypothesis asserts that viruses are remnants of cellular organisms; and 3. the virus-first hypothesis states that viruses predate or coevolved with their current cellular hosts.
The Progressive Hypothesis
A schematic diagram shows the steps in the replication of a retrotransposon. DNA and RNA molecules are depicted as different colored rectangles, and transcription, translation, and reinsertion steps are shown with arrows.
Figure 3 {illegible}
A schematic diagram shows the life cycle of a retrovirus as it infects a eukaryotic host cell, replicates, and releases its progeny. The virus's primary structural features are shown along with a simplified outline of a host cell with a nucleus. The steps in the process are depicted with key molecules and labeled arrows. Bar-headed lines indicate points where treatments can inhibit steps in the pathway.
Figure 2 {illegible}
According to this hypothesis, viruses originated through a progressive process. Mobile genetic elements, pieces of genetic material capable of moving within a genome, gained the ability to exit one cell and enter another. To conceptualize this transformation, let's examine the replication of retroviruses, the family of viruses to which HIV belongs.
Retroviruses have a single-stranded RNA genome. When the virus enters a host cell, a viral enzyme, reverse transcriptase, converts that single-stranded RNA into double-stranded DNA. This viral DNA then migrates to the nucleus of the host cell. Another viral enzyme, integrase, inserts the newly formed viral DNA into the host cell's genome. Viral genes can then be transcribed and translated. The host cell's RNA polymerase can produce new copies of the virus's single-stranded RNA genome. Progeny viruses assemble and exit the cell to begin the process again (Figure 2).
This process very closely mirrors the movement of an important, though somewhat unusual, component of most eukaryotic genomes: retrotransposons. These mobile genetic elements make up an astonishing 42% of the human genome (Lander et al. 2001) and can move within the genome via an RNA intermediate. Like retroviruses, certain classes of retrotransposons, the viral-like retrotransposons, encode a reverse transcriptase and, often, an integrase. With these enzymes, these elements can be transcribed into RNA, reverse-transcribed into DNA, and then integrated into a new location within the genome (Figure 3). We can speculate that the acquisition of a few structural proteins could allow the element to exit a cell and enter a new cell, thereby becoming an infectious agent. Indeed, the genetic structures of retroviruses and viral-like retrotransposons show remarkable similarities.
The Regressive Hypothesis
In contrast to the progressive process just described, viruses may have originated via a regressive, or reductive, process. Microbiologists generally agree that certain bacteria that are obligate intracellular parasites, like Chlamydia and Rickettsia species, evolved from free-living ancestors. Indeed, genomic studies indicate that the mitochondria of eukaryotic cells and Rickettsia prowazekii may share a common, free-living ancestor (Andersson et al. 1998). It follows, then, that existing viruses may have evolved from more complex, possibly free-living organisms that lost genetic information over time, as they adopted a parasitic approach to replication.
Viruses of one particular group, the nucleocytoplasmic large DNA viruses (NCLDVs), best illustrate this hypothesis. These viruses, which include smallpox virus and the recently discovered giant of all viruses, Mimivirus, are much bigger than most viruses (La Scola et al. 2003). A typical brick-shaped poxvirus, for instance, may be 200 nm wide and 300 nm long. About twice that size, Mimivirus exhibits a total diameter of roughly 750 nm (Xiao et al. 2005). Conversely, spherically shaped influenza virus particles may be only 80 nm in diameter, and poliovirus particles have a diameter of only 30 nm, roughly 10,000 times smaller than a grain of salt. The NCLDVs also possess large genomes. Again, poxvirus genomes often approach 200,000 base pairs, and Mimivirus has a genome of 1.2 million base pairs; while poliovirus has a genome of only 7,500 nucleotides total. In addition to their large size, the NCLDVs exhibit greater complexity than other viruses have and depend less on their host for replication than do other viruses. Poxvirus particles, for instance, include a large number of viral enzymes and related factors that allow the virus to produce functional messenger RNA within the host cell cytoplasm.
Because of the size and complexity of NCLDVs, some virologists have hypothesized that these viruses may be descendants of more complex ancestors. According to proponents of this hypothesis, autonomous organisms initially developed a symbiotic relationship. Over time, the relationship turned parasitic, as one organism became more and more dependent on the other. As the once free-living parasite became more dependent on the host, it lost previously essential genes. Eventually it was unable to replicate independently, becoming an obligate intracellular parasite, a virus. Analysis of the giant Mimivirus may support this hypothesis. This virus contains a relatively large repertoire of putative genes associated with translation — genes that may be remnants of a previously complete translation system. Interestingly, Mimivirus does not differ appreciably from parasitic bacteria, such as Rickettsia prowazekii (Raoult et al. 2004).
The Virus-First Hypothesis
A schematic diagram shows hypothetical evolutionary relationships between viruses and the three domains of life if viruses existed before cells. The ancient virosphere is depicted as a green oval in the center of the diagram, and contains the ancestors of modern viruses and the last universal common ancestor (LUCA) of all life. A blue, red, and yellow cone branch out from the LUCA; each cone represents a branch of the modern virosphere and contains a smaller inner cone that represents one of the three domains of life. The blue cones contain bacteria and bacterial viruses. The red cones contain Archaea and Archaeal viruses. The yellow cones contain Eukarya and eukaryotic viruses. A black arrow points from the blue bacterial cone to the red Archaeal cone. A question mark above the arrow suggests there is uncertainty regarding the evolutionary relationship between the modern bacterial and archaeal viruses.
Figure 4 {illegible}
The progressive and regressive hypotheses both assume that cells existed before viruses. What if viruses existed first? Recently, several investigators proposed that viruses may have been the first replicating entities. Koonin and Martin (2005) postulated that viruses existed in a precellular world as self-replicating units. Over time these units, they argue, became more organized and more complex. Eventually, enzymes for the synthesis of membranes and cell walls evolved, resulting in the formation of cells. Viruses, then, may have existed before bacteria, archaea, or eukaryotes (Figure 4; Prangishvili et al. 2006).
Most biologists now agree that the very first replicating molecules consisted of RNA, not DNA. We also know that some RNA molecules, ribozymes, exhibit enzymatic properties; they can catalyze chemical reactions. Perhaps, simple replicating RNA molecules, existing before the first cell formed, developed the ability to infect the first cells. Could today's single-stranded RNA viruses be descendants of these precellular RNA molecules?
Others have argued that precursors of today's NCLDVs led to the emergence of eukaryotic cells. Villarreal and DeFilippis (2000) and Bell (2001) described models explaining this proposal. Perhaps, both groups postulate, the current nucleus in eukaryotic cells arose from an endosymbiotic-like event in which a complex, enveloped DNA virus became a permanent resident of an emerging eukaryotic cell.
No Single Hypothesis May Be Correct
Where viruses came from is not a simple question to answer. One can argue quite convincingly that certain viruses, such as the retroviruses, arose through a progressive process. Mobile genetic elements gained the ability to travel between cells, becoming infectious agents. One can also argue that large DNA viruses arose through a regressive process whereby once-independent entities lost key genes over time and adopted a parasitic replication strategy. Finally, the idea that viruses gave rise to life as we know it presents very intriguing possibilities. Perhaps today's viruses arose multiple times, via multiple mechanisms. Perhaps all viruses arose via a mechanism yet to be uncovered. Today's basic research in fields like microbiology, genomics, and structural biology may provide us with answers to this basic question.
Summary
Contemplating the origins of life fascinates both scientists and the general public. Understanding the evolutionary history of viruses may shed some light on this interesting topic. To date, no clear explanation for the origin(s) of viruses exists. Viruses may have arisen from mobile genetic elements that gained the ability to move between cells. They may be descendants of previously free-living organisms that adapted a parasitic replication strategy. Perhaps viruses existed before, and led to the evolution of, cellular life. Continuing studies may provide us with clearer answers. Or future studies may reveal that the answer is even murkier than it now appears.
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.8. RELATIONSHIP BETWEEN EXOSOMES AND VIRUSES
What is a good article that explains the relationship between exosomes and viruses?
One good article that explains the relationship between exosomes and viruses is “Exosomes and Their Role in the Life Cycle and Pathogenesis of RNA Viruses” 1. This article discusses how exosomes, which are small vesicles secreted by cells, can play a role in the life cycle and pathogenesis of RNA viruses 1.
{So following is that article.}
Exosomes and Their Role in the Life Cycle and Pathogenesis of RNA Viruses
Abstract
Exosomes are membrane-enclosed vesicles actively released into the extracellular space, whose content reflect the physiological/pathological state of the cells they originate from. These vesicles participate in cell-to-cell communication and transfer of biologically active proteins, lipids, and RNAs. Their role in viral infections is just beginning to be appreciated. RNA viruses are an important class of pathogens and affect millions of people worldwide. Recent studies on Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), human T-cell lymphotropic virus (HTLV), and Dengue Virus (DENV) have demonstrated that exosomes released from infected cells harbor and deliver many regulatory factors including viral RNA and proteins, viral and cellular miRNA, and other host functional genetic elements to neighboring cells, helping to establish productive infections and modulating cellular responses. Exosomes can either spread or limit an infection depending on the type of pathogen and target cells, and can be exploited as candidates for development of antiviral or vaccine treatments. This review summarizes recent progress made in understanding the role of exosomes in RNA virus infections with an emphasis on their potential contribution to pathogenesis.
1. Introduction
Exosomes are lipid bilayer membrane-enclosed nano-sized (30–100 nm) vesicles with a density of 1.13–1.19 g/mL, secreted by virtually all cell types, and formed during the maturation of endosomes upon invagination and budding of the limiting membrane of late endosomes as intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). Exosomes were first observed in the early 1980s in the culture media of reticulocytes [1,2,3]. In their study, Harding et al. (1983) reported that clathrin-coated, pit-mediated endocytosis led to enrichment of gold-labeled transferrin on vesicles inside non-lysosomal multivesicular endosomes [1]. They observed that these endosomes, commonly known as MVBs, fused with the plasma membrane and released their inner vesicles by exocytosis. Johnstone, in 1987, coined the term “exosomes” for such vesicles, which are released from cells by exocytosis [1]. Exosomes are released by almost all cell types and have also been confirmed in all bodily fluids such as blood, urine, saliva, breast milk, bronchial lavage, cerebral spinal fluid, and amniotic fluid [4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19]. In order to best adapt to the surrounding microcosm and execute their functions and duties, continuous communication, achieved through methods like direct cell–cell contact or transfer of secreted molecules, is essential for cells and tissues. Although initially it was proposed that secretion of exosomes was a mechanism of discarding cellular waste [16,20,21], in recent years exosomes have emerged as an important tool for intercellular communication through the transfer of biologically active proteins, lipids, and RNAs [22]. Exosomes have been implicated in normal as well as pathophysiological conditions, such as lactation, immune response, neuronal function, development and progression of liver disease, neurodegenerative diseases, cancer, and viral infections [16,22,23,24,25,26,27]. Exosome-mediated extracellular delivery of nucleic acids and proteins among virally infected and uninfected bystander cells have been shown to play an important role in viral pathogenesis and control of host immune responses to infection [28,29,30,31]. This certainly suggests a crucial role for exosomes in the viral life cycle and this review focuses on the important role exosomes play in the life cycle of RNA viruses with an emphasis on their potential contributions to pathogenesis.
2. Molecular Composition of Exosomes
Exosomes are essentially cytoplasm enclosed in a lipid bilayer with exposed external domains of transmembrane proteins. Exosomes may contain all types of biomolecules like proteins, carbohydrates, lipids, and also a nucleic acid signature of source of origin. New purification methods providing highly pure preparations of exosomes have allowed the use of proteomic and molecular techniques to understand the molecular composition of exosomes. The presence of cellular proteins in exosome preparations from various cellular sources has been analyzed by various methods like western blot, fluorescence-activated cell sorting (FACS), ELISA, and mass spectrometry [11,32,33,34,35,36,37,38,39]. Extensive analyses involving quantitative RT-PCR and RNA deep sequencing to identify RNA species that are present in exosomes have also been carried out [40,41,42,43,44,45,46]. These extensive and in-depth analyses revealed that a defined subset of cellular proteins, probably involved in exosome biogenesis and maybe in some common exosome functions, is targeted specifically to exosomes. This may include cytoskeletal components such as actin and actin-binding proteins, tubulin, and proteins involved in intracellular membrane fusions and transport such as Annexins and Rab (Ras (rat sarcoma) genes from the rat brain) proteins [47,48]. Molecules involved in signal transduction such as protein kinases (14-3-3) and heterotrimeric G proteins, heat shock proteins (HSPs), such as HSP70 and HSP90, and MHC class I and II molecules are also part of this defined but common set of exosome proteins. Tetraspanins including CD9, CD63, CD81, and CD82, as well as cytoskeletal components such as actin, are among the most abundant proteins in exosomes from virtually any cell type. Since exosomes are generated through invagination of late endosomes, a variety of host proteins that participate in vesicle formation and trafficking such as apoptosis linked gene (ALG), 2 interacting protein X (ALIX), and tumor susceptibility gene 101 (TSG101) are also incorporated into the exosomes [49]. All of these proteins have been considered as consensus markers for exosomes [50,51,52].
Exosomal lipid composition has also been characterized and exosomes are rich in sphingomyelin, gangliosides, phosphatidylserine, and cholesterol [53]. Nucleic acid signature is the other important component of exosomes and recent studies have focused on exosomal nucleic acid content. It has been demonstrated that exosomes carry biologically active mRNA, miRNA, other non-coding RNA, and a limited amount of DNA coding for ribosomal RNA [54,55,56,57,58]. However, the RNA and protein composition of exosomes varies in both quantity and type of molecules, depending on the origin and physiological/pathological state of the cells, suggesting that recruitment of RNA and protein into exosomes is a regulated process [55,59]. The exosome structure is graphically represented in Figure 1.
Figure 1
Structure and composition of exosomes. Exosomes contain a plasma membrane-derived phospholipid bilayer membrane. The composition depends on the cell type of origin, state of health of the host, and extracellular stimuli. Exosome contents include mRNA, miRNA, DNA, and proteins like annexins, tetraspanins, Alix, TSG101, MHC molecules, Rab proteins, cytoskeletal proteins, enzymes, and signal transduction proteins. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase PGK: 3-phosphoglycerate kinase, PK: pyruvate kinase, EGFR: epidermal growth factor receptor, CDC42: cell division control protein 42, PI3k: phosphatidylinositide 3-kinases, ARF1: ADP-ribosylation factor 1, MUC1: Mucin 1, vRNA: viral RNA, vmiRNA: viral miRNA.
2.1. Biogenesis of Exosomes
Exosome biogenesis starts with the endocytosis and formation of early endosomes. The early endosome develops into the late endosome upon maturation, which is characterized by the formation of intraluminal vesicles (ILV) inside the lumen of the endosome. The ILVs, 30–100 nm in diameter, are formed by inward budding of the endosomal membrane, randomly engulfing portions of the cytosol and incorporating transmembrane and peripheral proteins into the invaginating membrane; this leads to formation of multivesicular bodies (MVBs) [5,60,61]. Although endocytosis and trafficking of plasma membrane receptors into MVBs is responsible for their degradation upon fusion with lysosomes [62], the fate of the MVBs may vary and not all MVBs are degraded in lysosomes, with a subset fusing with plasma membrane and resulting in generation of exosomes. The process of exosome biogenesis and cargo sorting is still not well understood and many studies suggest that the mechanisms of exosome biogenesis can be cell specific [63]. Exosomes are mainly secreted by two different mechanisms, constitutive release via the Trans-Golgi network and inducible release [64,65].
In the vesicle generation process, the endosomal sorting complexes required for transport 0 (ESCRT0) ubiquitinate proteins for MVB delivery and also recruit ESCRTI to endosomal membrane, which in turn recruits ESCRTII and ESCRTIII [66,67]. ESCRTIII mediates formation of polymeric filaments, which leads to membrane invaginations and eventually results in ILV formation [68]. The presence of ESCRT components in exosomes was identified using high throughput protein analysis methods, and downregulation of key components of ESCRT system abrogates ILV formation and release of exosomes [69]. Various studies also suggest ESCRT-independent mechanisms of exosome biogenesis and release. For example, in oligodendroglial cells exosome generation is regulated by the production of a lipid ceramide [70]. Recently, a CD63 tetraspanin-mediated mechanism of cargo sorting and ILV formation was reported, which is independent of ESCRT and ceramide [71]. Once the MVBs are formed, the soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE) proteins and GTPases mediate their fusion with plasma membrane. Rab 35 has been recently shown to be part of the MVB docking to the membrane and depletion of Rab35 significantly decreased exosome release [72]. Although exosomes and ILVs are similar and generated through common mechanisms, cells have different populations of vesicles [47,73,74] and the mechanisms that contribute to exosome formation and cargo sorting within these vesicles is still not well understood. The process of exosome biogenesis is summarized in Figure 2.
Figure 2
Schematic representation of exosome biogenesis and release. Exosome generation starts with early endosome formation during endocytosis. The membrane proteins are internalized through clathrin-coated vesicles and delivered to early endosomes. This leads to intraluminal vesicles (ILVs) formation by inward budding of the limiting membrane and multi vesicular bodies (MVBs) are formed. Upon maturation the exosome-filled MVBs are either sent to lysosomes for degradation or fused with the plasma membrane to release exosomes to the extracellular milieu. RER: Rough Endoplasmic Reticulum, GC: Golgi complex.
2.2. Exosome Characterization
Various viruses like paramyxoviruses, HBV, HCV, rhabdoviruses, herpersviruses, filoviruses, and arenaviruses utilize or need the ESCRT pathway for their release [75,76,77]. Characterization and investigation of exosomes derived from virus-infected cells is a tough task as these vesicles often are of similar density and fall in the same size range as many of these viruses, making it necessary and at the same time challenging to separate the two populations. Conventionally, the exosomes are isolated from culture media or bodily fluids using a sequential sucrose-gradient ultracentrifugation procedure [78]. Other methods of exosome isolation/purification include microfiltration technologies, microfluidic devices [79,80,81,82], exosome precipitation reagents like ExoQuick™ (System Biosciences, Mountain View, CA, USA), and Total Exosome Isolation reagent (Life Technologies Grand Island, NY, USA), as well as antibody-coated magnetic bead-based immunopurification [38,83]. Similar sequential centrifugation and ultracentrifugation methods are used to purify many enveloped or non-enveloped viruses [84,85,86,87]. For instance, exosomes derived from HIV or HCV-infected cells cannot be readily distinguished or separated from infectious viral particles by conventional biophysical techniques as they share similar buoyant densities and sedimentation velocities [22,88,89,90]. Hence, when isolating exosomes from virus-infected cells, it becomes critically important to make sure that the pelleted material is exosomes and not virus particles. To address this, various exosome characterization methods have been developed including measurement and analysis of size distribution using NanoSight nanoparticle tracking analysis system, visualization of exosomes using electron microscopy, and immunoblot analysis of universal exosome protein markers like CD63, CD81, TSG101, Annexin5, ICAM1, FLOT1, and Alix (Figure 3).
Figure 3
Schematic representation of exosome isolation strategies. Graphical representation of exosome isolation by both ultracentrifugation- and precipitation reagent-based isolation procedures, and analytical tools for exosome identification.
When using immunoblot analysis, the exosome marker chosen for characterization should be selected with caution, as some viruses have been shown to incorporate exosome proteins. For example, proteomic studies utilizing liquid chromatography and tandem mass spectrometry (LC-MS/MS) found that influenza virus incorporates exosome markers ICAM1, Annexin A3, CD81, and CD9, while CD63 and ALIX were not present [91]. Similarly, exosomes and retroviruses share a variety of molecules like MHC-II [92,93], integrins (CD11a, CD18), co-stimulatory molecules (CD28, CD54), and complement neutralizing molecules (CD55, CD59) [5,35,94,95]. Various other host molecules/proteins acquired by enveloped viruses are reviewed in Cantin et al. [95]. Hence, the enriched exosomes isolated by ultracentrifugation or precipitation reagent should be further subjected to immunopurification methods like CD63 immunomagnetic bead isolation or other efficient virus purification strategies to obtain contamination-free populations of exosomes (Figure 4).
Figure 4
Schematic representation of exosome immune-isolation. To obtain exosomes free of contaminants, exosomes can be subjected to immunomagnetic selection using anti-CD63 antibody conjugated beads and then characterized by immunoblot, electron microscopy, and size determination.
3. Transfer of Viral and Cellular Components by Exosomes and Their Role in Virus Replication and Transmission
Although the field of exosomes and their contribution to replication and pathogenesis of RNA viruses remain largely unexplored, a few RNA viruses have been investigated, such as HIV-1, HTLV-1, HCV, and Dengue virus. Findings from these studies have demonstrated that exosomes released from virus-infected cells contain a variety of viral and host cellular factors that are able to modulate recipient host cell responses and lead to the establishment of productive infection.
3.1. Exosomes as Carriers of Virus and Host RNA Species
It has been reported that exosomes carry various cellular regulatory RNAs, including miRNAs, sncRNAs, and siRNAs [96]. Exosomes derived from virus-infected cells have been shown to carry viral components including viral mRNA, miRNA, and genomic RNA, as well as genetic regulatory elements. Among the RNA viruses, HIV-1 was the first one to be studied somewhat in detail in the context of modulation of exosome formation. Exosomes derived from HIV-1 infected cells or patients with HIV infection incorporate the viral transactivating response (TAR) element transcribed from the integrated provirus, which has been suggested to enhance HIV replication in the recipient cells via downregulation of apoptotis [42]. Unspliced HIV-1 RNA species are recruited to exosomes and the presence of a stretch of sequences within the 5' end of the Gag p17 open reading frame is sufficient for this recruitment, while single- or double-spliced HIV-1 RNA is not incorporated into exosomes. The incorporation of genomic HIV-1 RNA in exosomes is further increased if the producer cells express HIV-1 defective for viral genome packaging [97].
Exosome-like microvesicles isolated from serum during infection with human Pegivirus (an RNA virus within the Pegivirus genus of the Flaviviridae previously known as Hepatitis G virus) have been shown to carry viral RNA and to be able to transmit viral RNA to peripheral blood mononuclear cells in vitro, resulting in productive infection [98]. Exosomes released from HCV-infected cells contain HCV RNA, which can be successfully transferred to dendritic cells to establish productive infection [99]. Two subsequent studies confirmed that exosomes derived from HCV-infected hepatocytes contain the complete HCV genome and they have the ability to mediate transfer of replication-competent subgenomic HCV RNA to permissive naive cells, leading to viral RNA replication and productive infection [100,101]. Exosomes derived from HCV-infected patients have also been found to contain negative sense HCV RNA (replication competent viral RNA), in association with Ago2, HSP90, and miR-122 [102].
A recent study reported that exosomes derived from Human T-lymphotropic virus type 1 (HTLV-1)-infected cells contain the Tax, HBZ, and Env gene mRNA transcripts, suggesting that exosomes can serve as vehicles to deliver functional HTLV-1 mRNA to recipient cells [103].
Exosomes secreted from HIV-1-infected primary alveolar macrophages carry viral microRNAs vmiR88, vmiR99, and vmiR-TAR; these miRNAs have also been detected in exosomes purified from the sera of HIV-infected individuals. Viral microRNAs vmiR88 and vmiR99 were shown to stimulate signaling in macrophages, resulting in robust release of TNFα through macrophage endosomal TLR8 [104]. Expression of HIV Nef protein in macrophage-like cells results in selective recruitment of 47 miRNAs into exosomes, with two miRNAs selectively retained in the same cells [105], suggesting that modulation of exosomal RNA composition during a viral infection is a regulated process and that specific mechanisms exist to recruit or retain specific miRNAs/RNAs. Astrocytes exposed to a combination of HIV protein Tat and opiate drugs secreted exosomes with elevated levels of miR-29b. In human neurons exposed to miR-29b-enriched exosomes, platelet-derived growth factor-B expression was repressed and neuron viability was correspondingly decreased [106]. The presence of Ago2, an essential protein of RNA-induced silencing complex (RISC) for mediating miRNA-targeted gene suppression, and cellular miRNAs in exosomes secreted from HTLV-1-infected cells suggested that HTLV-1 could manipulate mRNA translation in recipient cells [107].
3.2. Exosomes as Viral Protein Carriers
Exosomal protein composition varies depending on cell type and disease state. Several RNA viruses have been shown to modulate not only host cell protein composition of exosomes but also to recruit their own proteins into exosomes. Exosomes from HIV-1-infected cells have been shown to incorporate both Gag [108] and Nef proteins [109,110]. The latter is incorporated into exosomes upon anchoring into lipid raft microdomains through its N-terminal myristoylation and a stretch of basic amino acids residing in its alpha-helix-1 [111], as well as upon interaction with the host cell protein Mortalin [112].
Similar to HIV, the HTLV-1 transactivator protein Tax, a critical factor for proliferation and transformation of T cells, is incorporated in exosomes secreted from virally-infected cells [103]. Several cellular proteins also seem to be recruited to exosomes in a Tax-dependent manner, many of them involved in protein synthesis and antigen presentation [103]. Exosomes derived from HCV-infected cells have been shown to carry HCV core protein, in addition to apolipoproteins ApoE and ApoB [101]. In HCV-positive patients, the cellular membrane protein CD81 has been shown to associate with the envelope glycoprotein E2. Extracellular release of E2-CD81 within microvesicles is associated with increased virus fusing ability and infectivity of naive cells [113]. Table 1 enlists the protein and RNA species of RNA viruses recruited to exosomes.
Table 1
Viral protein and RNA species present in exosomes derived from RNA virus-infected cells.
Protein and RNA Species of RNA Viruses Present in Exosomes
Viral Proteins HIV: Nef and Gag proteins
HCV: HCV core protein
HTLV-1: Transactivator protein Tax
Viral RNA and microRNA HIV: HIV-1 transactivating response (TAR) element RNA, microRNAs vmiR88, vmiR99 and vmiR-TAR, unspliced HIV-1 RNA species,
HCV: HCV genomic RNA
HTLV-1: HTLV-1 Tax, HBZ, and Env gene mRNA transcripts
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3.3. Role of Exosomes in Pathogenesis
Immature dendritic cells capture HIV-1 and can transfer these captured HIV-1 particles to T cells. Wiley and Gummuluru, back in 2006, reported that exosomes derived from HIV-1 containing immature dendritic cells can transfer HIV-1 to T cells without de novo infection. Exosomes isolated with HLA-DR-1-conjugated magnetic beads from the supernatant of DCs exposed to HIV-luc reporter viruses incubated with Jurkat T cells resulted in productive infection of cells. They also reported that endocytosed virus particles were the main contributors to exocytosed virus fraction, as treatment of virus-exposed DCs with trypsin had no or a negligible effect on the amount of virus particles precipitated by HLA-DR-1-conjugated magnetic beads [114].
Although the role of exosomes in HIV infection has not yet been fully understood, increasing evidence suggests that exosomes facilitate both enhancement and inhibition of infection and replication, depending upon the cells of origin. For instance, exosomes derived from HIV-infected cells have been shown to contain the HIV coreceptors CCR5 and CXCR4, and transfer of these coreceptors to uninfected, non-permissive cells may enhance susceptibility to HIV infection [115,116]. In a recent study, Kadiu et al. reported that a portion of HIV virions shed from monocyte-derived macrophages is associated with exosomal aggregates and these entrapped virions demonstrate improved infectivity toward CD4+ target cells, compared to purified HIV-1 virus particles [117]. HIV-infected and viremic individuals exhibit elevated levels of plasma cytokines. Many cytokines have been found to be markedly enriched in exosomes from HIV-positive individuals relative to negative controls and exposure of naive peripheral blood mononuclear cells to exosomes purified from HIV-positive patients induces CD38 expression on naive and central memory CD4+ and CD8+ T cells, probably contributing to inflammation and viral propagation via bystander cell activation [118].
HIV accessory protein negative factor (Nef) is one of the earliest and most abundantly expressed viral proteins. Nef is also released in exosomes. Lenassi et al. (2010) reported that Nef not only stimulates its own export through exosomes, but these Nef exosomes also facilitate the depletion of CD4+ T cells that is a hallmark of acquired immunodeficiency syndrome (AIDS) [109]. In fact HIV Nef, through exosomes, induces the activation of resting CD4+ T lymphocytes, rendering these quiescent CD4+ T lymphocytes permissive to HIV-1 replication and thus stimulating viral spread [119]. HIV-1 Nef promotes viral replication and pathogenesis by mediating depletion of CD4 and MHC-I molecules. Nef binds to the cytosolic tail (CT) of CD4 and MHC-I and disrupts the intracellular trafficking of these proteins targeting them to multivesicular bodies (MVBs), and ultimately to lysosomes for degradation. In a recent study Carvalho et al. (2014) reported that exosomes secreted by CD4+ T cells, but not CD4− T cells, efficiently inhibit HIV-1 infection in vitro, suggesting that exosomal CD4 can bind to envelope proteins of HIV-1, hindering virus interaction with target cells and eventually reducing the infection [110]. They also showed that this effect could be reversed by depleting CD4 exosomes released by CD4+ T cells expressing Nef. The remaining exosomes have a reduced capacity to inhibit HV-1 infection in vitro [110]. HIV Nef also modulates exosomal miRNA composition, further suggesting a role for exosomes in HIV pathogenesis and viral replication [110].
Exosomes play an important role in HTLV-1 infection as well, probably by delivering functional HTLV-1 Tax protein, proinflammatory mediators, and viral mRNA transcripts of Tax, HBZ, and Env proteins. Along with other host proteins, major histocompatibility complex (MHC) class I A and class I E precursor were also identified in exosomes derived from HTLV-1 infected cells. The addition of C81 cell-derived exosomes (containing Tax protein) to myeloid dendritic cells resulted in a significant increase in the levels of IL-2, IL-5, and IL-6 cytokines. In fact, cell-free Tax could induce IL-10, IL-12, IL-17A, IFN-γ, and G-CSF secretion from dendritic cells. The findings of Jaworski et al. (2014) suggest that exosomes play a crucial role in signal transduction and may contribute to pathogenesis of HTLV-1 infection [103].
Human cytidine deaminase APOBEC3G (A3G) is part of a cellular defense system against HIV-1 as well as other retroviruses. In a recent study, Khatua et al. (2009) found that APOBEC3G secreted by cells in exosomes can confer resistance to both defective and wild-type HIV-1 infection in exosome recipient cells [120]. Esser et al. demonstrated that CD45, CD86, and MHC Class II molecules present in exosomes derived from HIV-infected cells may help in silencing immune response, therefore favoring virus replication [121]. Exosomes derived from HIV-1 infected CD8+ T cells suppressed replication of both CCR5- and CXCR4-tropic HIV-1 strains in vitro by inhibiting HIV-1 transcription in both acute and chronic models of infection [122]. Exosomes can not only transmit HCV to naive cells but also offer some degree of protection from HCV neutralizing antibodies. By making use of transmission electron microscopic imaging, Liu et al. demonstrated that HCV was present in both exosome-free and exosome-associated forms and the association with exosomes conferred the ability to resist anti-HCV antibody mediated neutralization, suggesting that HCV may utilize transmission via exosomes as an immune evasion mechanism [101,123].
Recently a new set of proteins called IFN inducible transmembrane proteins 1, 2, and 3 (IFITM1, 2, and 3) have been shown to display antiviral activities induced by IFN, conferring host cells resistance to various viral pathogens [124]. In their study Zhu et al. (2015) found that the IFITM3 protein level in host cells inversely correlates with their susceptibility to Dengue Virus-2 (DENV-2) infection [124]. Exosomes derived from HUVEC or HepG2 cells contain IFITM3 protein and can transfer this protein to neighboring cells. Investigating the functional aspect of this exosome-mediated transfer of IFITM3, they found that in recipient cells DENV-2 infection was effectively suppressed by the IFITM3-containing exosomes in a dose-dependent manner. The authors suggested that the IFITM3-containing exosomes did not affect the binding or post-entry steps during DENV-2 infection, but reduced the penetration of DENV-2 into cells, demonstrating an important role for exosomes in DENV-2 infection [124]. There is also evidence that some viruses harness exosomes to avoid immune recognition in the bloodstream or as reservoirs of virus latency. Hepatitis A virus (HAV), a non-enveloped virus, was found to be encapsulated into vesicles derived from endosomal membranes [47]. These enveloped HAV particles were fully infectious. They resembled exosomes and their biogenesis was dependent on ESCRTs and ESCRT effectors [47]. Their membrane cloak protected them from antibody neutralization and virus-specific antibodies appear only after 3–4 weeks of infection.
4. Potential Applications in Viral Infections
Exosomes appear to be an important tool of intercellular communication, as discussed above. However, their further use in various other processes is also being evaluated. The exosomes can be used as a diagnostic marker, as vaccines, and as a drug delivery vehicle for targeted or systemic delivery. Since exosomes have been detected in all bodily fluids, can be easily purified, and have a composition that varies in normal and diseased conditions, they can be exploited as diagnostic markers of diseases. However, the use of exosomes as a diagnostic marker for viral infection has not yet been explored adequately.
Targeted delivery is another area where the potential of exosomes to carry therapeutic cargo to specific organs or tissues is being evaluated. Expression of receptor-specific ligand molecules on the exosome surface through genetic engineering can transform exosomes into potent delivery vehicles that can deliver a drug/siRNA/miRNA based therapeutic moiety to cells or tissues of choice. In fact, the ability of exosomes to deliver therapeutic moiety or genetic material can be further improved by incorporating selected viral proteins into exosomes as virus-encoded envelope proteins exhibiting superior binding and entry specificity (reviewed in [125]). For instance, exosomes engineered to express a 29-mer peptide derived from the rabies virus glycoprotein (RVG), which specifically binds to acetylcholine receptors expressed on the brain cells, were exploited by Alvarez-Erviti et al. (2011) to transport small interfering RNAs to the brain [126]. The immature dendritic cells (DCs) were transfected with plasmids encoding exosomal protein Lamp2b, fused with the 29-mer RVG peptide. Exosomes were purified from DC cultures, loaded with GAPDH or BACE-1 siRNA, and injected intravenously through tail vein injection. The targeted delivery resulted in specific knockdown of GAPDH and BACE-1 in the mouse brain [127]. In other studies, DCs were transduced with adenoviral vector to express Interleukin (IL)-10, IL-4, or FasL, and the engineered exosomes were used to treat autoimmune disorders and inflammatory diseases (reviewed in [128]). Similar methods can be utilized to engineer exosomes to deliver siRNAs to control viral infections such as West Nile Virus or other viral infections of the brain or other organs like the liver or lungs. Exosomes are also being evaluated as vaccines in the field of infectious diseases. Aline et al. (2004), investigated the efficacy of DC2.4 cell line-derived exosomes to mount a protective immune response against toxoplasmosis [129]. They found that Toxoplasma gondii-pulsed DC-derived exosomes transferred to the spleen, elicited a strong systemic Th1-modulated Toxoplasma-specific immune response in vivo, and were able to protect the animals against Toxoplasma infection [129]. This suggests that exosomes can also be used for immunoprophylaxis against viral pathogens; however, systemic studies need to be conducted to evaluate the therapeutic or protective role engineered exosomes can play in the field of infectious diseases. Exosomes offer many advantages, including but not limited to being natural transport body vehicles of antigens and signals between cells, providing a stable environment for nucleic acids and proteins by protecting them from DNase, RNase, and proteinases, efficient association/interaction with antigen-presenting cells, and offering better molecular distribution capabilities as are present in all bodily fluids [125,130].
5. Conclusions
Various studies have demonstrated that exosomes are crucial intercellular communication channels and highlighted their potential role in viral transmission and modulation of immune responses, as viruses exploit the exosomal pathway for their assembly/budding, transfer of viral RNAs, and suppression of immune activation. In addition, exosomes could be utilized as diagnostic markers in viral infections and for targeted drug delivery. Since exosome research related to viral infections is still in an early stage, more studies are required to decipher the interplay between exosome biology and viruses, as a comprehensive understanding of exosome biology and its involvement in viral infections would permit the development of new strategies to interfere with viral replication and disease development.
L.O.L.
Show me ONE experiment where a 'virus' has been isolated directly from a sample taken from a person.
ONE.