virus
holtaprg_08_c19_final.pdfCHAPTER 19: VIRUSES
A Borrowed Life
-Viruses invade host cells by injecting their genetic information into them and hijacking metabolic machinery for production of more viruses to further the infection.
-Compared to eukaryotic and even prokaryotic cells, viruses are smaller and less complex.
-Virus: infectious particle consisting of little more than genes packaged in a protein coat.
-Because viruses cannot reproduce or carryout metabolism without a host cell, they are not considered living.
-Viruses are used by researchers to transfer genes in gene therapy studies.
19.1: A virus consists of a nucleic acid surrounded by a protein coat
The Discovery of Viruses: Scientific Inquiry
-Mayer: discovered that he could transmit tobacco mosaic disease from one plant to another by rubbing extracted sap from diseased leaves onto heathy ones.
-failed to find a microbe to blame for this, concluded this particular microbe was unusually small
-Ivanowsky: filtered sap to remove bacteria, but leaves still infected with disease after sap rubbed on healthy leaves
-concludes bacteria small enough to pass through filter or produced a toxin that passed through filter
-Beijerinck: infectious agent in sap could replicate within the host plant, could not be cultured on media in lab
-concludes caused by particle much smaller and simpler than a bacterium.
-Stanley: crystallized the infectious particle, now known as tobacco mosaic virus.
-viruses can be seen with electron microscope.
Structure of Viruses
-Viruses are infectious particles that consist of a nucleic acid enclosed in a protein coat and, for some viruses, surrounded by a membranous envelope.
Viral Genomes
-Viral genomes may consist of double-stranded or single-stranded DNA, or double-stranded or single-stranded RNA.
-viruses containing DNA are called DNA viruses.
-viruses containing RNA are called RNA viruses.
-The genome is organized as a single linear or circular molecule
-viruses can contain between 3 and 1000 genes.
Capsids and Envelopes
-Capsid: protein shell surrounding the viral genome.
-may be rod-shaped, polyhedral or more complex in shape.
-built from a large number of capsomere protein subunits, generally there are very few different types of capsomere proteins composing the capsid.
-Viral Envelope: accessory structure derived from plasma membrane of host cell that surround the capsid
-because they are derived from host cell mmb, consist of phospholipids and membrane proteins.
19.2: Viruses Replicate only in host cells
- Viruses lack metabolic enzymes and equipment for making proteins, such as ribosomes.
-Each particular virus can infect only a limited number of host species, this is called the host range.
-this is the result of the evolution of recognition systems by the virus.
-viruses usually recognize host by a "lock and key" fit between viral surface proteins and specific receptor molecules on the outside of cells.
-certain viruses have very broad host ranges, while others are limited to one species
-Viral infection of multicellular eukaryotes is usually limited to particular tissues.
- (i.e. : AIDS virus binds cells in immune system, human cold viruses bind only cells in the upper respiratory tract)
General Features of Viral Replicative Cycle
-A virus begins when virus binds host cell and viral genome enters cell.
-the mechanism of entry varies among viruses
-Once the viral genome is inside, the proteins it encodes can commandeer the host, reprogramming the cell to copy the viral genome and manufacture viral proteins.
-the host provides the nucleotides, enzymes, ribosomes, tRNAs, amino acids, ATP, etc.
-Many DNA viruses use the DNA pol of the host cell to synthesize new genomes
-RNA viruses use virally encoded RNA polymerases that can use RNA as a template. (uninfected cells make no such enzymes)
-After viral nucleic acid molecules and capsomeres are produced, they spontaneously assemble into new viruses within the host cell.
-The simplest of virus replicative cycles ends with the exit of hundreds or thousands of viruses from a host cell, which often damages or destroys the cell.
-The viral progeny that exit the cell have the potential to infect additional cells, spreading the viral infection.
Replicative Cycles of Phages
-Some double-stranded DNA viruses can replicate by two mechanisms: the lytic cycle and the lysogenic cycle
1-phage binds to the surface of the cell and injects their linear DNA genome.
2 -within the host, the phage DNA forms a circle
3-From this point it depends on which replicative mode is employed by the phage.....
- Lytic Cycle: a phage replicative cycle that culminates in death of the host cell, the term refers to the last stage of infection, during which the bacterium lyses (breaks open) and releases the phage progeny.
4- viral genes immediately turn the host cell into a phage producing factory.
5- the cell soon lyses and releases the virus progeny
-The phages that are released from the cell can then infect other healthy cells
-Successive cycles can destroy an entire bacterial population in a short time.
- Phages that only replicate by this mechanism are called virulent.
- Bacteria have defenses from these phages:
- natural selection favors bacterial mutants with surface proteins that are no longer recognized by phages as receptors. (However, natural selection also selects for phage mutants that can bind the altered receptors)
- phage DNA is also recognized by bacterial restriction enzymes and cut up into pieces, making it so the phage
cannot replicate.(However, natural selection also selects for phage mutants that are resistant to restriction enzymes)
- Some bacteria coexist with phages in a state called lysogeny.....
-Lysogenic Cycle: allows replication of the phage genome without destroying the host.
4- the DNA molecule is incorporated into a specific site on the host chromosome by viral proteins that break both circular DNA molecules and join them to each other.
-The viral DNA is known as a prophage when it is incorporated into the bacterial chromosome
-one prophage gene codes for a protein that prevents transcription of most other prophage genes,
making the phage genome mostly silent.
5- The viral DNA is replicated with the host DNA, and when the host divides, the virus DNA is present in the offspring in prophage form.
-Sometimes a few prophage genes are expressed during lysogeny.
- these genes may alter the host's phenotype.
-diphtheria,botulism and scarlet fever all cause the host bacteria to produce toxins, which make people ill.
-Phages capable of both replicative cycles are called temperate.
- temperate phages can be induced to exit the bacterial chromosome and initiate a lytic cycle.
Replicative Cycles of Animal Viruses
-viruses that cause illness in humans an animals can only replicate inside host cells, as well.
-One key variable is the nature of the viral genome, double-stranded or single-stranded DNA or RNA.
- this is the basis for common classification of viruses.
-Single- stranded RNA viruses are further broken down into 3 classes based on how the RNA genome functions inside a host cell.
-Few bacteriophages have an envelope or RNA genome, but many animal viruses have both.
-Viral envelopes:
-a viral envelope is an outer membrane, used to enter a host cell.
- on the exterior of the viral envelope are viral glycoproteins that bind to specfic receptor molecules on the surface of the host cell.
-see process outlined below
-The viral envelope is usually derived from the host cell's plasma membrane, though the molecules that make it up are specified by the viral genes.
-this replicative cycle does not kill host cells.
-Some envelopes are not derived from plasma membrane:
- i.e. herpes virus derive their membranes from the host's nuclear envelope, that is then shed and traded for one made from
the golgi's membrane.
-RNA as Viral Genetic Material:
-There are 3 types of single-stranded RNA genomes found in animal viruses.
- class IV: can directly serve as mRNA and thus can be translated into viral protein immediately after infection.
-class V: the RNA genomes serves instead as a template for mRNA synthesis.
-the RNA genome is transcribed into complimentary RNA strands, which function both as mRNA and as templates for the
synthesis of additional copies of genomic RNA
-class VI: retroviruses, equipped with enzyme called reverse transcriptase which transcribes an RNA template into DNA.
-HIV (human immunodeficiency virus) is the retrovirus that causes AIDS (acquired immunodeficiency syndrome).
-retroviruses contained 2 single strands of RNA and 2 molecules of reverse transcriptase. (see life cycle below)
- provirus: integrated viral DNA
-Evolution of viruses:
-Although viruses cannot replicate or carry out metabolic activities independently, their use of the genetic code shows an evolutionary connection with the living world.
-There are viruses that infect every type of living organism
-Because viruses need a host to replicate and metabolize, it is likely that viruses evolved after the first cells appeared.
-It is theorized that viruses originated from naked bits of cellular nucleic acids that moved from one cell to another possibly
through injured cell surfaces.
- The evolution of genes coding for capsid proteins may have allowed viruses to bind cell membranes, thus facilitating
the infection of uninjured cells.
-Candidates for the original sources of viruses are plasmids and transposons because they are all mobile genetic elements.
-plasmids are small, circular DNA molecules found in bacteria and in the unicellular eukaryotes called yeasts.
-plasmids exist apart from and can replicate independently of the bacterial chromosome, and are occasionally
transferred between cells
-transposons are DNA segments that can move from one location to another within a cell's genome.
-Some viral genes are essentially identical to genes of the host.
-Recent sequencing has also shown that the genetic sequences of some viruses are very similar to seemingly distant viral relatives.
- some animal viruses share
General Characteristics:
¨ virus means poison; someone once called them "a piece of bad news wrapped in a protein;"
¨ obligate intracellular parasites (can reproduce/replicate only inside a host cell)
¨ not cells; debate over whether or not they are considered “alive” (see below)
¨ consist of nucleic acids (DNA or RNA) in a protein coat, called a capsid (no cell membrane)
¨ they insert themselves into a host cell & direct the host cell's metabolic machinery to make more virus; the virus supplies information (the plan) in the form of its nucleic acid - raw materials and driving force (ATP & reducing power) are supplied by the host cell.
¨ all cellular organisms can be attacked by viruses; however, viruses are very specific for the organisms & cells they infect.
Are Viruses Alive?
¨ Characteristics of living things: reproduction, metabolism, organized as cells, contain all organic molecules (lipids, enzymes, nucleic acids, carbs), evolution & adaptation to changing environments.
¨ Viruses have some of these char's: they can evolve, they contain some macromolecules, they direct their own reproduction; However, they are not cells - they do not have cytoplasm, a cell membrane, organelles, ribosomes, or a nucleus. They have DNA or RNA, unlike prokaryotic and eukaryotic cells, which have both. In addition, they lack a metabolism of their own (they cannot produce ATP, etc.) - raw materials and driving force (ATP & reducing power) are supplied by the host cell.
I. HOW ARE THEY CLASSIFIED? (4 ways: size, structure, host range, life cycles)
A. Size - range from about 1/10th to 1/3rd the size of a small bacterial cell.
B. Structure - basic structure of a virus is a nucleic acid surrounded by a protein capsid; a membrane envelope may also be present outside of the capsid, but this is acquired from host cell. A complete viral particle (= capsid + nucleic acid + envelope if it is present) is called a virion.
1. Nucleic Acid - Viruses can store their genetic info. in different types of nucleic acid (each virus has only type). Viruses can have DNA or RNA. Their nucleic acid can be double stranded (ds) or single stranded (ss); they can even have double stranded RNA! RNA viruses can have a (-) sense strand or a (+) sense strand of RNA.
(+) sense RNA acts like mRNA and can be translated into proteins by the host cell’s ribosomes. (-) sense RNA does not make sense to the host cell’s ribosomes. After the virus containing this type of RNA enters the host cell, a complementary (+) sense strand is made from its (-) sense strand. Only (+) sense strand RNA can be read by the host cell’s ribosomes!
2. Capsids - protein coat that surrounds the nucleic acid; the constituent protein molecules making up the capsids are called capsomeres; there are 3 basic shapes based on how the capsomeres are arranged. See diagrams of these shapes!!
a. helical - proteins fit together as a spiral to form a rod-shaped structure.
b. polyhedral - proteins are arranged in equilateral triangles that fit together to form a geodesic dome-shaped structure; some appear almost spherical; you may have seen architectural structures that have this shape.
c. complex - combination viruses with a helical portion (tail) attached to a polyhedral portion (head); ex. many bacteriophages; may also have a tail sheath(participates in injecting the viral nucleic acid into the host cell), plate, pins, & tail fibers (help virus attach to host cell).
3. Viral Envelopes - pieces of the host cell's cell membrane that the virus acquires as it emerges from its host cell; the virus pushes out of the cell membrane, forming a bud that encloses the virus - then the bud pinches off behind, resealing the cell - as a result the host cell is not lysed. Glycoprotein spikes from the host cell’s glycocalyx may stick out of the envelope. Viruses that lack envelopes are called naked viruses. Because envelopes are acquired from host’s cell membranes, viruses may be hidden from attach by the host’s immune system. Envelopes also help viruses infect new cells by fusion of the envelope with the host’s cell membrane. On the other hand, enveloped viruses are damaged easily by physical and chemical antimicrobial agents.
C. Host Range - defined as the spectrum of organisms a virus attacks; viruses exhibit considerable specificity for hosts and even cells within that host; viral specificity is determined by whether or not a virus can attach to a cell. Attachment depends on the presence specific receptor sites on the surface of host cell and on specific attachment structures on the viral capsid or envelope. Examples of receptor sites are proteins, LPS’s, glycolipids, or glycoproteins.
D. Life Cycles of Bacteriophages (viruses that infect bacteria – means “bacteria eating”)
1. Replication [= Lytic Cycle] See diagram Events:
a. Adsorption - the virion attaches itself to a specific receptor site on the surface of the host cell.
b. Penetration - the viral nucleic acid penetrates the host cell
c. Uncoating - removing the capsid & envelope; basically 2 ways it can happen:
1.) during penetration, the virion disassembles so that only the viral nucleic acid enters host cell
2.) the entire virion enters the host cell & uncoating occurs later
d. Viral Synthesis (Latent Period) (also called biosynthesis) - more viral components
(nucleic acids & proteins for capsids) are synthesized by the host cell.
d. Maturation (Assembly) - components are assembled into new viruses
f. Release (Burst Period) - hundreds of intact virions exit host cell; 2 ways:
1.) If the virus is of the naked type, an encoded protein, lysozyme, dissolves the cell membrane &/or cell wall of the host cell, causing the cell to lyse & releasing the hundreds of viruses inside it.
2.) If the virus is to be an enveloped virus, it pushes out the cell membrane, forming a bud that encloses the virus - then the bud pinches off behind, resealing the host cell; as a result the host cell is not lysed.
2. Lysogenic Cycle (Lysogeny or Temperance) - Temperence involves the capacity of certain viruses to set up long-term relationships with their host cells - the virus remains latent for many cellular generations by becoming integrated into a host cell's chromosome (the integrated viral DNA is called a prophage). In this case no new viral components are synthesized & the host cell is not harmed. The virus may remain latent for long periods of time before initiating a lytic cycle. The problem with this type of cycle is that the viral nucleic acid that becomes integrated into the host cell's chromosome gets replicated along with the host cell's chromosome and is passed to daughter cells during cell division. In the prophage state, some viral genes are expressed, which may slightly change the host cell's phenotype (ex. only lysogenic strains ofCorynebacterium diphtheriae cause the disease diphtheria because the disease-causing toxin is encoded in the prophage of the infecting virus). Something (ex. temperature change) may trigger prophages to go into the lytic cycle. Released virions cannot infect cells that are carrying the same prophage - it makes the cell immune to attack by a virion of the same phage.
II. TAXONOMY
¨ Family names all end in viridae ; family names are often converted into English (ex. Retroviridae are called retroviruses). Genus names end in virus - species names are English words.
Ex. Retroviridae, Lentivirus, Human Immunodeficiency Virus (HIV)
¨ Groupings reflect only common characteristics and are not intended to represent evolutionary relationships.
A. Cultivating Animal Viruses & Diagnosing Viral Illnesses
¨ At one time animal viruses had to be cultivated & counted by infecting animals.
¨ In the 1930's it was discovered that embryonated chicken eggs could be used to culture animal viruses; embryonated eggs are inoculated with dilutions of a virus sample to determine the highest dilution that kills the embryo; this procedure was more economical & efficient than using adult animals.
¨ In the 1950's cell culture & tissue culture methods were developed. This solved the problem of viral specificity. Ex. Before cell cultures it was impossible to culture viruses in mice or chicken eggs that only infected humans (ex. HIV); continuous cell lines are usually derived from cancerous tissue & grow indefinitely in culture; regular cell lines grow increasingly slowly after 20-30 subcultures & eventually lose their ability to support viral replication; the most famous c.c.l. is the HeLa cell line(named after Helen Lack, the donor - from cervical cancer).
¨ Important Note: Physicians rely on symptoms to diagnose most viral illnesses. Culturing viruses takes too long & antibodies in the blood can usually be detected only after patient has recovered.
¨ Viral infections sometimes affect human cells in ways that can be seen under the microscope. For ex. the measles virus causes the membranes of neighboring cells to fuse, creating giant, multinucleated cells. Some virus-infected cells can be id. because they contain inclusion bodies, collections of viral components such as capsids and nucleic acid, waiting to be assembled into new viral particles. For example, the rabies virus produces inclusion bodies called negribodies in infected nerve cells (this is what we look for in suspected cases of rabid animals - have to look for negribodies in brain - animals have to be euthanized).
B. Replication of Animal Viruses (Lytic cycle) - proceeds through similar stages as bacteriophage replication.
1. Adsorption - Proteins in cell membrane act as receptor sites for a virus; remember, no cell walls in animal cells; adsorption is largely responsible for tissue specificity of animal viruses - only cells with a complementary receptor are attacked by a particular virus.
2. Penetration can occur in 3 ways:
a. viral envelope fuses with cell membrane, emptying the rest of the virion inside the cell.
b. other enveloped viruses enter by being phagocytized by a host cell
c. most naked animal viruses enter as most bacteriophages do - the capsid adsorbs to cell surface & only the viral nucleic acid enters cell.
3. Uncoating - Envelopes/capsids are often removed in the penetration process; viruses that enter the cell partially or completely intact are uncoated inside the cell by the host cell's own hydrolytic enzymes, sometimes those in its lysosomes.
4. Viral Synthesis - The specifics of this process depend on which of the 5 types of nucleic acids is present in the virus.
5. Maturation - Assembly not really understood
6. Release - Enzymes cause lysis of the host cell or viruses "bud." Viruses that kill the host cell by causing lysis are called cytocidal. Viruses that damage the host cell but do not kill it are called cytopathic. Persistent viral infections can last for years, producing new virus particles by budding without killing the infected cell.
C. Latency (similar to temperance or lysogeny) - Sometimes the viral nucleic acid is integrated in the host cell's DNA (called a provirus), allowing the infected animal cells to function normally for years (just as a lysogenic bacteriophage or prophage does).
Ex. Typical of DNA viruses belonging to Herpesvirus family - herpes simplex 1 (causes fever
blisters) causes a symptomless latent infection of nerve cells of mouth & lips - infection can be reactivated by a fever, a cold, too much sun, or stress.
Ex. Varicella Zoster (another Herpsevirus) causes chickenpox as the primary infection &
shingles as the reactivation.
Ex. HIV (Human Immunodefiency Virus) – belongs to the Retrovirus family; causes AIDS
(Acquired Immune Deficiency Syndrome).
D. Some Animal RNA Viruses
Retroviruses (Retroviridae)
¨ large group of RNA viruses; includes HIV (Human Immunodeficiency Virus) which causes AIDS (acquired immune deficiency syndrome); infects T cells (type of white blood cell).
¨ capsid contains 2 copies of the same (+) sense RNA molecule (called a diploid virus); capsid also contains the enzyme reverse transcriptase.
¨ Retro means "backward." This virus uses the enzyme reverse transcriptase to make DNA from its RNA. This DNA can be integrated into the host cell's chromosome. The proviral DNA can now be transcribed into mRNA and translated into viral proteins to assemble new viruses for release; As with prophages, the provirus can stay in a latent stage in which it is replicated along with host cell DNA, causing the host cell no damage.
¨ AZT (azidothymidine), which is used against HIV, helps stop reverse transcription by targeting the enzyme reverse transcriptase.
Flaviviridae
¨ enveloped; polyhedral capsid; (+) sense RNA
¨ includes Yellow Fever (hemorrhagic fever)
Togaviridae
¨ enveloped; polyhedral capsid; (+) sense RNA
¨ includes Rubella virus (Rubella or German measles)
Picornaviridae
¨ naked; polyhedral capsid
¨ includes Enterovirus (causes polio); Rhinovirus (common cold); Hepatovirus (Hepatitis A)
Orthomyxoviridae - Influenza Viruses
¨ Flu viruses; 3 types (A, B, C); A is the most common, infecting many species of animals, including humans; A is responsible for many pandemics (worldwide epidemics); B & C only infect humans & do not cause pandemics; Outbreaks of B occur every 2-3 years; C causes mild cold-like illnesses.
¨ enveloped RNA viruses; protein spikes in envelope; its (-) sense RNA is divided into 8 separate pieces, each one packaged in a helical capsid
¨ This virus exhibits antigenic shift- sudden changes in properties that id. the virus as a foreign invader to the defenses of the human immune system; occurs from genetic changes that can occur when 2 different flu viruses infect the same cell; when this happens it is likely that the RNA molecules of the 2 infecting virions recombine in various ways among the new virions, producing a virus that is significantly different from either of the original infecting strains. This is why you can get the flu over and over again!
Rhabdoviridae
¨ enveloped; helical capsid; (-) sense RNA
¨ includes Rabies virus
Paramyxoviridae
¨ enveloped; helical capsid; (-) sense RNA
¨ includes viruses that cause Mumps, Measles, Viral pneumonia, Bronchitis
Bunyaviridae
¨ enveloped; segmented RNA; (-) sense RNA
¨ includes Hantavirus (“4 corners disease”)
Filoviridae
¨ enveloped; filamentous capsid; (-) sense RNA
¨ includes Ebola virus
Reoviridae
¨ naked; polyhedral capsid; ds RNA
¨ includes Rotavirus (most common cause of diarrhea in infants and young children under the age of 2)
E. Some Animal DNA Viruses
Adenoviridae
¨ naked; polyhedral capsid; ds DNA
¨ mainly responsible for human respiratory diseases; also causes diarrhea in babies and young children
Herpesviridae - enveloped; polyhedral capsid; dsDNA
¨ Simplex virus – Herpes simplex 1 (oral) and 2 (genital & neonatal)
¨ Varicellovirus – Varicella zoster – chicken pox and shingles
¨ Roseolovirus – Roseola infantum – roseola in infants (rash and fever)
¨ Lymphocryptovirus – Epstein Barr virus – causes infectious mononucleosis and Burkitt’s lymphoma; also linked to Hodgkin’s disease.
Poxviridae
¨ enveloped; brick shaped capsid; ds DNA; largest of all viruses
¨ includes Orthopoxvirus – small pox & cow pox
Papovaviridae
¨ naked; polyhedral capsid; ds DNA; replicate in nuclei of host’s cells.
¨ Includes Papillomavirus – warts (some associated with cervical cancer)
Hepadnaviridae
¨ enveloped; mostly ds DNA; hepa = liver
¨ Hepatitis B virus
Parvoviridae
¨ naked; ssDNA; uses a helper virus to supply necessary component to produce more viruses.
¨ Includes Canine parvovirus – causes severe and sometimes fatal gastroenteritis in dogs.
¨ Also includes Erythrovirus (B19) – causes 5th disease (erythema infectiosum) – deep red rash on children’s cheeks and ears and both a rash and arthritis in adults; can cross placenta and damage fetus.
F. Viruses and Cancer
1. Tumors - uncontrolled growth of tissue (cells are dividing out of control); most are benign (non-life threatening); some are malignant (they spread or metastasize to surrounding tissues).
2. Cancer – malignant tumors that metastasize to surrounding tissues.
3. Cause – Most human cancers arise form genetic mutations or cellular damage caused by environmental factors (chemicals - nicotine, pesticides; radiation - UV, X-rays, etc.; diet). About 15% are attributed to viral infections.
4. Examples:
a. Human T-cell leukemia (blood cancer),
b. Epstein-Barr virus causes Burkitt's lymphoma
c. Hepatitis B virus causes hepatocellular carcinoma (liver cancer)
d. human papillomavirus causes skin & cervical cancers.
e. Kaposi’s sarcoma – thought to be associated with Herpesvirus
5. How? Some tumor viruses are retroviruses; they convert a normal cell to a tumor cell by introducing an oncogene into it (oncogenic provirus). Some researchers believe even normal retroviruses might cause tumors - merely inserting a normal provirus into the host chromosome near a normal gene might alter its expression and convert it to an oncogene.
G. Viruses and Teratogenesis
Teratogenesis – the induction of defects during embryonic development. A teratogen is a drug or other agent that induces such defects. Viruses are teratogens that can be transmitted across the placenta and infect the fetus. Cytomegalovirus (CMV), Herpes Simplex virus (HSV), and Rubella account for a large number of teratogenic effects. TORCH series is a series of blood tests used to detect antibodies to these viruses.
IV. VIROIDS
¨ Defined - a circular molecule of ssRNA without a capsid; don’t produce proteins
¨ Cause several economically important plant diseases; none known to infect animals
¨ 1/10 the size of the smallest plant virus
¨ How it causes disease is a mystery; one theory is that it interacts in some way with the host genome, changing the expression of the host genes to cause disease.
V. PRIONS
¨ Defined: infectious agent composed only of protein
¨ Affect the central nervous system.
¨ Ex. scrapie of sheep, Creutzfeldt-Jakob disease (CJD) of humans, mad cow disease.
¨ Not known exactly how it causes disease.