THE VIRUSES LECTURE THREE (3) Viruses cause about 60% of all human infections.
Characteristics of Viruses
a. b. c. d. e. f. g. h. i. Acellular (noncellular agent). No cytoplasmic membrane. No cytosol present. Genome is DNA or RNA, never both. Cannot grow in or respond to the environment. Submicroscopic. Have a capsid protein coat. An obligate intracellular parasite. No membrane bound organelles.
Outside the host cell, these entities are called Virions. Within a host cells, these entities are called Viruses. Viruses have a genome of either DNA or RNA. The DNA and RNA also vary in their makeup. a. dsDNA – Herpes and Chickenpox viruses are examples. b. ssDNA – Erythrovirus (Parvovirus 5th’s Disease). c. dsRNA – Rotavirus (Acute Infantile Diarrhea). d. +ssRNA – Flavivirus (Hepatitis C). e. –ssRNA – Filovirus (Ebola or Marburg Hemorrhagic Fever). Whatever the genetic material is, it will be inside a protein Capsid. The genome of the virus and the capsid make up the Nucleocapsid. Some virions will have an envelope covering the nucleocapsid. The envelope is for protection of the genome and it can contain sites on it’s surface that will recognize complementary chemicals on the surface of host cells.
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VIRAL GENETIC MATERIAL Either DNA or RNA These genomes can be linear or circular. The Influenzavirus which causes the Flu has a genome that is in eight linear segments of –ssRNA. The Lassavirus also has a –ssRNA single molecule that is in segments. The Lentivirus has a +ssRNA genome that is in segments. The Poliovirus has a ssRNA genome that is a single molecule. VIRUS HOSTS Most viruses are Host cell specific. It is according to the virion’s surface proteins or glycoproteins and the proteins or glycoproteins of the host cell. Viruses do not look for a particular host, but a specific host cell. HIV’s specific host cell is the T4 cell of the immune system in humans. This virus will not infect any other tissue cell of the human body if it does not have the T4 marker on the cell’s surface. Other than this, the HIV has no affinity for any other type host cell. Some viruses are Generalists, which means they can infect various cells in a lost of different host. A good example of this is the Lyssavirus, which causes Rabies. A virus or complex virus that specifically infects a bacterium is called a
Bacteriophage.
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VIRUS SIZES The Latin word for Virus is “poison”. This term was coined back in the 1800’s for these little agents that were smaller than a bacterium that caused disease. It was Dmitri Ivanowsky in 1892 (a Russian Microbiologist) was the first person to demonstrated that viruses were acellular. Ivanowsky was working with the Tobacco Mosaic Virus. He used a porcelain filter to try and filter out the causing agent that was destroying tobacco crops. The agent passed right through his filter. When the filtrate was placed back on a tobacco plant, it once again caused the dreaded disease. Finally in 1935, Wendell Stanley, an American Chemist isolated and characterized the virus by the invention of the Electron Microscope. This was when viruses were seen for the first time in history. MORPHOLOGY OF THE CAPSIS Protein subunits called capsomeres make up the capsid of a virus. This protein is of one single type. SHAPES OF VIRUSES Shape is one characteristic to classify a virus. There are three distinct shapes: a. Helical – this capsid has capsomeres that bond together with a spiral form to encircle the genetic material. b. Polyhedral – a modified spherical form called an “icosahedral”. This form has 20 facets, like a 20-sided dice. c. Complex – more than one shape here. The bacteriophages are complex in their make up of a polyhedral head, a sheath, base plate and tail fibers. Smallpox has several layers & do identifiable capsid.
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VIRAL ENVELOPE Many viruses have envelopes surrounding their capsids. This is a lipid bi-layer type structure. A virus will be surrounded by this envelope when it buds out of a host cell once it is replicated and released. The envelope being lipid in nature, can also have proteins within it that are glycoproteins that were virally coded for. These integral type proteins protrude through the envelope and rise above the top of it. The host cell’s DNA coded for the phospholipids and some proteins, but the viral genome coded for the specific integral proteins. It is these viral integral proteins that have the recognition sites for host cell surface proteins for attachment. CLASSIFICATION OF VIRUSES Viruses are classified on three criteria: a. Type of genetic material. b. Enveloped or naked. c. Shape. Latin names are not given to viruses at the species level. The specie name is given for their common English designation. The Family is the highest taxonomic group as of now. Then there is the Genus and then the species. Kingdom, Division and Class have not yet been determined. EXAMPLES: Family Genus species Common Cold Coronaviridae 5th’s Disease Parvoviridae
Coronavirus
“Severe Acute Respiratory Syndrome”
Erythrovirus
“Erythema Infectiosum”
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VIRAL REPLICATION With virions entering host cells, they become viruses. Once within the host cell, the viruses being obligate intracellular parasites, the virus takes over the host cell’s metabolic machinery. Once infected, the host cell is under the viral genome’s control. The host cell now cannot synthesize proteins or enzymes for it’s self. Everything that is synthesized is specifically for the new virions. These items are capsomeres, genome, viral proteins & viral enzymes. As the replication cycle is near completion, this will usually cause the host cell to be lysed out and killed. There are two cycles for viral replication: THE LYTIC CYCLE One example of this type of replication is between a dsDNA bacteriophage called a Type 4 or T4 phage. 1. Attachment: By chance and random collision, a bacteriophage comes into contact with a bacterium. There has to be an exact fit between the proteins on the phage tail fibers and the complementary receptor proteins contained within the cell wall of the host cell. Bacteriophages are bacteria specific, like enzymes. A specific phage will infect only a specific bacterium, like the T4 phage that will infect a specific strain of Escherichia coli, like Strain B. 2. Entry: Lysozyme is released by the T4 phage. This weakens the peptidoglycan layer by breaking the B-1-4 linkage between the NAM-NAG bond. The tail sheath now contracts and this inserts a conduit through the cell wall and membrane and into the cytoplasm.
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The dsDNA of the phage now moves down the hollow tube and into the cytoplasm of the bacterium host. 3. Synthesis: Viral enzymes now destroy the host genetic material. The genome is broken down into it’s nucleotides. The viral genome is now in charge and only viral constituents are synthesized for more viral replication. Synthesized here are the viral proteins, capsomeres for the capsid, base plate, tail fibers and DNA polymerase. 4. Assembly: Spontaneously, the capsomere protein sub-units come together, attach to one another and form a new capsid. The sheath assembles and is attached to the icosahedral and the tail fibers. Once the capsid is synthesized, enzymes help to place the genome within the newly formed capsid. 5. Release: Lysozyme now degrades an area of the cell wall, causing a stress in that area as the bacteria breaks open and the newly phages are released. The bacterial cell dies as a result of this. THE LYSOGENIC CYCLE Lysogenic Replication or Lysogeny is the cycle where the phage’s DNA can be spliced into the bacterium’s genome and can stay there for an indefinite period of time. The phages in this cycle are called Temperate Phages or Lysogenic phages. The main phage that carries out this cycle is called the Lambda phage. It contains a dsDNA linear genome within a complex capsid that is an icosahedral in shape and is attached to a sheath that has a tail but NO tail fibers. This phage is also an E. coli parasite. 1. Attachment: The Lambda phage comes into contact with E. coli and attaches to it’s surface. The phage injects it’s dsDNA into the bacterium’s cytoplasm.
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The bacteria genome is not degraded and the phage does not at this time take over the metabolic processes of the bacterium. The phage remains inactive. The phage dsDNA will now splice it’s self into the bacterium chromosome and lie dormant. In this form, it is called a Prophage! 2. Entry: The prophage will synthesize a protein that suppresses the genes of the phage and this causes it to lie dormant. This protein also keeps other phages of the same type from trying to infect this bacterium at this time. 3. Prophage in Chromosome: When the bacterium goes into fission, it’s chromosome is replicated and the phage dsDNA replicates as well. 4. As this bacterium in the prophage form replicates, it’s descendents remain a part of the bacterial chromosome and this can go on for generations. 5. Something may activate the phage and it will excise it’s self out of the prophage form. This causes the cell now to go into the Lytic Cycle. 5. Induction: When the phage dsDNA is excised from the genome, this is called “Induction”. What can cause induction are chemical & physical agents, carcinogenic chemicals, x-rays and ultraviolet light. After induction, steps 6, 7 & 8 are like the lytic cycle of synthesis, assembly and release. ANIMAL VIRUSES AND THEIR REPLICATION
Attachment of Animal Viruses
For a virus to enter a specific cell, the glycoproteins and proteins on
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the virus have to be complementary to the same type structures on the host cell. These attachment viral proteins or glycoprotein spikes can be found either on the capsid or within the viral envelope. VIRAL ENTRY INTO HOST CELLS 1. Direct Penetration : usually occurs with Naked Viruses. There is fusion of the capsid to the host cell’s cytoplasmic membrane and the viral genome enters the host cell’s cytoplasm. The Poliovirus is an example that uses direct penetration. 2. Membrane Fusion: Usually occurs with enveloped viruses. The viral envelope fuses with the host cell’s membrane and becomes a part of it and the viral capsid enters the cytoplasm of the host cell, then uncoats and the genome is released into the cell’s cytoplasm. Examples are the Measles and Mumps viruses. 3. Phagocytosis: The viral envelope fuses with the cytoplasmic membrane. The membrane then surrounds the viral envelope, like in “Phagocytosis” and the complete viral particle has entered the host cell’s cytoplasm. The envelope is degraded, releasing the capsid which is degraded and then the genome is released into the cytoplasm of the host cell. SYNTHESIS OF ANIMAL VIRUSES 1. dsDNA Viruses – after entry into the host cell and the capsid is degraded, the genome (dsDNA) is released. The genome now moves into the cell’s nucleus. The DNA codes for mRNA in the nucleus and then moves into the cytoplasm where it codes for capsomere proteins, using the host ribosomes.
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The capsomere protein subunits now enter the nucleus where they will assemble spontaneously. Examples of this type virus are the Papillomavirus and the Herpes
Simplex viruses.
2. ssDNA Viruses – when this genome enters the host cell and goes to the nucleus, host cell polymerase synthesizes a complementary strand of DNA to make a dsDNA molecule. A mRNA is coded for, enters the cytoplasm and synthesizes proteins for new viral particles. An example of this type genome is the Parvovirus. 3. +ssRNA viruses – this piece of genetic material can act directly as a mRNA. From this + strand, a – complementary strand is synthesized by RNA polymerase. Example of this type genome is the Poliovirus. Only Viruses can transcribe RNA into RNA! 4. Retroviruses – these are also +ssRNA viruses. This type genome of retroviruses is not used directly as mRNA. A enzyme that the virus brought with it into the cell, is called reverse transcriptase, and it will transcribe a single strand of DNA from the RNA strand. The DNA can then give rise to more +ssRNA molecules. The DNA can also enter the host cell’s nucleus and splice into one of the host cell’s chromosomes, creating a Provirus. Example of this type virus is HIV. 5. –ssRNA Viruses – a ribosome does NOT recognize –RNA genomes. When this type virus enters the host cell, it brings with it an enzyme called RNA-dependent RNA transcriptase. This enzyme transcribes +RNA from the –RNA strands. the +RNA or mRNA can now code for viral proteins and enzymes. Examples of this type virus are the Flu virus and Rabies virus.
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6. dsRNA Viruses – the positive strand of this genome will serve as a mRNA to synthesize proteins and enzymes. One needed enzyme is RNA polymerase, needed to transcribe more mRNA molecules. An example of this type virus is the Rotavirus, which causes Acute Infantile Diarrhea. ASSEMBLY & RELEASE OF ANIMAL VIRUSES DNA viruses component parts are assembled within the nucleus and are released from the nucleus into the cytoplasm. RNA viruses assemble their component parts within the cytoplasm. How many viruses are released from the host cell depends on how many were constructed within the cell. This depends on the viruses size and it’s type. The health of the host cell is also a factor. The Herpes viruses will need about 24 hours to replicate. Where as the T4 Bacteriophage replicates within 25 minutes.
“Budding” is the mechanism used by enveloped viruses to escape from
the host cell after they have been assembled.
When the virion is released, it attaches to the cytoplasmic membrane, which becomes it’s envelope when the virus is totally released. As the virus is attached to the cytoplasmic membrane, it will synthesize some proteins that become the glycoprotein spikes that are on the surface of the envelope, which will later be used for attachment. Naked viruses will be released by Exocytosis or through Lysis of the host cell’s membrane, which will kill the host cell.
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LATENCY OF ANIMAL VIRUSES If a viruses enters a host cell and lies dormant, this is called Latency. Viruses that carry out this process are called Latent viruses or Proviruses. Some latent viruses do not have their genome spliced into the host cell’s genome. Some do. HIV is one virus that does splice it’s genome into one of the host cell’s chromosomes.
This creates a Provirus. When this happens in HIV, it is a permanent condition and induction
never occurs. As this infected cell, with the provirus present, replicates, all progeny will also contain the provirus form. TISSUE CULTURES FOR VIRUSES Viruses cannot be grown on any type of agar media. They have to be placed into a medium or broth that contains cells. Tissue and Cell cultures are synonymous, they mean the same. Only one type of cell is used in a cell culture. There are two types of cell cultures: 1. Diploid Cell Cultures – these are the type of cells that come from an embryonic animal, plant or human cell. These are pure cells that have been isolated and placed under the correct growth conditions. Cells in these type of cultures are good for approximately 100 generations and then the cells die. 2. Continuous Cell Cultures – tumor cells are used here. Tumor cells are Neoplastic cells, that provide a never ending supply of new cells. Henrietta Lacks, died of cervical cancer in 1951. Her cells are the semi-standard for human cell (tissue) culture.
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These cells are also used for doing studies on cell metabolism, aging as well as viral infection. In the research community, her cells are called HeLa cells, which are the first two letters of her first and last name. This cell line has been around for over 50 years now. Some of the chromosomes have been long and are no longer diploid and other characteristics are now missing as well. But these cells are still being used in research. The lady is dead, but her cells will live on for many years.
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