Viruses Flashcards
What are the stages of virus replication cycle?
- Attachment/adsorption
- Penetration
- Uncoating
- Genome replication and amplification
- mRNA synthesis and translation
- Virion assembly
- Maturation and release
What makes influenza unique?
- Replicates in the nucleus
- Most RNA viruses replicate in the cytoplasm
What receptors may be used for attachment?
Protein - ICAM-1 for most rhinoviruses (high altitude, high levels of ICAM-1, increased chance of infection, alcohol downregulates ICAM-1
Carbohydrate - Sialic acid for influenza virus
What are some mechanisms that are used for uncoating? This also involves penetration
- Uncoating at the plasma membrane (fuse their envelope with the cell plasma membrane) -> HIV is an example of a virus whose contents is released directly into the cytoplasm (fusion occurs at the plasma membrane)
- Uncoating within endosomes
Alphavirus (Ross River virus) are examples of of viruses that enter in this way - Uncoating at the nuclear membrane - non-envelope - can’t fuse but can still be taken up via endocytosis - lysis occurs at the endosome - docking onto nuclear membrane - uncoating
How does fusion occur at the plasma membrane?
Native trimer - CD4 binding - coreceptor binding - membrane fusion
After receptor (CD4) and co-receptor (CXCR4 or CCR5) binding a hydrophobic fusion region of gp41 of the HIV surface spike become exposed and initiates fusion of the two membranes
Where do RNA and DNA replicate?
DNA viruses replicate in the nucleus and RNA viruses replicate in the cytoplasm.
What are the early proteins and late proteins like?
Early proteins usually non-structural (DNA or RNA polymerases)
Late proteins usually structural (capsid proteins - building blocks of the virion)
What is the difference between +ve sense mRNA and -sense mRNA?
+ve sense mRNA can be translated directly (looks like mRNA)
- ve sense mRNA cannot
Why do RNA viruses encode RNA dependent RNA polymerases? What are the properties of the RNA polymerases?
Eukaryotic cells do not have the ability to copy RNA- RNA or copy RNA- DNA. Therefore, all RNA viruses must encode for their own RNA dependent RNA polymerases.
Eukaryotes have the ability for DNA replication, transcription and translation.
Like all polymerases, RNA dependent RNA polymerases, require a template, always copy 5’ to 3’ -> will always bind to the 3’ (mechanism is the same as with transcription and replication)
- A double stranded replication intermediate is formed
- Newly formed RNA can then be used for translation or packaged into virions
What occurs for negative sense viruses?
Negative sense viruses include influenza and this RdRp must be carried in with the virus particle - this is also true for dsRNA viruses
What is reverse transcriptase?
It is an enzyme that is able to copy RNA to DNA, can digest RNA, can copy DNA to DNA and it enables viruses, like HIV, to integrate their RNA genome into the host DNA chromosome
What is involved in amplication of the viral genome and viral proteins?
- Poliovirus
- Non-structural proteins such as polymerase enzymes must be made before the genome can be replicated
+ ve sense RNA looks like mRNA and can be directly translated
-ve sense RNA has to be transcribed first
To commence replication, each virus must produce mRNA from its genome, then separate proteins. There are many different strategies for this.
- Translation of proteins -> carried out by ribosomes in the host (host dependent- viruses do not encode for ribosomal subunits)
- Post translational cleavage of polyproteins (virus and host dependent)
- Glycosylation of envelope glycoproteins occurs in the RER and Golgi (host dependent)
Viral assembly and release
• Non-enveloped animal viruses:
- All have an icosahedral structure- there are many different strategies for assembly of such structures
- One simple strategy is the spontaneous assembly of the capsid proteins around the nucleic acid genome
- The virus particle may require proteolytic cleavage to induce the final conformation in the capsid proteins of the mature infectious virion- this is carried out by host or viral proteases
- Virions accumulate in the cytoplasm or nucleus and are only released when the cell eventually lyses
• Enveloped virus:
- For many enveloped viruses release may take place by budding from the cell surface
- Patches of viral envelopes glycoproteins accumulate in the plasma membrane
- Capsid proteins and nucleic acid condense directly adjacent to the cell membrane
- The membrane surrounding the nucleocapsid then bulges out and becomes nipped off to form the new enveloped virion
- Some enveloped viruses utilise the cellular secretory pathway to exit the cell. Virus particles bud into Golgi-derived vesicles and are released to the outside of the cell when the transport vesicle fuses with the cell membrane
What is an example in this case of all of this process?
Dengue virus
- targets immune cells
- enveloped
- two important receptors involved: cognate and Fc receptor
- receptor mediated endocytosis
- lowered pH -> release of viral DNA
- positive sense strand RNA -> be directly translated
• Dengue virus is an RNA virus
• It is covered with envelope proteins surrounding a lipid bilayer envelope
• Inside the envelope is a capsid shell containing the RNA virus’s genome
• Immune cells are target by the dengue virus
• There are two cell surface receptors involved that are important in dengue infection
• The cognate receptor is involved in normal infection and the Fc receptor is involved in antibody-dependent enhancement
• The virus’ envelope protein binds to the cognate receptor and triggers a cellular process called receptor mediated endocytosis
• The virus is internalised in a bubble like structure called the endocell -> lower the pH of the interior -> the virus responds to the lowered pH by changing the conformation of the envelope proteins to form spike like structures
• The tips of the spikes are hydrophobic which allows them to penetrate the endosomes membrane
• They bend until the endosomes’ membrane and the virus’ membrane fuse together and release the capsid into the cytoplasm
• The capsid breaks apart and releases the viral RNA
• The RNA travels to the rough endoplasmic reticulum - it is a positive sense strand and can be directly translated into proteins
• The ends of the RNA form structures that bind to translation initiation proteins
• The complex attaches to the ribosome to initiate translation
• Translated as a long polypeptide chain - the capsid protein is on the cytoplasm side of the ER
• The envelope protein and the pre-membrane protein are in the lumen side and are activated by the host’s peptidase enzyme
• In the cytoplasm, one of the viral proteins, protease enzyme, activates all the other proteins in the polyprotein chain
• These proteins aggregate to form the replication complex
• The viral RNA is synthesised in multiple steps
• First the ends of the viral RNA fold up and the RNA forms a circle
• The RNA then attaches to the replication complex to start the first round of synthesis
• Using the virus’ positive sense RNA strand as the template, a negative sense copy is made -> forms a double helix
• The RNA then becomes a circle again
• This time the negative sense RNA strand acts as a template to make a positive sense strand -> many copies are made by repeated cycles of RNA synthesis
• Some of these strands are translated to make more of these proteins
• Eventually enough is made to assemble new viruses
• The envelope proteins aggregate in the lumen of the ER and the capsid proteins aggregate on the cytoplasmic side
• A viral RNA binds to the capsid protein and is packaged into a new virus particle that buds off from the ER- the virus is still immature
• Its pre-membrane proteins are on the tips of the envelope proteins
• The virus buds off and travels through the Golgi and continues towards the cell surface
• Before reaching the surface, the pre-membrane proteins are processed and the virus becomes mature
New Dengue viruses are released from the cell ready to infect other cells
How can we study viruses?
- Cell culture
- Cytopathic effect
- CPEs include:
- Rounding up and detachment of cells
- Cell lysis
- Swelling of nuclei
- Formation of fused cells (syncytia)
- Infection of the embryonated egg may produce local tissue lesion (pack)
- Inclusion bodies (high-power microscope required)
- Duplication of membranes (high-power microscope required)
- Fragmentation of organelles (high-power microscope required)
- However many viruses also multiply in cells without causing an obvious CPE (retroviruses, hepatitis C)
Plaque assay
- We can use the cytopathic nature of viruses to:
- Identify whether they are present or not
- Quantify the amount of virus in a sample
- Cytopathic viruses can be quantitated by the number of plaques or packs that they cause on susceptible cell monolayers
- Each hole or plaque is caused by 1 virus particle killing the cells it has infected
CPE: Syncytium
- During replication of the virus, expression of the fusion protein at the cell membrane can result in the fusion of neighbouring cells, and the formation of multi-nucleate cells or syncytia
- Effect of viral replication on the host cell membrane
- Virus infection alters membrane integrity
- Viral surface proteins mediate fusion of many infected cells
CPE: Inclusion bodies
- Can be observed by both light and electron microscopy
- Dense aggregation of viral and host proteins in cells
- Generate areas where viral proteins can be made
CPE: Cell transformation
• Some viruses encode oncogenes whose expression in the virus infected-cell is associated with tumour production
• Most oncogenes code for proteins with growth promoting properties and their expression can lead to uncontrolled proliferation of the infected cell and tumour development
Other viruses cause tumours because their replication affects the cellular version of an oncogene
