M: Virology 2 - Week 11 Flashcards
What is the eclipse period?
the time between infection by (or induction of) a bacteriophage, or other virus, and the appearance of mature virus within the cell; an interval of time during which viral infectivity cannot be recovered.
What is the latent period?
the period between infection with a virus or other microorganism and the onset of symptoms
Define Uncoating
The release of viral nucleic acids/genome from the capsid that covers them
What is the one-step growth curve?
Describes the production of progeny virus over a period of time following infection under one-step conditions
When do one-step conditions exist?
When all cells are infected simultaneously so as to prevent secondary cycles of infection
What is a viral plaque?
a visible structure formed within a cell culture, demonstrating a region of cell destruction
What is a plaque forming unit (PFU)?
A measure of the number of particles capable of forming plaques per unit volume, such as virus particles
What happens to PFU during the Eclipse Period of viral replication? (in one-step growth curve of the unenveloped virus)
No change
What happens to PFU during the Latent Period of viral replication?
No change for most of it, then a logarithmic increase near the end
What happens to PFU during the Uncoating Phase of viral replication?
No change
What happens to PFU during the Synthetic Phase of viral replication?
logarithmic increase
In the one-step growth curve for viral replication, at what times after viral absorption do the following periods/phases occur?
- Eclipse
- Uncoating
- Latent
- Synthetic
Eclipse: 0-12 hours
Uncoating: 0-12 hours
Latent: 0-16 hours
Synthetic: 12-16 hours
In the one-step growth curve: what happens to the virus 16 hours after viral absorption?
extracellular bursting out of new viruses (happens for the next 28 hours until hour 44)
Name the 6 steps of viral replication
Attachment/Adsorption Penetration Uncoating Replication Assembly Release
How do viruses attach?
Viral attachment protein binds specifically to a receptor on the cell plasma membrane
This interaction defines and limits the host species as well as the type of cell that is infected
What receptors might the cell plasma membrane have for viruses? (2) Provide an example for each
Protein (e.g. ICAM-1 for most rhinoviruses)
Carbohydrate (e.g. sialic acid for influenza)
Can viruses use multiple different receptors on the same host cell?
Yes. Some viruses do that
What is referred to as the “one-two punch” for viruses to get into the cell
Some viruses use 2 different receptors on the same host cell: for initial attachment, then a coreceptor for closer attachment and entry
How do viruses penetrate the cell membrane? (2)
The coat of enveloped viruses may fuse with the host cell membrane and release the virus nucleocapsid into the host cytoplams
Alternatively,
other viruses may enter the cell via “endocytosis”
What does viral endocytosis involve?
invagination of the cell membrane to form vesicles in the cell cytoplasm which becomes acidified
What is the purpose of viral uncoating?
enables the nucleic acid to be transported within the cell and transcribed to form new progeny virions
What is direct fusion?
is when viruses initiate fusion and penetration of their core or nucleocapsid directly through the cell’s surface (plasma) membrane at neutral or alkaline pH
Several viruses do this.
Name 2 examples of viruses that undergo direct fusion
Paramyxoviruses (e.g. measles)
Retrovirus HIV
How do paramyxoviruses undergo direct fusion?
via a fusion (F) glycoprotein on their envelope
How does retrovirus HIV undergo direct fusion?
gp120 binds sequentially to 2 receptors: CD4 receptor and CCR5 (chemokine co-receptor)
- this exposes gp41, which drives fusion of envelope and plasma membrane
Describe the steps involved in the entry of HIV-1 by membrane fusion
- Native trimer: Attachment through non-specific cell receptors
- CD4 binding: structural changes in gp120 after binding CD4 receptor
- Coreceptor binding: binding to newly exposed chemokine coreceptor sites promotes gp41 fusion peptide insertion
- Membrane fusion: structural rearrangement of gp41 trimers drives membrane fusion
In the entry of HIV-1, which chemokine coreceptor sites become exposed? (2) Which chemokine is predominantly used during the process of transmission?
CCR5 and CXCR4. CCR5 is the predominantly used on for transmission
What does the entry and uncoating of influenza viruses require? (2) Why?
requires endocytosis and acidification - to induce a conformation change in HA that reveals a fusion domain on HA
What is HA? What does it stand for? (in relation to influenza virus)
Influenza Haemagglutinin. is a homotrimeric glycoprotein found on the surface of influenza viruses and is integral to its infectivity
Describe the assembly phase of the viral life cycle
proteins assemble around the nucleic acids to form new viral particles
Where do DNA viruses replicate? Are there any exceptions?
in the nucleus. (poxyviruses are an exception)
Where do RNA viruses replicate?
in the cytoplasm
What does mRNA encode?
viral proteins that are translated by the host cel
Where is mRNA transcribed from?
viral DNA
Where is mRNA formed directly from?
some RNA viruses
What is the difference between transcription and translation? (2)
Transcription: the synthesis of RNA from a DNA template where the code in the DNA is converted into a complementary RNA code
Translation: the synthesis of a protein from an mRNA template where the code in the mRNA is converted into an amino acid sequence in a protein
Are “early” viral proteins structural or non-structural? What about “late” viral proteins?
early: usually non-structural
late: structural
What do “early” viral proteins assist with?
replication (e.g. polymerases)
Provide one example for “early” and “late” viral proteins?
early: polymerases
late: capsid proteins
Name 3 viruses that can directly translate their genome to proteins. Why can they do this?
Picornaviridae
Togaviridae
Flaviviridae
Can do this because their genome is m(+)RNA. This is needed before we can translate but since these viruses already have it there is no need to undergo transcription to get it.
Can negative sense viral RNA act as mRNA?
No. Only positive-sense do that (i.e. only positive sense can act as mRNA to be translated into protein in the host cell)
What does RDRP stand for? What does it do?
RNA-dependent RNA polymerases. Enzyme that produces complementary strands from the template
Which of the following contains a RNA-dependent RNA polymerase in its capsid?
- Positive sense ssRNA or Negative sense ssRNA?
Negative-sense
How do (+)ssRNA viruses overcome their inherent lack of RNA-dependent RNA polymerase?
they use their genome as an mRNA to make an RNA-dependent RNA polymerase (or REPLICASE) upon infection
How does replication of ssRNA work when starting with (+)ssRNA? What if we start with (-)ssRNA?
Minus strands are copied form plus strands, then more plus strands from the minus strand template
(vice-versa if starting with (-)ssRNA)
What is the replicative intermediate?
refers to the complex formed when RNA-dependent RNA polymerase produces the complementary strand for ssRNA
How do (-)ssRNAs make mRNA?
They use their replicase (i.e. rna-dependent rna polymerase) to make mRNA using the -ve sense genome as a template
Can DNA viruses make DNA?
Yes. They have a DNA genome so they can just transcribe and make RNA
True/False: Most dsDNA viruses have viral enzymes within them
False. They make viral enzymes when they arrive from the copy of DNA genome
ds DNA viruses use cellular enzymes in the nucleus for what purpose? (2)
to express mRNA
to replicate DNA
What do DNA viral proteins modulate?
normal cellular control mechanisms to favor virus replication
i.e. DNA viruses will have proteins that greatly favour the replication of viral proteins
Name 4 DNA viruses
Adenovirus
Herpes virus
Pox virus
Papo virus
What do ssDNA viruses need to do to further their genome replication?
They need to make a copy of the second strand
How and where is translation of structural and non-structural proteins carried out?
Carried out by ribosomes in the host cell cytoplasm
Once translated, what do the translated proteins almost invariably undergo?
Post-translational cleavage of polyproteins or trimming of structural proteins
What does post-translational cleavage of polyproteins or trimming of structural proteins require?
virus-coded proteases
Where does glycosylation of envelope glycoproteins occur? (2)
RER and Golgi vesicles
What are viral structural proteins designed to assemble into?
Capsids
What might the virus particle require to induce the final capsid conformation?
Proteolytic cleavage
Where do virions accumulate following assembly? (2) and where are they released? (1)
Accumulate in the cytoplasm or nucleus and are released by lysis
How may virus particles be released following assembly? (3)
Particles may be released from the cell by:
movement through the ER (exocytosis),
breakdown of the cell,
or by budding
Which method of viral release from the cell is the main method for enveloped viruses?
By budding
What is viral budding?
Nucleocapsids assembled or in the process of being built induce formation of a membrane curvature in the host cell membrane and wrap up in the forming bud which is eventually pinched off by membrane scission to release the enveloped particle
Are enveloped or non-enveloped viruses released by exocytosis after assembly?
Can be either. However most often tends to be non-enveloped viruses
What is viral exocytosis?
Viral progeny are synthesized within the cell and the host cell’s transport system is used to enclose them in vesicles; the vesicles of virus progeny are carried to the cell membrane and then released into the extracellular space.
Does viral exocytosis (via movement through ER) kill the infected cell?
No
What is the primary mode of viral exit from the cell for non-enveloped viruses?
cell lysis (to violate the integrity of the bilayer). This kills and ruptures the cell obviously
Name 2 drugs that target the attachment of viruses
Maraviroc
Fuzeon
Name 1 drug that targets the uncoating of viruses
Amantadine
Name 2 drugs that target the genome replication of viruses
Acyclovir herpesviruses
Zidovudine (AZT)
Name 2 drugs that target the RNA synthesis of viruses
Ribavirin
Interferons
Name 3 drugs that target the protein synthesis of viruses
Interferons
Saquinavir
Darunavir
Name 2 drugs that target the release of viruses from the cell
Tamiflu
Relenza
Name the 3 classes of Interferons
IFN-alpha
IFN-beta
IFN-gamma
What is the role of interferons?
induce proteins (IRPs) that inhibit viral replication or modify immune responses
Describe the antiviral actions of interferons
Wide variety of antiviral actions - block viral protein synthesis
Are interferons active against DNA viruses?
No. Not very active
Do Interferons cause side effects? If so, how broad?
Yes. Wide ranging effects
How many phosphate groups do nucleosides have? What about nucleoside analogues
none for both
What is the substrate for DNA polymerisation?
Nucleoside triphosphate
What is Acyclovir?
A guanosine analogue used to treat herpesviruses
Why aren’t nucleoside analogues able to form a DNA polymer?
they lack the 3’ hydroxyl group required
What does the synthesis of acyclovir monophosphate require? (1)
thymidine kinase (which adds a phosphate group to acyclovir)
What is Ribavirin?
Guanosine analogue
What is the function of Ribavirin?
Inhibits replication of many DNA and RNA viruses in vitro
How does Ribavirin work? Describe its forms and what function each have (3 + 2)
Intracellular phosophorylation gives it mono-, di- and tri-phosphate forms.
Mono-phosphate form: reduces nucleic acid synthesis
Tri-phosphate form: alters viral mRNA formation
Name 4 conditions you can use Ribavirin for
Use for: respiratory syncitial virus bronchiolitis + pneumoniae influenza haemorrhagic fevers hepatitis C
How do you administer Ribavirin for treatment of influenza and respiratory syncitial virus bronchiolitis and pneumoniae?
Aerosolised, IV (intravenous)
How do you administer Ribavirin for treatment of hemorrhagic fevers? (2)
oral or IV
How do you administer Ribavirin for treatment of hepatitis C?
oral; in combination with interferon
Describe the HIV replication cycle (3)
HIV is an RNA virus
HIV consists of an enzyme inside the particle – the enzyme converts the 2 copies of (+) viral RNA and reverse transcribes them into a double-stranded copy of DNA
The double-stranded DNA is taken into the nucleus and gets stitched into the host cell DNA to form pro-virus DNA
Name 4 targets in the HIV replication cycle for anti-retroviral therapy
Fusion/Entry inhibitors
Reverse Transcriptase inhibitors
Integrase inhibitors
Protease inhibitors
What is Zidovudine?
A reverse transcriptase inhibitor
What are reverse transcriptase inhibitors (RTIs)? What can they be used to treat? (3) What is reverse transcriptase required for? (2)
are a class of antiretroviral drugs used to treat HIV infection or AIDS, and in some cases hepatitis B. RTIs inhibit activity of reverse transcriptase, a viral DNA polymerase that is required for replication of HIV and other retroviruses.
What does NRTI (or NARTI) stand for? Name one example
Nucleoside analogue reverse-transcriptase inhibitors; a type of reverse-transcriptase inhibitor
e.g. Zidovudine
What type of nucleoside analogues can NRTIs be? (3)
Analogues of:
Thymidine
Cytidine
Guanine
How are NRTIs incorporated into the viral DNA? (so that they can inhibit reverse-transcriptase)
In order to be incorporated into the viral DNA, NRTIs must be activated in the cell by the addition of 3 phosphate groups to form NRTI triphosphates
How are NRTIs phosphorylated?
via intra-cellular kinase enzymes
Can reverse-transcriptase inhibitors eliminate HIV virus completely?
No. They can reduce the infection of new cells but cannot eliminate the virus completely
When might you use combination therapy with reverse-transcriptase inhibitors?
When you have drug resistant mutant viruses
What nucleoside analogue is Zidovidine?
a Thymidine analogue
What does NNRTI stand for? Do NNRTIs have close structural similarity to nucleosides?
Non-nucleoside reverse-transcriptase inhibitors. No they do not!
What type of anti-HIV drugs have a close structural similarity to the amino acid recognition sequence?
Protease inhibitors
What features of a virus in general help confer drug resistance? (2)
High replication rate
High mutation rate (RNA>DNA)
How does viral drug resistance typically emerge? How can we overcome this?
by “selective pressure”. Overcome by using combination therapy
What is a “pre-existing” viral mutant
is a viral resistance mutation that exists before the patient has had any kind of treatment
How often do “pre-existing” viral mutations occur? (in comparison to non-pre-existing)
often
What is HAART?
Refers to any HIV treatment that uses a combination of 2 or more drugs
What are the advantages of HAART? (3)
Synergistic potency
Durable anti-viral response
Minimise development of drug resistance
Define Viral hepatitis
“inflammation of the liver”. a viral infection that causes liver inflammation and damage.
Is viral hepatitis acute or chronic?
Can be either (Acute = weeks to months) (Chronic = years to lifetime)
How does acute viral hepatitis present? [symptoms] (3)
non-specific, flu-like symptoms
jaundice
dark urine
How does chronic viral hepatitis present? [symptoms] (3)
general malaise
cirrhosis
liver cancer
In what body fluids is the concentration of blood born viruses high? (3)
blood (obviously)
serum
wound exudates
In what body fluids is the concentration of blood born viruses moderate? (3)
semen
vaginal fluid
saliva
In what body fluids is the concentration of blood born viruses low? (5)
urine faeces sweat tears breastmilk
What proteins were used in HIV treatment a few years ago and are being phased out? Why are they being phased out?
Interferons (IFNs). They are being phased out because they made the treatment unpallatable.
Now we have IFN-free DAA combination treatment
What does DAA stand for?
Direct acting antivirals
