Virus Replication Flashcards
permissive cell
cell in which a virus is able to replicate; cell machinery supports replication of the virus
non-permissive cell
cells in which a factor or factors necessary to viral reproduction is not present or one detrimental to viral reproduction is present (e.g. absence of appropriate receptors)
MOI
multiplicity of infection; # virions added per cell during infection
one step virus growth curve
collect aliquots of cells and cell-culture fluid at different time intervals for measuring titer of intracellular and extracellular virions
adsorption
virus attaches to and enters cells, and the titer of free virus in the medium may actually decline
eclipse period
time interval between uncoating and appearance, intracellularly, of first infectious progeny virions
- no infectious virus detected during this time
- 2-12 hours for most virus families
latent period
time before new infectious virus appears in the medium (time from uncoating to just prior to release of first extracellular virions)
- no extracellular virions detected in this phase
burst size
# infectious virions released per average cell *height of curve
Which step of virus replication is the “kiss of death”?
attachment
receptor
structure on surface of a cell (or inside) that selectively receives and binds a specific substance, and mediates its entry or action into the cell
Is binding to a cellular receptor always sufficient for infection?
No, sometimes an additional cell surface molecule (co-receptor) is required for entry
Whats an example of a virus that uses more than one host cell receptor?
HIV
What are the possible methods of virus penetration?
- endocytosis
- surface fusion
- pore-mediated penetration
- antibody-mediated penetration
receptor-mediated endocytosis occurs with which kind of viruses?
naked viruses and most enveloped viruses
During endocytosis, what is the vesicle coated in?
clathrin (CCP)
What acts to pinch off the clathrin-coated pit from the host membrane to create a clathrin-coated vesicle (CCV)?
dynamin
Once the clathrin is removed and the vesicle delivers viral content to endosomes, what has to happen for the viral genome to be released?1
The pH in the endosome changes to acidic
How can the viral genome be released for non-enveloped viruses
- lysis when a viral capsid induces rupture of endosomal membrane
- induce local permeabilization of host endosomal membrane to allow virus capsid penetration into the cytoplasm
Other types of receptor-mediated endocytosis (just know names)
- caveolin-mediated endocytosis of virus by host (specialized lipid rafts)
- clathrin and caveolin independent endocytosis of virus by host
Membrane fusion (surface fusion) can only occur for which types of viruses?
enveloped viruses only
After surface fusion occurs, what remains on the cell surface and is antigenic?
viral glycoproteins -> cell can become target of immune system of the host
What is ADCC?
antibody-dependent cell mediated cytotoxicity
- antibody binds antigens on target cell
- Fc receptors on NK cell recognize
- NK cell kills target cell
Which protein acts to facilitate membrane fusion?
fusion protein (F)
- pH independent in HIV and measles
- pH dependent in influenza virus
What type of viruses use pore-mediated penetration?
non-enveloped viruses
antibody mediated attachment and penetration
- FIP virus
- enters host macrophage by attachment of spike proteins to CD13 receptor
- antibodies against spike proteins can’t clear the virus, so when they bind spike proteins they facilitate viral entry through antibody IgG-Fcgamma receptor
What happens during the eclipse period so that the virion can no longer be detected?
viral uncoating
What in the endosome promotes fusion of the virus envelope with the endosomal membrane to uncoat the virus?
low pH
Where does virus uncoating occur?
nuclear membrane
*poliovirus begins uncoating after binding to external receptor but not completely uncoated until inside the cell
Example of a virus in which uncoating requires a complex series of steps involving both host and viral gene products?
poxvirus
2 viruses in which first stages of the viral replication cycle actually occur inside the capsid?
retroviruses and reoviruses
Which step causes the loss of infectivity of virions?
uncoating
What is the role of DNA-dependent RNA polymerase?
transcribes the (-) DNA strand to form (+) mRNA and then translates that to create a viral protein
What is the role of DNA-dependent DNA polymerase?
Copies both + and - strands to form dsDNA viral genome that can be passed down
replication of ssDNA
- DNA-dependent DNA polymerase copies + strand to form dsDNA intermediate
- DNA-dependent RNA polymerase transcribes (-) strand to form (+) mRNA and translates that to viral protein
- DNA-dependent DNA polymerase copies the (-) DNA strand to form (+) ssDNA viral genome
replication of dsRNA
- RNA dependent RNA polymerase copies both strands to form viral genome
- RNA dependent RNA polymerase transcribes (-) strand to form (+) mRNA and translates to viral protein
replication of ssRNA (-)
- RNA dependent RNA polymerase transcribes (-) strand to (+) mRNA and translates to viral protein
- RNA dependent RNA polymerase copies (-) strand to form (+) ssRNA
- RNA dependent RNA polymerase copies (+) strand to form (-) ssRNA viral genome
replication of ssRNA (+)
- (+) ssRNA directly translated into viral protein
- RNA dependent RNA polymerase copies (+) strand to (-) ssRNA
- RAN dependent RNA polymerase copies (-) strand to form (+) ssRNA viral genome
- RNA dependent RNA polymerase also transcribes (-) ssRNA to form (+) ssRNA and translates to viral protein
Why is reverse transcriptase necessary for replication of retroviruses?
They have to synthesize cDNA which will integrate into the host genome
- begin with (+) ssRNA RT copy to form (-) ssDNA which is then copied to form (+) ssDNA -> come together to form cDNA
- the cDNA then used to form (+) ssRNA both for genome and protein production
What is special about Hepadnaviridae?
- have circular dsDNA which is incomplete and require RT to form RNA intermediate to replicate
- hepatitis B is most well-known
*probably arise from a deletion from a retrovirus !
What has to happen to pre-RNA before it can be translated?
- capping
- addition of 3’ poly-adenylated tails
How are caps synthesized?
- by host cell enzymes (retroviruses, adenoviruses)
- by viral enzymes (poxviruses, reoviruses)
- cap snatching: steals caps from host mRNAs (influenza)
What is capping?
addition of 7-methylguanosine to 5’ end of RNA
Why does capping occur?
- stability of mRNA
- binding of mRNA to ribosomes
- mark mRNA as “self”
Why is adding a tail important?
polyA tails interact with polyA-binding protein which is important for translation
What is the major signal for 3’ cleavage to add a tail?
AAUAAA
*cleavage occurs 10-35 nucleotides downstream from signal
What is splicing?
RNA splicing is process that removes introns and joins exons in a primary transcript
What is an exon?
portion of a gene that codes for amino acids
What is an intron?
portion of a gene that doesn’t code for amino acids (bye felicia)
constitutive splicing
every intron spliced out and every exon spliced in
alternative splicing
all introns spliced out and only select exons spliced in -> mRNAs that have different coding info derived from a single gene
monocistronic mRNA
encodes one polypeptide
*one ORF and single initiation and stop codon
polycistronic mRNA
encodes several polypeptides; multiple ORF
How does polycistronic mRNA become functional protein?
- translated to polyprotein and protease forms functional proteins
- endonuclease forms monocistronic mRNAs which are translated into functional proteins
Is polyprotein functional?
No -> need protease to separate into individual functional proteins
- occurs in Flaviviridae
- HIV has protease inhibitors -> proteins won’t be free and functional
Important viral proteins include:
- enzymes
- structural proteins
- viral nonstructural proteins
- regulatory proteins
- inhibitors
Where does assembly and maturation occur?
- nucleus
- cytoplasm
- plasma/cell membrane (most enveloped viruses)
How do naked virions release progeny?
lysis of host cell
How do enveloped virions release progeny?
budding
budding
- proteins that are to become spikes of virus attach to cytoplasmic membrane
- matrix protein coats inside of cytoplasmic membrane
- nucleocapsid enclosed by viral envelope, composed of host’s cytoplasmic membrane
- pinches off
Which viruses mature by budding through membranes of Golgi or ER?
Flaviviruses Arteriviruses Coronaviruses Bunyaviruses *vesicles with virus then migrate to plasma membrane and are relased by exocytosis
replication of retroviruses
- enter cell and RT RNA -> DNA
- integrase puts viral DNA into host genome
- transcription to viral RNA
- translation to viral proteins
- budding to form mature virions
extracellular spread of viruses
released viruses in extracellular environment and travel to new host cell -> same replication cycle repeats
intercellular spread of viruses
- spread cell to cell without contact with extracellular milieu -> rapid virus dissemination, evasion of immune system, persistent infections
HIV, herpesvirus, measles
types of intercellular transmission of viruses
- cell-cell plasma-membrane fusion followed by movement of infectious viral material into uninfected target cell (herpesvirus, paramyxovirus, retrovirus)
- passage of virions across tight junction (herpesvirus)
- movement of virions across neural synpase (rabies)
- viral induction of actin or tubulin containing structures (poxvirus)
- viral subversion of actin containing structures, form filopodial bridges (retroviruses)
- membrane nanotube subversion (HIV1)
- virological synapses (retroviruses)
nuclear spread of virus genome
viral genome integrated into host cell genome and passed down to next progeny or generation of host cells (retrovirus)
