Virus Replication Flashcards
What is the one step growth curve?
growth curve used to study viral replication by observing the time of a complete process of infection by a given virus in a permissive host cell until the release of new viral progeny
- one cycle of growth is observed
What 7 steps are taken to prepare viruses for one step growth curve analysis?
- inoculate cell culture with virus
- incubate virus-infected cells in CO2 incubators
- examine the viral infected cells daily under inverted microscope
- harvest cell lysates at various time intervals
- serially dilute the collected cell lysates
- do plaque assay and calculate the number of plaques at each time point
- analyze data and create viral one step growth curve
What are the 2 main phases of the viral one step growth curve?
- ECLIPSE PHASE: time from entry of the virus into the host cell until the assemble of new progeny viruses (1-20 hrs)
- EXPONENTIAL GROWTH PHASE: number of produced viruses increases exponentially until reaching a plateau where no more viral particles are produced
How does the bacterial growth curve compare to the viral one step growth curve?
BACTERIAL - lag phase, logarithmic phase, stationary phase, death
VIRAL - attachment, eclipse, assembly/maturation, release
Despite the high variability among viruses, they replicate in a standard of 7 consecutive steps. What are they?
- attachment/absorption
- penetration
- uncoating of envelope/capsid
- synthesis of viral proteins (translation)
- synthesis of viral nucleic acids
- assembly and maturation
- release of progeny virus
How do viruses attach to the target cell?
ligands on the viral surface, called virus attachment proteins, bind to the receptors (proteins, glycoproteins, carbohydrates, lipids) on the plasma membrane of the target cells
What is tropism? How is it affected by the presence of viral receptors? What are the 2 types of tropism in viruses?
the affinity of a virus to attach to specific target cells over other ones - the way the virus responds to external stimuli in order to attach to infect cells
viral receptors can be the primary determinant of tropism
- TISSUE TROPISM: virus is more likely to bind to cells of specific tissues more than others (measles and skin cells; mumps and salivary gland cells)
- SPECIES TROPISM: virus is more likely to bind to cells of a certain species more than other species (BHV-1 and cattle)
How do Influenza viruses attach to target cells? HIV?
hemagglutinins on the viral cell bind to sialic acid on the target cells (sialic acid is rich in respiratory cells!)
GP-120 on the viral cell binds to CD4+ receptors on the target cells (on immune cells!)
What 5 factors affect the efficacy of virus attachment to target cells?
- density of receptors on the host cells
- density of ligands on the surface on the virus
- ratio of virus/host cells
- temperature and pH
- presence of specific ions, like calcium
What tissues do neurotropic, pneumotropic, viscerotropic, dermotropic, and pantropic viruses target? What are some examples of each?
NEUROTROPIC: nervous tissue, brain and spinal cord; Rabies, AEV
PNEUMOTROPIC: respiratory tissue, lung, trachea, bronchi; influenza, rhinovirus, SARS-CoV-2
VISCEROTROPIC: GIT tissues; Rotavirus, NDV, Enterovirus
DERMOTROPIC: skin; Poxvirus, LSDV, HPV, cattle papillomavirus
PANTROPIC: many tissues and organs; SARS-CoV-2, NDV, YFV
How do Picornaviruses inject their genomic RNA into their host?
create a pore into either at the plasma or endosomal membrane and release their RNA
What are 2 strategies for keeping viruses from attaching to host cell receptors?
- removal of cellular receptors on host cell (neuraminidase can remove sialic acid)
- application of monoclonal antibodies to attach to cellular receptors and block viruses
What are the 4 overall steps of endocytic viral entry/penetration via vesicle formation?
- binding of the virus to the host cell receptors
- invagination of the membrane
- formulation of an endocytic vesicle
- release of the virion into the cytoplasm
(L)
How do enveloped viruses undergo receptor-mediated fusion?
- attach to host cell
- viral and cellular envelopes fuse
- capsid contianing viral nucleic acids is released inside the host cell
- viral envelope forms a patch on the plasma membrane of the host cell
(R)
How do naked viruses tend to enter host cells? What are the 3 steps?
receptor-mediated endocytosis
- viral ligand-cell surface interaction causes a clathrin-coated pit formation/invagination at the cell surface
- the clathrin-coated pit (CCP) buds off to form vesicles
- CCP sheds and the virus containing vesicle fuses with lysosomes
Mechanism of viral fusion with host cells:
“drill” enzymes create a pore in the host plasma membrane and negative pressure pushes viral genome into host cells
How do DNA viruses undergo transcription?
utilize the cellular DNA-dependent DNA polymerase
- dsDNA can be directly transcribed into mRNA using cellular DNA polymerase II
- ssDNA must first have cellular DNA polymerase I create a complementary strand to make it double-stranded, and then transcription into mRNA can occur using cellular DNA polymerase II
once mRNA is made, translation can occur
How do positive sense single-stranded RNA viruses undergo translation?
the viral genome itself acts as RNA, so it is directly translated into proteins using an RNA-dependent RNA polymerase
How do negative sense single-stranded RNA viruses undergo translation?
virus must utlilize its own RNA-dependent polymerase, a transcriptase, to creat a complementary (positive) strand of RNA first, and then it can be translated by the same enzyme into viral protein
How do double-stranded RNA viruses undergo translation?
its segments of RNA are translated by their own RNA-dependent RNA polymerase that is able to convert each segment into mRNA that can be translated by the RNA-dependent RNA polymerase
What are ambisense viruses? How do they undergo transcription and translation?
viruses containing a genome that has RNA that is both positive and negative sense
these viruses use their own RNA-depdendent DNA polymerase (reverse transcriptase) to turn the (+)ssRNA segments into (-)ssDNA, from there, the DNA is able to be transcripted into mRNA using cellular RNA polymerase II and translated into viral proteins
Summary of virus replication strategies:
How are viral envelopes acquired? Where is the nucleocapsid located?
viral glycoproteins migrate from the ribosomes via ER toward the cell membrane and grouped into cylindrical spikes, allowing the virus to bud off
underneath the plasma/nuclear membrane of host cell
What are the 2 ways that viruses are able to release from infected cells based on the type of virus?
- naked viruses tend to cause the host cell to lyse (aka cytopathic viruses); caused by accumulation of the viral particles in the nucleus and cytoplasm, triggering of apoptotic pathways, or cell necrosis due to the viral release of lysosomal enzymes
- enveloped viruses tend to bud off the host cell by moving into the extracellular fluid while covered with the plasma membrane of the host cell —> undergoes second replication cycle in a new cell
