Virology Chapter 10-12: Retroviruses Flashcards
What is the structure of retroviruses?
- enveloped
What is the genome structure of retroviruses?
two identical single-stranded RNA genome
- (+) sense
- 5’ cap
- 3’ polyA tail
(nucleocapsid)
What do retroviruses package?
- RT – to copy their RNA genome into double-stranded DNA
- integrase – to integrate their DNA into host cell’s DNA
Why do retroviruses integrate their DNA into host cell’s DNA?
allows retroviral genome to be transmitted to both daughter cells when infected cell divides
What transcribes virus’ genome, and produces genome for new virus particles?
host cell RNA polymerase
What is the matrix protein (MA, p17)?
lines inner surface of envelope
What does the envelope of HIV contain?
- trans-membrane glycoprotein (TM, gp41) joined to glycoprotein (SU, gp120) by disulphide bond
- matrix protein (p17)
- conical capsid
What does the capsid of HIV contain?
- made of virus protein p24 (CA)
- two (+) RNA covered with nucleocapsid (NC, p7) proteins
- enzymes involved in early stages of genome replication – RT and integrase (IN)
- protease
Where are protease proteins found?
inside and outside capsid
What is Vpr?
regulatory protein
What is the genome (nucleocapsid) packaged with)
RT and IN
What does gp120 do?
binds to host cell protein CD4
binds to chemokine receptor
What does gp41 do?
fusion protein – results in viral envelope fusing with cell’s plasma membrane, releasing capsid into cell cytoplasm
Does the (+) sense RNA serve as mRNA immediately upon entry into host cell?
NO
What is the primer for synthesis of DNA?
specific tRNA (derived from host cell) associated with genome
What is the genome synthesized and processed by?
host cell mRNA “handling machinery” – after virus has integrated its DNA into host cell chromosome in previous round of infection
What are the 3 major genes of HIV that are common to all retroviruses?
5’ - gag - pol - env - 3’
How many genes does HIV have?
9
- 3 major genes common to all retroviruses
- 2 genes for regulatory proteins
- 4 genes for accessory proteins
What does gag gene encode?
structural proteins – from Gag polyprotein
- CA
- MA
- NC
What does pol gene encode?
enzymes needed in genome replication – from Pol portion of Gag/Pol polyprotein
- PR
- RT
- IN
What does env gene encode?
envelope proteins needed to bind to host cells – from Env polyprotein
- gp120
- gp41
When does PR get activated? What does it do?
after assembly of virus particle
cleaves Gag and Gag/Pol polyproteins at specific sites
Reverse Transcription
What does reverse transcriptase (RT) do?
- RNA dependent DNA polymerase
- RNase (enzyme to degrade RNA)
- DNA dependent DNA polymerase
Reverse Transcription – Key Points
- RT (RDRP activity) uses tRNA as primer to synthesize DNA copy of RNA genome
- creates DNA:RNA hybrid molecule - RNase H removes most of RNA strand (this particular form of RNase can digest RNA in hybrid molecules)
- Remaining RNA is used as primer for synthesis of complementary strand of DNA
- RNA is copied to yield double-stranded DNA molecule
Reverse Transcription
Where and when does this occur?
in capsid, after capsid has been released into cytoplasm
Reverse Transcription – Process
- Part of tRNA molecule hybridizes to complementary site on RNA genome called primer binding site (PBS)
- RT reads sequence of genomic RNA in 3’ to 5’ direction, adding complementary DNA nucleotides in 5’ to 3’ direction
- When end of strand is reached, RNase H digests this portion of the RNA template
- DNA product is transferred to site near 3’ end of RNA template
- Newly-made (-) strand of DNA binds to repeat (R) sequence on RNA template
- RT reads sequence of genomic RNA in 3’ to 5’ direction, adding complementary DNA nucleotides in 5’ to 3’ direction to complete synthesis of (-) strand of DNA
- Most of RNA is digested, except for poly-purine tract (PPT)
- RT uses PPT on RNA strand as primer, and reads (-) strand of DNA as template in 3’ to 5’ direction, adding complementary DNA nucleotides in 5’ to 3’ direction
- RNase H activity digests PPT region of RNA template
- Newly made fragment of (+) strand DNA is transferred to 3’ end of (-) strand DNA, and RT completes synthesis of double-stranded DNA
Reverse Transcription
Compare the length of the double-stranded DNA product to the single-stranded RNA template when reverse transcription is complete.
double-stranded DNA product is longer
- LTRs are generated from the process
- R-U5 (repeat sequence unique sequence 5’ end) and U3-R from genomic RNA have now both become U3-R-U5 in viral DNA – gives it the required LTR at each end
Reverse Transcription
What does the 3’ LTR contain?
R site, that contains cleavage site and polyadenylation site used for transcription of viral mRNA
Reverse Transcription
RT Activity
RDDP:
- uses RNA strand as template to continue DNA synthesis
RNase H:
- continues to remove RNA, leaving short segments to be used as primers for DNA synthesis
DDDP:
- uses RNA primers to synthesize (+) strand DNA
What is the primer for synthesis of the minus strand of cDNA? For the positive strand of DNA?
(-) strand: tRNA that was packaged with genome
(+) strand: small stretch of original RNA genome
Why is RNase H important in the HIV replication cycle? Could HIV complete the reverse transcription without RNase H? If no, what specific step would be impaired?
- RNase H is important for removing RNA from RNA:DNA hybrid so that full DNA:DNA unit can be created
- RNA:DNA units cannot be integrated into host’s genome, therefore if RNase H was defective, the virus would not be able to make DNA to integrate into the cell, and no virus progeny will be produced
Attachment and Entry of Virus Genome
- HIV glycoprotein gp120 binds to CD cell surface marker on host cell – T helper, or macrophages and DC
- gp120 undergoes conformational change
- Co-receptor molecule (CXCR4 or CCR5) is recruited to site, and also binds to virus
- Fusion of virus envelope with host cell membrane
- Virus’ capsid is released into cell’s cytoplasm
Reverse Transcription
What does the capsid structure do as reverse transcription occurs in capsid?
- Uses microtubule network to traffic towards cell nucleus
- Pre-integration complex (virus DNA (provirus) + integrase) traffic through nuclear pore complex
- Virus genome is uncoated (removal of nucleocapsid protein from RNA)
Integration of Genome – Process
(following nuclear transport)
- Integrases recognize specific sequences, and use endonuclease activity to make a staggered cut into host cell DNA
- Integrase transfers proviral DNA so that it is joined to host cell DNA
- Host cell DNA polymerases repair the single-stranded DNA of the target site
- Results in duplication of the target site
Integration of Genome
Is this process specific?
yes – requires site-specific insertion sequences that are not recognized by integrase protein
however, sites of integration into host cell genome are not specific – multiple copies of this insertion sequence in the genome
Integration of Genome
Is this process reversible?
no – host cell is now permanently affected
Integration of Genome
What does integrase do?
catalyzes integration of provirus into host cell DNA at random sites – uses endonuclease activity to make staggered cuts into host cell DNA
Transcription of Genome (mRNA and genomic RNA)
Where is HIV transcription initiated?
promoter site in 5’ LTR of virus’ DNA integrated in the genome
begins at first base of R region of LTR
Transcription of Genome (mRNA and genomic RNA)
What is each LTR composed of?
- U3 (unique 3’ seqeunce)
- R (repeated sequence)
- U5 (unique 5’ sequence)
Transcription of Genome (mRNA and genomic RNA)
What does the U3 region contain?
binding sites for cellular transcription factors
Transcription of Genome (mRNA and genomic RNA)
When does polyadenylation occur?
immediately after the last base of R region in LTR
Transcription of Genome (mRNA and genomic RNA)
What does the primary RNA transcript contain?
many sequences that can be recognized by cell’s RNA-splicing enzymes
Transcription of Genome (mRNA and genomic RNA)
What types of mRNAs are generated?
- unspliced mRNA
- spliced mRNA
many are polycistronic (contain coding info of more than one gene product)
Transcription of Genome (mRNA and genomic RNA)
What does the type of RNA produced depend on?
splicing sites used
Transcription of Genome (mRNA and genomic RNA)
What are the 3 types of RNAs produced?
- unspliced RNA
- incompletely spliced RNA
- fully spliced RNA
Transcription of Genome (mRNA and genomic RNA)
What is unspliced RNA used for?
- used as mRNA that is translated by cytoplasmic ribosomes to generate Gag and Gag-Pol polyproteins
- RNA transcripts can be packaged into new virus particles to serve the genomic RNA
Transcription of Genome (mRNA and genomic RNA)
What is incompletely spliced RNA?
- they use splice donor site located closer to 5’ end of HIV RNA genome, in combination with any of the splice acceptors located in central region of the virus
- can be translated to generate Env polyprotein, or some of the accessory or regulatory proteins
Transcription of Genome (mRNA and genomic RNA)
What is fully spliced RNA used for?
can be translated to generate some of the accessory and regulatory proteins
Transcription of Genome (mRNA and genomic RNA)
How do mRNAs leave the nucleus?
fully spliced viral mRNAs exit the nucleus by the export pathway, following by the majority of cellular mRNAs
Translation of mRNAs
What is unspliced mRNA translated into?
two polyproteins
- Gag
- Gag-pol
Translation of mRNAs
Gag polyprotein translation
ribosome stops translation at stop codon in Gag ORF
polyprotein is cleaved into:
- capsid protein (CA)
- nucleocapsid protein (NC)
- matrix protein (MA)
Translation of mRNAs
Gag-pol polyprotein translation
ribosomal frameshift (in the 5’ -1 direction) required to induce the ribosome to skip over gag stop codon, and enter into pol gene reading frame
- this programmed change in reading frame occurs at heptanucleotide slippery sequence that is follow by spacer region and downstream RNA stem-loop structure
- structure causes ribosome to stall during translation, and slip back on nucleotide and continue translation in the -1 reading frame
polyprotein is cleaved into:
- RT
- IN
- PR (protease)
- all Gag polyprotein products (CA, NC, MA)
Translation of mRNAs
Does ribosome shifting occur often?
no – for each new virus particle, many structural proteins are needed, as opposed to only once copy of the polymerase enzymes
Translation of mRNAs
What serves as the mRNA for envelope proteins?
some spliced mRNAs
Translation of mRNAs
Env polyprotein translation
- mRNA is translated by ER ribosomes
- during transit through Golgi, Env polyprotein is glycosylated, and cleaved by host cell enzyme furin to form gp120 and gp41
- gp41 is anchored in host cell membrane
- gp120 is attached to gp41 by disulfide bond
Translation of mRNAs
What is mRNA for accessory and regulatory proteins translated by?
cytoplasmic ribosomes
Assembly and Egress of Virus Particle
What is the function of Gag polyprotein in assembly of HIV particle?
- forms dimers (and other oligomers)
- can interact with plasma membrane at sites where envelope proteins gp120 and gp41 are present
- oligomers (may additionally contain Gag-pol polyprotein) can selectively capture HIV genomic RNA, and serve as nucleation point for HIV assembly at plasma membrane
- recruit additional Gag polyproteins to plasma membrane, and induce curvature into plasma membrane, resulting in HIV bud formation
- recruits ESCRT (endosomal sorting complex required for transport) machinery needed for membrane fission
Assembly and Egress of Virus Particle
How is the virus particle released?
membrane fission
Assembly and Egress of Virus Particle
When is the ESCRT pathway used?
in pathways used in cellular processes such as endocytosis, where:
- membrane constricts away from cytoplasm
- membrane fission is catalyzed by cytoplasmic dynamin, which acts from outside of the bud neck
Assembly and Egress of Virus Particle
What are the requirements when viruses bud?
- membrane must be constricted towards cytoplasm
- cytoplasmic host factors that catalyze membrane fission must work from within the bud
Maturation of Virus Particle
When does this occur?
during, or shortly after budding
Maturation of Virus Particle
What mediates this process?
viral-encoded protease that is part of Gag-Pol polyprotein
Maturation of Virus Particle – Process
- Protease cleaves specific sites in Gag and Gag-Pol polyprotein
- CA protein reassembles to form capsid of virus structure
- MA remains associated with envelope
- Gag-Pol polyprotein is cleaves so that RT and IN are functional when virus infects new cell
- Enzymes remain inactive until virus enters cytoplasm of host cell, and gains access to nucleotides to allow for reverse transcription, then integration
How does the retroviral RNA genome acquire its 5’ methylated cap and poly(A) tail?
done by host cell enzymes (same enzymes used by cell to make its own mRNA)
Once integrated, what enzyme is responsible for the synthesis of HIV RNA?
host cell RNA polymerase II is responsible for transcribing HIV genome
- can do this because HIV’s genome was converted to DNA and integrated into host DNA
The env gene is dependent on a different mechanism for expression that the pol gene. What is this mechanism?
dependent on transcript splicing
- Gag-pol-env are transcribed into a single nascent mRNA, but then host splicing enzymes cleaves gag-pol out, leaving an env only transcript
- this transcript is then translated by ER ribosomes – necessary because ribosomes would not be able to assemble in middle of mRNA
How is HIV-1 transmitted?
direct contact of mucus membrane or bloodstream with biological fluid containing HIV-1
- sexual contact
- pregnancy, child-birth, breast feeding
- injection drug use
- blood transfusion / organ transplant
What are CD4 T cells required for?
- activation of macrophages to kill intracellular pathogens
- activation of B cells for antibody production to neutralize viruses, or opsonize or kill bacteria
What are the 4 stages of the clinical course of HIV-1?
- incubation period
- acute infection
- latency stage
- AIDS
What is the stage of infection of HIV-1 determined by?
measuring:
- CD4 T cell count
- level of HIV-1 in blood
Incubation Period
- time period
- symptoms
- CD4 T cell count
time period:
- 2-4 weeks
symptoms:
- asymptomatic
CD4 T cells:
- slightly declines
Acute Infection
- time period
- symptoms
- CD4 T cell count
- level of HIV-1 in blood
time period:
- 4 weeks
symptoms:
- non-specific – therefore not recognized as signs of HIV-1 infection
- frequently misdiagnosed as influenza
CD4 T cells:
- drops
level of HIV-1 in blood:
- abundant
What marks the beginning of the latency stage of HIV-1?
seroconversion – production of antibodies to the virus
- body’s immune system activates CTLs that kill some virally infected cells (resulting in decline in CD4 T cells)
- body’s immune system activates some B cells resulting in antibody production
- antibodies against several HIV protein can be detected (ie. anti-gp120 antibodies)
Latency Stage
- time period
- what HIV-1 is doing
- level of HIV-1 in blood
time period:
- 2 weeks, to decades
what HIV-1 is doing
- actively replicating within lymphoid organs
level of HIV-1 in blood:
- low
- immune response may be controlling the amount of circulating virus
AIDS
- how it occurs
- level of HIV-1 in blood
how it occurs:
- CD4 T cell numbers decline below critical level
- HIV-1 positive individuals become highly susceptible to infections with variety of opportunistic bacteria, viruses, fungi, and parasites that are normally controlled by elements of immune system that HIV-1 has damaged
level of HIV-1 in blood:
- very high
What is the HIV virus load test?
measures amount of HIV in bloodstream
- done on blood sample
- measure amount of virus nucleic acid by employing various hybridization methods with or without RNA amplification (ie. RT-PCR)
- patient may have their HIV viral loads measured several times during a year
What is the limit of detection of HIV virus load tests?
depends on the test
- some have detection level of 5 copies/ml
- some have detection level of 20 copies/ml
What is the main goal of anti-viral drugs?
to reduce viral load to an undetectable level (below level of detection)
- does not mean virus has been eliminated
- slows or stops disease progression, and prevents transmission to sex partners
What are methods to monitor CD T cell count?
- optical or fluorescence microscopy – in labs that have low sample throughput, or where resources are limited (antibodies to CD3 and CD4 proteins required)
- flow cytometry (FACS) – in labs that have high sample throughput
What is the current method to prevent HIV-1 infection?
limit spread of HIV-1 between individuals
- vaccines
- anti-viral medications
- RT inhibitors
- protease inhibitors
- integrase inhibitors
- pre and post-exposure prophylaxis
Why are there problems in developing a HIV vaccine?
- immune system produces antibodies and CTL responses during acute phase of HIV-1 infection, but these immune responses do not offer protection (do not eliminate the virus)
- lack of animal model for testing
Why are attenuated, live virus vaccines not suitable?
potential reversion to the virulent form
Why are inactivated virus vaccines not suitable?
- not suitable if inactivation procedure denatures the epitope on the glycoprotein that would need to be neutralized by antibodies to prevent viral attachment
- safety concerns regarding the complete inactivation of the virus
Why is developing anti-viral medications that target virus infections more difficult than antibiotics that target bacterial infections?
virus is using host cell for replication
- medications would have to enter host cell cytoplasm and selectively interfere with virus replication cycle without harming uninfected host cells
- ie. medication that targets ribosomes would not be suitable because protein synthesis is essential for life
- goal is to try to identify virus-specific molecules or processes (ie. proteins, enzymes etc.) that can be targets of these antiviral compound
How do anti-retroviral medications work?
prevent HIV from multiplying, reducing amount of HIV in the body
When do anti-retroviral medications work?
early in replication cycle:
- RT inhibitors
- IN inhibitors
- prevent virus from permanently infecting cell
later in replication cycle:
- PR inhibitors
- prevent virus from maturing into infectious particle
What is the treatment for HIV?
anti-retroviral therapy (ART)
- take a combination of anti-viral medications everyday
- suppress plasma HIV-1 RNA levels to < 50 copies/ml (below detection level)
What are the categories of antiviral medications currently used against HIV-1?
- RT inhibitors – main
- IN inhibitors – main
- PR inhibitors – main
- fusion inhibitors
- CCR5 antagonists
- post-attachment inhibitors
many of the drugs are used as combination therapeutics to elicit a synergistic response
ie. two nucleoside analogue RT inhibitors + PR inhibitor
Why is RT an obvious target for anti-viral therapy?
has unique enzyme activity – synthesizing DNA from RNA template
What are the two types of approaches used in affecting RT function?
- nucleoside analogs – terminates DNA synthesis
- non-nucleoside analogs – bind somewhere on RT enzyme and change enzyme shape, making it non-functional
What do protease inhibitors do?
inhibit HIV-1 protease, preventing maturation of viruses by blocking cleavage of Gag-Pol polyprotein to release RT and integrase
results in non-infectious particle – virus may bind to another host cell, but reverse transcription will not occur when capsid enters into cytoplasm
What type of drugs are protease inhibitors?
transition state analogues
plug drugs
What is prophylaxis?
use of medicine to protect against infectious
What is pre-exposure prophylaxis (PrER)?
- what is it
- when is it taken
- who is it for
what is it:
- anti-HIV medicines
when is it taken:
- everyday, before possible exposure
who is it for:
- people who don’t have HIV, and are at risk of getting HIV from sex or injection drug use
What is post-exposure prophylaxis (PER)?
- when is it taken
- who is it for
when is it taken:
- after HIV exposure
- within 72 hours
who is it for:
- people who don’t have HIV but may have been exposed
Several anti-viral drugs that are effective against HIV are nucleotide analogs. In the laboratory, the replication process of the viral genome is terminated when it is incubated the presence of these drugs. Why is it possible that these drugs can be used without disrupting normal host functions?
human cells do not have enzyme that can read RNA as a template for synthesis of any type of nucleic acid
Human cell polymerases, both DNA polymerase and RNA polymerase, read DNA as the template
What do chemokine receptor blockers do?
causes gp120 to not able to achieve the conformational change that triggers the conformational change in the gp41 protein – fusion
peptide is not revealed
Why is chemokine receptor blocking a more challenging method?
it could impair the host’s normal function (although there is some redundancy in chemokine receptors and their mode of action)
