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+ MICB 202 Final > Virology Chapter 10-12: Retroviruses > Flashcards

Virology Chapter 10-12: Retroviruses Flashcards

1
Q

What is the structure of retroviruses?

A
  • enveloped
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2
Q

What is the genome structure of retroviruses?

A

two identical single-stranded RNA genome

  • (+) sense
  • 5’ cap
  • 3’ polyA tail

(nucleocapsid)

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3
Q

What do retroviruses package?

A
  • RT – to copy their RNA genome into double-stranded DNA

- integrase – to integrate their DNA into host cell’s DNA

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4
Q

Why do retroviruses integrate their DNA into host cell’s DNA?

A

allows retroviral genome to be transmitted to both daughter cells when infected cell divides

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5
Q

What transcribes virus’ genome, and produces genome for new virus particles?

A

host cell RNA polymerase

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6
Q

What is the matrix protein (MA, p17)?

A

lines inner surface of envelope

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7
Q

What does the envelope of HIV contain?

A
  • trans-membrane glycoprotein (TM, gp41) joined to glycoprotein (SU, gp120) by disulphide bond
  • matrix protein (p17)
  • conical capsid
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8
Q

What does the capsid of HIV contain?

A
  • 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
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9
Q

Where are protease proteins found?

A

inside and outside capsid

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10
Q

What is Vpr?

A

regulatory protein

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11
Q

What is the genome (nucleocapsid) packaged with)

A

RT and IN

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12
Q

What does gp120 do?

A

binds to host cell protein CD4

binds to chemokine receptor

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13
Q

What does gp41 do?

A

fusion protein – results in viral envelope fusing with cell’s plasma membrane, releasing capsid into cell cytoplasm

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14
Q

Does the (+) sense RNA serve as mRNA immediately upon entry into host cell?

A

NO

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15
Q

What is the primer for synthesis of DNA?

A

specific tRNA (derived from host cell) associated with genome

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16
Q

What is the genome synthesized and processed by?

A

host cell mRNA “handling machinery” – after virus has integrated its DNA into host cell chromosome in previous round of infection

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17
Q

What are the 3 major genes of HIV that are common to all retroviruses?

A

5’ - gag - pol - env - 3’

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18
Q

How many genes does HIV have?

A

9

  • 3 major genes common to all retroviruses
  • 2 genes for regulatory proteins
  • 4 genes for accessory proteins
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19
Q

What does gag gene encode?

A

structural proteins – from Gag polyprotein

  • CA
  • MA
  • NC
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20
Q

What does pol gene encode?

A

enzymes needed in genome replication – from Pol portion of Gag/Pol polyprotein

  • PR
  • RT
  • IN
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21
Q

What does env gene encode?

A

envelope proteins needed to bind to host cells – from Env polyprotein

  • gp120
  • gp41
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22
Q

When does PR get activated? What does it do?

A

after assembly of virus particle

cleaves Gag and Gag/Pol polyproteins at specific sites

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23
Q

Reverse Transcription

What does reverse transcriptase (RT) do?

A
  • RNA dependent DNA polymerase
  • RNase (enzyme to degrade RNA)
  • DNA dependent DNA polymerase
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24
Q

Reverse Transcription – Key Points

A
  1. RT (RDRP activity) uses tRNA as primer to synthesize DNA copy of RNA genome
    - creates DNA:RNA hybrid molecule
  2. RNase H removes most of RNA strand (this particular form of RNase can digest RNA in hybrid molecules)
  3. Remaining RNA is used as primer for synthesis of complementary strand of DNA
  4. RNA is copied to yield double-stranded DNA molecule
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25
Q

Reverse Transcription

Where and when does this occur?

A

in capsid, after capsid has been released into cytoplasm

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26
Q

Reverse Transcription – Process

A
  1. Part of tRNA molecule hybridizes to complementary site on RNA genome called primer binding site (PBS)
  2. RT reads sequence of genomic RNA in 3’ to 5’ direction, adding complementary DNA nucleotides in 5’ to 3’ direction
  3. When end of strand is reached, RNase H digests this portion of the RNA template
  4. DNA product is transferred to site near 3’ end of RNA template
  5. Newly-made (-) strand of DNA binds to repeat (R) sequence on RNA template
  6. 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
  7. Most of RNA is digested, except for poly-purine tract (PPT)
  8. 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
  9. RNase H activity digests PPT region of RNA template
  10. Newly made fragment of (+) strand DNA is transferred to 3’ end of (-) strand DNA, and RT completes synthesis of double-stranded DNA
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27
Q

Reverse Transcription

Compare the length of the double-stranded DNA product to the single-stranded RNA template when reverse transcription is complete.

A

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
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28
Q

Reverse Transcription

What does the 3’ LTR contain?

A

R site, that contains cleavage site and polyadenylation site used for transcription of viral mRNA

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29
Q

Reverse Transcription

RT Activity

A

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

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30
Q

What is the primer for synthesis of the minus strand of cDNA? For the positive strand of DNA?

A

(-) strand: tRNA that was packaged with genome

(+) strand: small stretch of original RNA genome

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31
Q

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?

A
  • 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
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32
Q

Attachment and Entry of Virus Genome

A
  1. HIV glycoprotein gp120 binds to CD cell surface marker on host cell – T helper, or macrophages and DC
  2. gp120 undergoes conformational change
  3. Co-receptor molecule (CXCR4 or CCR5) is recruited to site, and also binds to virus
  4. Fusion of virus envelope with host cell membrane
  5. Virus’ capsid is released into cell’s cytoplasm
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33
Q

Reverse Transcription

What does the capsid structure do as reverse transcription occurs in capsid?

A
  1. Uses microtubule network to traffic towards cell nucleus
  2. Pre-integration complex (virus DNA (provirus) + integrase) traffic through nuclear pore complex
  3. Virus genome is uncoated (removal of nucleocapsid protein from RNA)
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34
Q

Integration of Genome – Process

A

(following nuclear transport)

  1. Integrases recognize specific sequences, and use endonuclease activity to make a staggered cut into host cell DNA
  2. Integrase transfers proviral DNA so that it is joined to host cell DNA
  3. Host cell DNA polymerases repair the single-stranded DNA of the target site
  4. Results in duplication of the target site
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35
Q

Integration of Genome

Is this process specific?

A

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

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36
Q

Integration of Genome

Is this process reversible?

A

no – host cell is now permanently affected

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37
Q

Integration of Genome

What does integrase do?

A

catalyzes integration of provirus into host cell DNA at random sites – uses endonuclease activity to make staggered cuts into host cell DNA

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38
Q

Transcription of Genome (mRNA and genomic RNA)

Where is HIV transcription initiated?

A

promoter site in 5’ LTR of virus’ DNA integrated in the genome

begins at first base of R region of LTR

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39
Q

Transcription of Genome (mRNA and genomic RNA)

What is each LTR composed of?

A
  • U3 (unique 3’ seqeunce)
  • R (repeated sequence)
  • U5 (unique 5’ sequence)
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40
Q

Transcription of Genome (mRNA and genomic RNA)

What does the U3 region contain?

A

binding sites for cellular transcription factors

41
Q

Transcription of Genome (mRNA and genomic RNA)

When does polyadenylation occur?

A

immediately after the last base of R region in LTR

42
Q

Transcription of Genome (mRNA and genomic RNA)

What does the primary RNA transcript contain?

A

many sequences that can be recognized by cell’s RNA-splicing enzymes

43
Q

Transcription of Genome (mRNA and genomic RNA)

What types of mRNAs are generated?

A
  • unspliced mRNA
  • spliced mRNA

many are polycistronic (contain coding info of more than one gene product)

44
Q

Transcription of Genome (mRNA and genomic RNA)

What does the type of RNA produced depend on?

A

splicing sites used

45
Q

Transcription of Genome (mRNA and genomic RNA)

What are the 3 types of RNAs produced?

A
  • unspliced RNA
  • incompletely spliced RNA
  • fully spliced RNA
46
Q

Transcription of Genome (mRNA and genomic RNA)

What is unspliced RNA used for?

A
  • 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
47
Q

Transcription of Genome (mRNA and genomic RNA)

What is incompletely spliced RNA?

A
  • 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
48
Q

Transcription of Genome (mRNA and genomic RNA)

What is fully spliced RNA used for?

A

can be translated to generate some of the accessory and regulatory proteins

49
Q

Transcription of Genome (mRNA and genomic RNA)

How do mRNAs leave the nucleus?

A

fully spliced viral mRNAs exit the nucleus by the export pathway, following by the majority of cellular mRNAs

50
Q

Translation of mRNAs

What is unspliced mRNA translated into?

A

two polyproteins

  • Gag
  • Gag-pol
51
Q

Translation of mRNAs

Gag polyprotein translation

A

ribosome stops translation at stop codon in Gag ORF

polyprotein is cleaved into:

  • capsid protein (CA)
  • nucleocapsid protein (NC)
  • matrix protein (MA)
52
Q

Translation of mRNAs

Gag-pol polyprotein translation

A

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)
53
Q

Translation of mRNAs

Does ribosome shifting occur often?

A

no – for each new virus particle, many structural proteins are needed, as opposed to only once copy of the polymerase enzymes

54
Q

Translation of mRNAs

What serves as the mRNA for envelope proteins?

A

some spliced mRNAs

55
Q

Translation of mRNAs

Env polyprotein translation

A
  • 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
56
Q

Translation of mRNAs

What is mRNA for accessory and regulatory proteins translated by?

A

cytoplasmic ribosomes

57
Q

Assembly and Egress of Virus Particle

What is the function of Gag polyprotein in assembly of HIV particle?

A
  • 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
58
Q

Assembly and Egress of Virus Particle

How is the virus particle released?

A

membrane fission

59
Q

Assembly and Egress of Virus Particle

When is the ESCRT pathway used?

A

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
60
Q

Assembly and Egress of Virus Particle

What are the requirements when viruses bud?

A
  • membrane must be constricted towards cytoplasm

- cytoplasmic host factors that catalyze membrane fission must work from within the bud

61
Q

Maturation of Virus Particle

When does this occur?

A

during, or shortly after budding

62
Q

Maturation of Virus Particle

What mediates this process?

A

viral-encoded protease that is part of Gag-Pol polyprotein

63
Q

Maturation of Virus Particle – Process

A
  1. Protease cleaves specific sites in Gag and Gag-Pol polyprotein
  2. CA protein reassembles to form capsid of virus structure
  3. MA remains associated with envelope
  4. Gag-Pol polyprotein is cleaves so that RT and IN are functional when virus infects new cell
  5. Enzymes remain inactive until virus enters cytoplasm of host cell, and gains access to nucleotides to allow for reverse transcription, then integration
64
Q

How does the retroviral RNA genome acquire its 5’ methylated cap and poly(A) tail?

A

done by host cell enzymes (same enzymes used by cell to make its own mRNA)

65
Q

Once integrated, what enzyme is responsible for the synthesis of HIV RNA?

A

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
66
Q

The env gene is dependent on a different mechanism for expression that the pol gene. What is this mechanism?

A

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
67
Q

How is HIV-1 transmitted?

A

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
68
Q

What are CD4 T cells required for?

A
  • activation of macrophages to kill intracellular pathogens

- activation of B cells for antibody production to neutralize viruses, or opsonize or kill bacteria

69
Q

What are the 4 stages of the clinical course of HIV-1?

A
  • incubation period
  • acute infection
  • latency stage
  • AIDS
70
Q

What is the stage of infection of HIV-1 determined by?

A

measuring:

  • CD4 T cell count
  • level of HIV-1 in blood
71
Q

Incubation Period

  • time period
  • symptoms
  • CD4 T cell count
A

time period:
- 2-4 weeks

symptoms:
- asymptomatic

CD4 T cells:
- slightly declines

72
Q

Acute Infection

  • time period
  • symptoms
  • CD4 T cell count
  • level of HIV-1 in blood
A

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

73
Q

What marks the beginning of the latency stage of HIV-1?

A

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)
74
Q

Latency Stage

  • time period
  • what HIV-1 is doing
  • level of HIV-1 in blood
A

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
75
Q

AIDS

  • how it occurs
  • level of HIV-1 in blood
A

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

76
Q

What is the HIV virus load test?

A

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
77
Q

What is the limit of detection of HIV virus load tests?

A

depends on the test

  • some have detection level of 5 copies/ml
  • some have detection level of 20 copies/ml
78
Q

What is the main goal of anti-viral drugs?

A

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
79
Q

What are methods to monitor CD T cell count?

A
  • 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
80
Q

What is the current method to prevent HIV-1 infection?

A

limit spread of HIV-1 between individuals

  • vaccines
  • anti-viral medications
  • RT inhibitors
  • protease inhibitors
  • integrase inhibitors
  • pre and post-exposure prophylaxis
81
Q

Why are there problems in developing a HIV vaccine?

A
  • 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
82
Q

Why are attenuated, live virus vaccines not suitable?

A

potential reversion to the virulent form

83
Q

Why are inactivated virus vaccines not suitable?

A
  • 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
84
Q

Why is developing anti-viral medications that target virus infections more difficult than antibiotics that target bacterial infections?

A

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
85
Q

How do anti-retroviral medications work?

A

prevent HIV from multiplying, reducing amount of HIV in the body

86
Q

When do anti-retroviral medications work?

A

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
87
Q

What is the treatment for HIV?

A

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)
88
Q

What are the categories of antiviral medications currently used against HIV-1?

A
  • 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

89
Q

Why is RT an obvious target for anti-viral therapy?

A

has unique enzyme activity – synthesizing DNA from RNA template

90
Q

What are the two types of approaches used in affecting RT function?

A
  • nucleoside analogs – terminates DNA synthesis

- non-nucleoside analogs – bind somewhere on RT enzyme and change enzyme shape, making it non-functional

91
Q

What do protease inhibitors do?

A

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

92
Q

What type of drugs are protease inhibitors?

A

transition state analogues

plug drugs

93
Q

What is prophylaxis?

A

use of medicine to protect against infectious

94
Q

What is pre-exposure prophylaxis (PrER)?

  • what is it
  • when is it taken
  • who is it for
A

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

95
Q

What is post-exposure prophylaxis (PER)?

  • when is it taken
  • who is it for
A

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

96
Q

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?

A

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

97
Q

What do chemokine receptor blockers do?

A

causes gp120 to not able to achieve the conformational change that triggers the conformational change in the gp41 protein – fusion
peptide is not revealed

98
Q

Why is chemokine receptor blocking a more challenging method?

A

it could impair the host’s normal function (although there is some redundancy in chemokine receptors and their mode of action)

+ MICB 202 Final (20 decks)

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