Micro - E4 Flashcards
1
Q
Bacterial virus
A
Bacterial virus = phage = bacteriophage
- Genome can be double-stranded or single-stranded DNA or RNA
- Capsid: protein coat
- Envelope: lipid coat (some may have this)
2
Q
Two Classes of bacteriophages
A
- Virulent or Lytic: results in cell lysis (cell death) and production of many progeny phage particles.
- Temperate or lysogenic: can either cause the cell to lyse and produce more progeny phage particles or can lead to maintenance of the virus genome within the living host cell resulting in a dormant state (lysogeny)
3
Q
Lytic Growth Cycle
A
1) Adsorption (attach to receptors on bacterium)
2) Penetration (injection and membrane fusion – if have envelope)
3) Gene expression
4) Nucleic acid replication
5) Synthesis of structural proteins
6) Assembly of virons
7) Lysis of bacterium and release of progeny phage
4
Q
Liberation of progeny bacteriophage
A
Lysis of the bacterial host and release of the newly synthesized bacteriophages coincides with activation of degrative enzymes that destroy the cytoplasmic membrane and the peptidoglycan cell wall.
- These enzymes are produced as “late proteins”
5
Q
T7 early genes (timing?) (transcribed by which polymerase?)
A
code for proteins required for viral replication (e.g., a specific DNA polymerase or polymerase component) (1-12 mins after phage injects its genome into the cytoplasm)
E. coli “host” RNAP recognizes early promoter and transcribes early mRNA, but doesn’t recognize late promoters.
6
Q
T7 late genes (timing?) (transcribed by which polymerase?)
A
code for the structural components (capsid, tail fibers) of the virus, and the lysis proteins (8minutes-end)
Transcribed by T7 polymerase
7
Q
Why does it take 8 minutes before the T7 late transcripts start to be synthesized?
A
phage polymerase, “T7 polymerase”, is the product of an early gene, called gene 1. (specific for the phage late genes because it only recognizes the late promoters)
takes ~8 minutes to transcribe the gene 1 gene and translate the gene 1 mRNA to make functional T7 polymerase proteins.
8
Q
Why does transcription of early T7 genes (and host genes) cease during late gene expression?
A
product of the late gene 2 (Gp2) binds to E. coli RNA polymerase holoenzyme and totally inhibits its activity.
–> shuts off all RNA synthesis from bacterial host and early phage promoters.
9
Q
Kinds of transcription terminators at end of early T7 mRNA at TE?
A
Both factor-dependent (has rho-attached to RNAP and factor-independent)
10
Q
Temperate vs. Lytic Phage
A
- Temperate: undergoes lytic or lysogenic life cycle (exp. Bacterial lambda)
- Lytic (virulent phages): always undergo lytic growth cycle, cannot “lysogenize” (a repressed viral state with viral genome inserted into host chromosome, ensuring stable inheritance & maintenance)
11
Q
Inherited phage genome
A
“prophage” or “provirus”
12
Q
Cell containing a prophage
A
lysogeny
13
Q
N protein
A
associates with RNAP and causes it to be blind to terminators (a transcriptional “antiterminator”) allowing RNA polymerase to bypass the EARLY terminators
14
Q
Q-protein
A
an antiterminator of transcription from Plate. Late genes encode phage structural components and the host lysis enzymes. (once you read through terminator, DESTINED for lysis)
15
Q
Which gene was not expressed during bacteriophage lambda lytic growth?
A
cI –> repressor of lytic growth
Recall: protein that binds to DNA at a site within or downstream of a promoter site –> blocks transcription from that promoter by competing with RNA polymerase for interaction with the DNA.
16
Q
To choose the lysogenic response, the phage needs to do two things quickly
A
o synthesize a high concentration of CI repressor
o synthesize the Int protein, a DNA recombination protein, that integrates the phage genomic DNA into the bacterial host chromosome at a specific site.
17
Q
CI gene is transcribed from two promoters:
A
PRE (promoter for repressor establishment)
PRM (promoter for repressor maintenance)
18
Q
CI protein
A
CI –> repressor that binds to PL and PR –> shuts off all early and middle gene expression of the phage (preventing lytic infection)
19
Q
CII protein
A
CII –> transcriptional activator that binds to Pre (promoter for repressor establishment), acts as + regulator, leads to high levels of CI repressor (λ repressor) expression.
20
Q
In order to maintain repression of the lytic cycle, phage λ has a second mechanism for synthesizing the repressor CI…
A
another promoter, PRM (promoter for repressor MAINTENENCE)
requires the λ CI repressor to act as a positive regulator for its own synthesis. (C1– negative reg of pL and pR but + regulator of its own synthesis)
21
Q
Conditions which bacteriophage lambda undergoes lytic growth
A
RICH MEDIUM –> HIGH PROTEASE –> CII degraded–> phage undergoes LYTIC GROWTH
22
Q
Conditions which bacteriophage lambda undergoes lysogeny
A
POOR MEDIUM –> LOW PROTEASE –-> CII activates Repressor Promoter –>Phage undergoes LYSOGENY
23
Q
How prophages escape from lysogenic state
A
INDUCTION (Elicted by DNA damage through the “SOS response”)
-The return to the lytic cycle can only occur if the repressor protein self-destructs or autoproteolyzes, which occurs upon DNA damage.
24
Q
How does SOS response affect a prophage?
A
DNA damage activates RecA.
Activated RecA triggers temperate phage repressors to self-destruct (autoproteolyze).
Loss of phage receptor –> initiation of prophage excision (imprecise) from bacterial chromosome and subsequent lytic growth.
- DNA damage “activates” the cellular protein RecA, which, in turn, interacts with certain bacterial repressors, like LexA (represses expression of binding of DNA repair genes) and causes them (LexA) to self-destruct. This is termed the “SOS” response.
- No LexA –> SOS DNA repair genes are expressed
- int and xis: carry out a recombination reaction that removes the phage DNA from the chromosome as a circle that is identical to the original phage DNA molecule that had previously integrate
25
imprecise excision or “misexcision” of the prophage
Int and Xis proteins recombine sequences within phage genes and nearby adjacent bacterial chromosome sequences (production of circles DNA containing mostly phage and some bacterial DNA.)
26
Specialized transduction
transfer of host bacterial chromosomal DNA limited to the flanking regions of a prophage integration site that was misexcised and packaged in phage particles and delivered to another bacterium. *ONLY TEMPERATE phages can do this
27
Lysogenic conversion
acquisition of a new property (phenotype) by a host bacterium due to establishment of lysogeny by a temperate phage
exp. Shiga-like dysentery toxins produced by E. coli O157:H7 and its relatives are encoded on λ-like temperate phages
28
Process of expression of Shiga-like dysentery toxins produced by E. coli O157:H7
Certain abx --> bacterial DNA damage --> activated RecA* protein --> phage repressor undergoes autoproteolysis --> phage induced (expression of toxin genes stx A and B, enhanced by Q protein and expressed upon induction of the prophage)
29
3 phage resistance mechanisms
1. Alter phage receptor (alter surface proteins used by virus to adsorb)
2. Restriction modification systems (Degradative endonucleases (restriction enzymes) cleave or “restrict” phage DNA genomes.)
3. CRISPR system: bacterial mechanism of adaptive immunity against invading nucleic acids (bacteria cleaves foreign DNA, can prevent any attacks from bacteriophages that they have previously encountered).
30
Pseudomonas aeruginosa
Gram-negative, highly motile, rod-shaped bacterium.
Adheres tightly to lung epithelium.
Produces extracellular polysaccharide (mucoidy).
Primary cause of death in CF patients.
31
Pseudomonas infection associated with cystic fibrosis
Sx: Bronchiectasis (abnormally dilated airways)
tx: IV abx therapy was initiated with a cephalosporin (Ceftazidime) and an aminoglycoside (Tobramycin)
32
Gram-negative bacterial envelope structure
Inner and outer membrane with periplasmic space between (periplasmic space: Hydrolytic enzymes, Chemoreceptors, Transport proteins)
33
Gram negative outer membrane
Phospholipid in inner leaflet
-Barrier to hydrophilic compounds.
LPS in outer leaflet
- Negatively charged surface
- Resistant to detergents and other hydrophobic molecules
PORES - allow entry of SMALL (< 700 MW) hydrophilic nutrient molecules through outer membrane
34
How is LPS (endotoxin or O-Ag) a potent antigen
Innate response: cytokine production, septic shock
Adaptive response: serotyping of strains, e.g. E. coli O157:H7
35
Adhesins
- on microbial cell surface
- bind to sugars or proteins on the host cell surface
- can be very specific: some bind only to a single type of epithelial cell in a single animal species
- some areas (human mouth) cell receptors and the adhesins that bind them vary from one tiny area to another
36
Adhesive structures of bacteria
- pilli (fimbriae)
- flagella
- capsules (see in mucoid material)
*PA has flagelli and pilli
37
Why are flagella are critical for P. aeruginosa infection mucous layer
Flagella act as adhesins and allow the bacteria to swim through the mucous layer and attack the intestinal epithelium
Can be polar or all over cell (peritrichous)
P. aeruginosa has a single, polar flagellum.
38
Bacterial chemotaxis and motility are controlled
| by...
"two-component” regulatory systems
(1) membrane-bound sensor histdine kinase detects specific molecules in envio and (2) a cytoplasmic response regulator that is activated by the kinase, causes the flagellar motor to turn clockwise (tumbling movement) or counterclockwise (directed movement).
The phosphorylated response regulator binds to the flagellar motor and determines its rotational mode.
39
Capsules
- Loose network of polymers surrounding cell
- Polysaccharide or protein or both
Functions:
Resists drying
Promotes adherence
Protects against phagocytosis
exp. mannuronate-guluronate co-polymer (alginate of Pseudomonas)
40
Important Encapsulated Pathogens
Streptococcus pneumoniae
Haemophilus influenzae
Neisseria meningitidis
Pseudomonas aeruginosa
41
Encapsulated bacteria often capable of causing septicemia and meningitis
Streptococcus pneumoniae
Haemophilus influenzae
Neisseria meningitidis
42
What contributes to intractable nature of Pseudomonas lung infection
Capsule- resistance of drying, promotion of adherence, protection against some environmental stresses.
viscous mucus of CF patients, biofilm formation
^contribute to intractable Pseudomonas lung infection.
43
Role of alginate production (mucoidy) in P. aeruginosa infection of CF patients
Alginate (mannuronate-guluronate co-polymer) – extracellular polysaccharide excreted by P. aeruginosa (there are non-mucoid phenotypes of P. a)
Bacteria that produce large amounts of alginate are said to be “mucoid”
Alginate coats the bacterial cells forming a capsule-like structure
Alginate capsule protects P. aeruginosa against host defenses and antibiotics
44
What controls production of alginate?
Alginate production depends on AlgU (alternative sigma factor) for transcription of alginate genes
45
MucA
MucA --> negative regulator of AlgU (sigma factor).
MucA normally holds Algu in an inactive state --> alginate genes not expressed
Cells sense cell wall stress --> MucA degraded by a protease in periplasm --> AlgU is freed and activated --> alginate genes expressed
46
Why is there so much alginate in the lungs of CF patients?
Lungs of CF patients accumulate mutant strains of P. aeruginosa that produce a truncated inactive MucA
AlgU --> always active --> high levels of alginate produced = mucoidy.
47
Biofilms
thick layers of the extracellular polysaccharide matrix in which the bacterial cells are embedded
Protected from abx, if burst, can release other bacteria --> spread infection
When ready for release, start to express Fla and Pil
48
How do bacteria know when to form a biofilm?
Intercellular Signaling (generally associated with population crowding)
- Within a species or across species lines
- Within populations of dispersed cells or in communities of cells
49
Quorum sensing signaling compounds
Acylated homoserine lactones (Gram-negatives)
- generated and sensed by bacteria, lipophilic so can diffuse out of cell
- if P. a mutants are unable to make homoserine lactone --> defective in biofilm formation
Short peptides (Gram-positives)
- produced intracellularly, longer precursors are then modified as they're pumped out of cytoplasmic membrane
- signaling peptides are taken into producer cell or other cells in vicinity by peptide transporters
50
Two-Component signal transduction system mechanism of regulating transcription (P. aeruginosa)
In absence of phosphorylated response regulator, unstable recognition of promoter -> no transcription of biofilm genes
In the presence of phosphorylated response regulator --> binds to DNA just upstream of RNAP and stabilizes RNAP binding to the promoter.
51
Crowded vs sparsely populated environments
In crowded environments --> lots of peptide --> pathogenic pathway genes turned on
In sparely envio --> peptides away from bacterium --> will not turn on bacterial genes
52
quorum sensing
Cell is sending out a signal that it then monitors. The signal can also be sensed by other bacteria of the same or related species.
In some cases --> inducer must be acted upon by some environmental factor before it can be sensed by the cell.
Other cases --> signal sent out is sensed without alteration. Cell is simply monitoring the concentration of the compound in the environment, not whether or not the inducer has been modified.
Sensing the concentration of a compound that is secreted by cells is called “quorum sensing.”
53
What are two features that distinguish Gram-positive from Gram-negative bacteria?
Gram-negative bacteria have an outer membrane and a periplasmic space, neither of which are found in Gram-positive bacteria.
54
Why is LPS not considered a classical toxin? How does it trigger toxic reactions?
The outer membrane, known as LPS or endotoxin. It is not a classical toxin because it is not a protein and does not have enzymatic activity. It causes a toxic reaction because when administered in high doses, it causes an outpouring of inflammatory cytokines.
55
What is a biofilm? Does their formation at infected sites affect treatment efficacy?
A biofilm is an architecturally complicated structure consisting of thick layers of the extracellular polysaccharide matrix in which the bacterial cells are embedded. Biofilm infections are particularly difficult to treat and often require physical removal for cure.
56
Two component regulatory systems are ubiquitous in bacteria. What are the two components and what do they do?
The component that senses the environment is a sensor histidine kinase. The sensor domain of this kinase is in the extracellular space, where it can receive environmental signals. The kinase domain is in the intracellular space, where it can phosphorylate a response regulator (2nd component of the two component system). The response regulator controls the expression of many genes and its DNA binding activity is altered by phosphorylation.
57
RNA viruses
Picornaviruses and Myxoviruses
58
DNA viruses
Papillomaviruses
59
retroviruses
HIV
60
Acute flaccid myelitis (AFM)
Rapid onset of flaccid weakness in one or more limbs and distinct abnormalities in spinal cord grey matter on magnetic resonance imaging (MRI)
Causing paralysis in children
* AFM cases began surging at the same time as a widespread outbreak of Enterovirus D68 was causing sever respiratory illness (2014)
* Related to Poliovirus (ie, both are Picornaviruses)
* Similar late summer, early fall spikes in incidence
61
Mammallian gene expression
1. transcription
2. capping and polyadenylation
3. splicing
4. transport to cytoplasm
5. translation
Proteins have signals that get them where they need to go
- Signal peptide targets the nascent polypeptide to the ER, where it gets translated and trafficked for secretion
- A trans-membrane sequences or modifications target proteins to membranes
62
SUMMARY: KEY FEATURES OF HUMAN CELLS THAT IMPACT VIRUS REPLICATION
1. Division of cells into membranous compartments (Nucleus, cytoplasm, ER)
2. Human genes are divided into exons and introns
3. Human mRNAs are monocistronic: one mRNA --> one protein
4. Translation can occur at the ER so that proteins are either secreted or embedded in the membrane
63
Virion
the physical particle, infectious or transmissible form, but cannot replicate
What gets delivered to target cell for infection. NO replication on its own
64
Replicative form
the viral genome, structural proteins, and enzymes necessary to generate new virions inside the host cell
What exists inside host cell
65
VIRION --> REPLICATIVE FORM --> MORE VIRIONS
^steps
* Identify permissive species and cell
* Cross the cell membrane
* Release genome, and get it where it needs to go
* Replicate the genome using viral and/or cellular polymerases (RNA virus MUST encode its own polymerase)
* Translate viral genes ALWAYS using cellular ribosomes
* Release new virions from cell
66
ALL VIRIONS HAVE 3 ESSENTIAL COMPONENTS
Capsid: ordered array of proteins that provides the structural integrity
Ribonucleic protein:
• Nucleicacidgenome: (+/-) RNA or DNA (never both), ss or ds
• Protein: aids in folding/condensation of the genome
(often a chaperone)
Enzymes: often polymerase, integrase
(some virons also have envelope (membrane derived from host cell)/envelope glycoprotein (glycosylated, often heavily, protein embedded in the envelope)
67
Papilloma Virus
Non-Enveloped virus
Capsid
dsDNA genome
68
Influenza
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Enveloped virus (host-derived membrane)
Genome (-) ssRNA
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69
VIRAL STRUCTURAL PROTEINS FORM HIGHLY SYMMETRIC PROTEIN LATTICES
Because...
1. limited coding capacity. Can use single gene, generate many copies over and over again to assemble into that particle.
2. strength: must form so encapsidates the genome, but tight enough to endure extreme conds (exp. surviving strongly acidic stomach)
70
VIRUSES CAN EXHIBIT TWO DISTINCT SYMMETRIES
Cylinrical (screw)
| Icosahedral (spherical)
71
Cylindrical symmetry
Exp. Ebola. capsid can form branches and knots
- Virions can incorporate any amount of volume—and potentially a variable amount of genetic material—by varying the length of the cylinder
• Virions can vary in size
72
ICOSAHEDRAL SYMMETRY
- Virions incorporate a fixed volume, and a fixed amount of genetic material
- Virions are highly homogeneous
73
HOW TO PRODUCE DIFFERENT VIRAL PROTEINS? (deal with 1 mRNA: 1 protein rule)
- Synthesize polyprotein off of one mRNA
- Protein is segmented
- Nested mRNA's
- Differential splicing
74
VIRUS REPLICATION
Attachment
• Interaction between virus protein and cell surface receptor.
Penetration
• Receptor-mediated endocytosis or direct interaction at the cell surface. • Fusion of virus and cell membranes.
Uncoating
• Release of nucleic acid (+/- enzymes) for expression.
Transport to Site of Replication Early Events
• Depend on virus group.
• Prepare for nucleic acid replication.
Late Events
• Nucleic acid replication. • Virion protein synthesis. • Virion assembly and release.
75
VIRUSES TAKE ONE OF TWO ROUTES INTO THE CELL
| which do enveloped/non-enveloped take?
1. Enter at membrane and meets receptor -->. RME --> from endoscope, releases genome into cytoplasm (some enveloped and ALL non-enveloped enter this way)
2. Enter at cell membrane and fuse at surface (enveloped viruses ONLY)
76
Retrovirus (+RNA)
| exp. HIV
Needs to be reverse transcribed into DNA.
Genes become a permanent fixture in the cell.
Infectious bc needs splicing
RNA-dependent DNA polymerase
77
Late events
Budding and release
78
E. coli 0157:H7
Pathogen whose virulence factors are carried on lambdoid phage
Gene product: shiga-like toxin
Phenotype: hemorrhagic diarrhea, hemolytic uremic syndrome
Severity of the illness increased w/ admin of certain antibiotics that target and damage bacterial DNA ( --> induction of prophages and massive rise in transcription and expression of the stx toxin genes)
79
How do dsDNA viruses replicate their genomes?
| When are host enzymes used, and when are are viral enzymes used?
early mRNA synthesis --> host RNAP
Viral protein synthesis --> host ribosomes
exp. papillomavirus (genome infectious)
80
How do +ssRNA viruses replicate their genomes?
| When are host enzymes used, and when are are viral enzymes used?
viral protein synthesis --> host ribosomes
Exp. picornavirus (polio) (genome infectious)
81
How do -ssRNA viruses replicate their genomes?
| When are host enzymes used, and when are are viral enzymes used?
mRNA synthesis --> virion enzyme (transcriptase)
protein synthesis --> host ribosomes
Exp. Myxovirus (influenza) (genome not infectious)
82
How do proviruses (+ssRNA) replicate their genomes?
| When are host enzymes used, and when are are viral enzymes used?
synthesis of viral DNA --> virion enzyme (reverse transcriptase)
Exp. Retrovirus (HIV) (genome not infectious)
83
How do reoviruses (+/-ssRNA) replicate their genomes?
| When are host enzymes used, and when are are viral enzymes used?
Synthesis of mRNA--> virion enzyme (transcriptase)
Viral protein synthesis--> cell ribosomes
Exp. reovirus (genome not infectious)
84
Picornaviruses
Small non-enveloped (+)RNA viruses (pico-RNA-virus)
Exp. Common cold (Rhinovirus)
* Hepatitis A (HAV)
* GI disease (Coxsackie virus and others)
* Heart disease (Cardiovirus)
* Foot (Hoof) and mouth disease (FMDV)
* Sever respiratory infection, and AFM? (Enterovirus D68)
* Poliomyelitis (Poliovirus)
85
Picornavirus Structure and Genome
Non-enveloped,
Icosahedral
60 copies of 1 protein complex forms capsule
*~1 ribosome
Genome: 7-8.8 kb, single-stranded
(+)-sensed RNA
* capped with VpG (all proteins translated at same time)
86
Translation of Picornavirus proteins
Once in cell, immediately recognized and bound by host ribosomes (independent of 5' mG)
87
Picornavirus Polyprotein Cleavage
Polyprotein is cleaved by viral proteases (2A and 3C) into 11-15 individual proteins
*circumventing 1mRNA:1protein rule
88
Picornavirus genome Replication
Picornavirus 3D polymerase --> both + and - RNA
89
Where do picornaviruses assemble
Cytoplasm (only +RNA is encapsidated)
90
Picornavirus genome is transcribed and replicated by...
viral polymerase
91
Myxovirus surface proteins
Subdivided by antigenicity of the surface proteins Hemagglutinin (HA) and Neuraminidase (NA) into serotypes: H1N1,H3N2, etc.
92
Myxovirus STRUCTURE AND Genome
Genome: 12kb, segmented -ssRNA.
No poly(A) tail or 5’m7G cap (not recognizable to cell)
Enveloped, cylindrical capsid,
*variable in size
93
Myxovirus - what is present with genome
Polymerase, neuraminidase, envelope (derived from host cell) 3 glycoproteins: hemagluttanin and neuraminidase, and M2 ion channel.
94
INFLUENZA ENTERS CELL THROUGH ENDOCYTIC PATHWAY
1. Attachment (hemagglutinin and sialic acid)
Receptor-mediated endocyticis
Acidification of the endosome triggers fusion of viral and cellular membranes
Fusion releases genome into the cytoplasm
RNA genome is transported
to the nucleus
95
MYXOVIRUS transcription and genome Replication
Capped used as primer but also not
96
viral genomic -RNA in RNP
import signals
97
Where are new visions assembled?
plasma membrane
98
Antigenic shift
Cell becomes infected with multiple different strains
Genome segments shuffle, generating many different variants
99
Antigenic drift
Mutations arise randomly --> loss or gain of fitness, and changes to antigenicity
100
What determines strain of myxovirus
Hemagglutinin (HA) and Neuraminidase (NA) --> found on the surface of the virion, determine the serotype
101
Myxoviruses
- 1918 influenza epidemic
| - recent H1N1 pandemic
102
Papillomaviruses
Cause of various diseases, predominantly warts, of humans and other animals
Pervasive.
All replicate in stratified epithelium (skin or mucosa) coordination with epithelial differentiation
Genital HPV --> most common STD
Most cervical cancer --> caused by HPV 16 or 18
103
HPV Structure and Genome
Non-enveloped, icosahedral
Genome: 8kb, circular dsDNA
Organized into chromatin using cellular histones (5 early (E) genes; 2 (L) late genes)
104
Where do papillomaviruses replicate?
turatigied epithelium (attach to HSPG on basement membrane, exposed at sites of trauma
105
PAPILLOMAVIRUS ENTRY INTO CELLS
Internalized via RME --> late endoscopes (sim to picornaviruses, but capsid disassembles
106
All enveloped viruses enter cells via
RME
107
Gene expression of papilloma viruses is tightly coupled to ...
differentiation state of epithelial cells
Early synthesis of E1 & E2 mRNA using cell-specific promoter @ differential splicing
E2 --> master regulator
108
Which binds p53
HPV E6
109
VIRAL DNA GENOME Replication
- maintained as plasmid
- E1/E2 recruit DNA rep machinery
- replicated as tho DNA
110
HPV late gene expression
occurs only in terminally differentiated keratinocytes
- L1/L2 transported back too nucleus where new visions are formed
111
Assembly of papillomavirions
Virions do not lyse the cell. They are released by normal ketatinocyte death and loss of membrane integrity
112
HPV16,18 Transformation in Cervical Epithelium
Selection of cells containing integrated E6 and E7, but not E2 production in transformed cells)
113
Retroviruses
Widespread in humans, all other vertebrates, and even some invertebrates
Human retroviruses include HIV, HTLV, Foamy viruses
Cause a variety of diseases: Cancers, Immunodeficiencies (AIDS), others
Transmitted by close contact: Sexual, Mother to Offspring, Blood
114
All retroviruses replicate through ...
a DNA intermediate (provirus) integrated into the host genome
Can also integrate into germline DNA to become inherited (endogenous) proviruses, which constitute about 8% of our genome
115
Retrovirus Structure and Genome
Enveloped, gag poly protein forms vivrion.
Genome: +ssRNA, 7-10kb
always gag, pol, and env
116
gag:
pol:
env:
(all retroviruses have these)
gag: structural protein. Forms virion
pol: viral enzymes: protease (PR), reverse transcriptase (RT), and integrase (IN)
env: envelope glycoprotein
117
retrovirus irion must contain the viral enzymes
RT, PR, IN
+ sense RNA but not readily translated
118
Early events in the Retrovirus replication cycle
virus attaches to cell surface receptor
envelope glycoprotein fusion w/ PM
RT immediately upon entry
Integration into host genome
119
Retroviral reverse transcription (DNA Synthesis)
Carried out by viral reverse transcriptase
Synthesis of (-)DNA to 5’ end using host tRNA primer
Transfer of new DNA fragment to 3’ end with continued synthesis of (-) strand
Completion of (-) and (+) strands of DNA
Completion of synthesis with U3RU5 as long terminal repeat (LTR). Transfer to nucleus
120
Retroviral reverse transcription (DNA Synthesis)
5’UTR --> R, U5… 3’ UTR --> U3, R
| After reverse transcription --> ID regions 5’ and 3’ *imp for integration
121
Retrovirus DNA Integration
Integration into (more or less) random sites of cellular DNA
Carried out by viral integrase
122
Retrovirus Provirus Expression
Viral genes are expressed as though they were host genes
Unspliced mRNA is used to express Gag and Pol
Env mRNA requires splicing*
Simple retroviruses require cell division
Complex retroviruses (HIV) traffic mRNAs out to cytoplasm
123
LATE EVENTS IN RETROVIRUS REPLICATION
ASSEMBLY AND RELEASE
- viral proteins (ER --> PM)
- viruses released
- oncogene --> alters shape
MATURATION: via gag & pro cleavage )via viral protease)
124
Retroviral oncogenesis: 2 mechanisms
1. Integrates upstream of protooncoge – LTR can deregulate expression of proto-oncogene *rare
2. Only when retrovirus contains oncogene in its genome (any cell infected with retrovirus becomes cancerous)
125
Features of malignant transformation (retrovirus)
Can transform and replicate in same cell
Integration is part of normal replication cycle
Oncogene is normal cell gene modified by the virus
Oncogene can be either in virus or cell genome
Oncogene product is part of normal signaling pathway
Many of the same oncogenes are altered by mutation in no-viral human cancers
126
Features of malignant transformation (papilloviruses/other DNA viruses)
Transformation leads to loss of virus production (only integrating pt of viral genome)
Integration is a rare chance event, and not part of normal virus life cycle
Viral oncogenes are also required for replication
Oncogenes are viral genes
Oncogene product interacts with members of the pathway that stimulates cells into S-phase DNA synthesis
127
Simple retrovirus (MLV)
gag: structural protein. Forms virion
pro: protease
pol: reverse transcriptase (RT) and integrase
env: envelope glycoprotein
128
Complex retrovirus - HIV-1
gag: structural protein. Forms virion
pro: protease
pol: reverse transcriptase (RT) and integrase
env: envelope glycoprotein
+ 6 adding gene products: counteract innate immune response, and nuclear import/export of viral nucleic acids that allow the virus to constantly replicate
129
HIV replication cycle
Attachment: env and CD4, 2 possible coreceptors: CCR5 or CXCR4
coreceptor triggers fusion --> capsid release into cytoplasm
Reverse transcriptase (via viral reverse transcriptase)
Integration ("randomly") in genome
mRNA synthesis as usual
virus assembly at PM
budding --> *need maturation before becoming infectious (cleavage --> Gag, Pro, and Pol)
130
HIV Env protein
SU (gp120)
- binds membrane receptor
TM (gp41)
- Responsible for fusion
HEAVILY glycosylated --> 50% molecular weight is carb
^ carried out by host enzymes in ER as envelope forms
131
Extensive genetic variation in HIV is due to...
Error-prone RT and constant replication --> mutations accumulate quickly --> genetic variation of the virus and to eventual loss of target CD4+ T cells, inevitably leading to AIDS
Minority of cells enter a resting state, which can last for decades, foiling efforts to cure the infection
132
Typical course of HIV
Primary infection usu single infectious event
-Rapid proliferation of virus, and corresponding loss of CD4+ cells. Flu-like symptoms
```
Chronic infection (few wks later)
-Partially effective immune response reduces viral load down to a set point, giving rise to clinical latency, which can last for several years. All the while, virus is constantly replicating, and CD4+ cells are depleting gradually.
```
In total, 10^8 – 10^9 infected CD4+ cells --> killed. At ~200 cells/ul --> progression to AIDS occurs, sx return, massive virus proliferation, opportunistic infections, death
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Best predictor of progression to AIDS
Plasma viral load is a better predictor of progression to AIDS and death than was the number of CD4+ cells
Why? A minority of infected cells survive for extended periods, becoming “latently infected”. Not necessarily producing virus. Only the cells where active replication is occurring --> AIDS
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Antiretroviral therapy (ART)
reduces this HIV replication >10,000 fold, but is not curative
(stably integrated provirus is not affected by antivirals. stable proviral intermediate, which can exist in long-lived memory CD4+ T cells creating a reservoir of provirus)
Virus load and infected cells persist at low levels (ie, below detectable levels in standard assays)
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Most HIV drugs target...
Target RT itself (RT is specific to HIV, not host)
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Virus Evolution in the Host and drug therapies
Under mono-therapy, the virus load is reduced to some extent. But pre-existing resistant mutants are selected for.
With a combination of three drugs, a virus that is resistant to all three drugs is extremely unlikely
* Drugs prevent new infections, but do nothing about existing infected cells. Stable provirus still exists in latently infected cells
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It has been found is that patients on ART often have clusters of identical virus sequences. What happens to the diversity?
Clonal proliferation of latently infected cells, despite effective inhibition of viral replication.
Means that the latent reservoir can be replenished, or potentially expanded, despite effective ART.
Only a small fraction of the proliferated clone may be producing virus at any one time
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Appearance of 3TC-Resistant Mutations
| in Treated Patients
Within the first couple weeks of treatment, a typical mutation occurs at position 184 of RT, which is in the active sight of the enzyme
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Virus load and infected cells persist at low levels (ie, below detectable levels in standard assays) , why?
Persistent low-level of virus is due to latently infected memory CD4+ T cells that periodically become reactivated, start producing virus, and die.
Upon removal of therapy, viral load rebounds in weeks
Quiescent infected CD4 cells can last for years
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HIV replication cycle
```
Reverse transcription,
integration,
transcription + splicing,
translation,
assembly,
budding,
maturation via protease
```
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Picornavirus Summary
ss(+)RNA genome that is translated immediately upon entry into the cell
Translation is initiated by 5’ cap-independent mechanism
A single mRNA is translated to form a polyprotein, which is cleaved into individual functional proteins
Genome is transcribed and replicated by viral polymerase
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Myxovirus Summary
ss(-)RNA genome, where each segment encodes a different protein
Enveloped virion that enters cells by fusing its membrane with the cellular endosomal membrane
Hemagglutinin (HA) and Neuraminidase (NA) are found on the surface of the virion, and determine the serotype
Virus diversity and evolution occurs through anitgenic drift and antigenic shift
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PAPILLOMAVIRUS Summary
Double stranded circular DNA genome
Replication in differentiating keratinocytes
E2 is the master regulator of transcription
Cellular transformation through activities of E6 and E7
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RETROVIRUS Summary
ss(+)RNA, but is not readily translated immediately upon entering the cell
Enveloped virion that enters through fusion with the plasma membrane
Genome is reverse transcribed by viral polymerase (reverse transcriptase) immediately after entering the cell
Replicates through a DNA intermediate (provirus) that is integrated into the host genome
Gag and Pol are translated as polyproteins, and cleaved by viral protease during maturation
