EXAM II Study Guide Virus/Phage + DNA + Immunology + Pathogenicity

VIRUS/PHAGE
Define viruses and phages. Differentiate Viroid, Prion, GTA
Virus

Genetic element containing either RNA/DNA surrounded by a protein capsid and that replicates
only inside host cells

Phage

Virus that infects only bacterial cells

Viroid

Naked molecules of RNA important infectious disease agents in plants

Prion

An infectious agent composed only of protein that is responsible for causing a variety of
spongiform encephalopathies (e.g., scrapie).

GTA*

Phage-like element produced by several bacteria that mediates horizontal gene transfer

*Gene Transfer Agent

Which of these is not UV sensitive?

UV sensitivity: Bacteriophage > Bacterium > Gene > Prions

Estimate the abundance of viruses in aquatic systems and postulate the significance of these pathogens in
terms of controlling primary productivity and horizontal gene transfer.
A. 106 - 109 ml-1 in seawater
B. Control 1 productivity: Each infection has the potential to introduce new genetic information into an
organism or progeny virus, thereby driving the evolution of both host and viral assemblages
a. Viruses kill ~20% of biomass/day
Contrast viruses with bacteria in terms of genome structure, metabolic activity, life cycle and size (in nm).
Bacteria

Virus

Cell wall

Peptidoglycan/LPS

No cell wall; protein coat

Reproduction

Fission; asexual

Lysogenic; lytic

Cellular organization

Complex

Simple

Ribosome

Enzymes

Some

DNA/RNA

Both DNA/RNA

DNA OR RNA

Life cycle
Size

Some obligate IC parasites Obligate intracellular parasites

1-5 m

10-400 nm

Viral Genome

Structure
o

Genetic information may be coded as DNA or RNA and in double-stranded or single-stranded form.

The viral genome may contain exotic bases.

Replication
o

Takes place only after successful infection of an appropriate host.

Proceeds when the injected viral genome subverts normal replicative processes of the host,
producing new virus particles.

Bacteriophage types
o

May be discerned by their mode of propagation.

Lytic phages quickly produce many copies of themselves as they kill the host.

Temperate phages can lie seemingly dormant in the host (prophage state), timing replication of
prophage genetic material to replication of the host cell. Various activation signals trigger the
prophage to enter a lytic cycle, resulting in host death and the release of new phages.

Sketch these viral morphologies:

Membrane = Peplomeres (spikes)

Virus
Morphology
TMV (Tobacco Mosaic Virus)
Helical
T4 Phage
Complex
HIV
Enveloped
HSV
Enveloped
Polyomavirus
Icosahedral

Explain the role of reverse transcriptase, RNA replicase, holin and endolysin in the life cycle of viruses and
phages.
Enzyme

Role

Type of Phage

Reverse transcriptase Generation of cDNA from RNA; integration of retrovirus into host
RNA Replicase
Holin
Endolysin

Retrovirus

Catalyzes replication of phage RNA in host

Lysogenesis

Host lysis; produces lesions in cell membrane

T4 phage protein

Attacks peptidoglycan

T4 phage protein

Contrast the biology of common phages:

Virus

Genome

Transmission
Cycle

Mode of Transmission

Morbidity

Mortality

Polio

+ ssRNA

Non-human
host

Fecal - oral

Endemic Pakistan

< 1%

WNV

+ ssRNA

Vector borne
zoonotic

Arthropod vector

Differential

4%

Ebola

- ssRNA

Zoonotic

Contact

Endemic (Africa)

High (50 90%)

influenza

- ssRNA
sgmtd

Zoonotic

Aerosols
Humidity

50,000/year; seasonal

Adults: 5-10%
Kids: 20-30%

HIV

retro

Human

STD

---

100% w/o therapy

HSV

dsDNA

Human

STD; mother fetus

50 60 million US

Low

smallpox

dsDNA

Zoonotic

Contact

eradicated

High

Discuss how the above examples maintain virulence and evade specific immunity through antigenic drift
and shift.
A. Antigenic drift: natural mutagenesis of viruses as viruses replicate results in altered antigenic profile
B. Antigenic shift: segmented genome of select viruses (influenza) allows reassortment of different
strains; occurs through infection of same host cells and new-intermixed viral genome
Sketch lytic and lysogenic cycles using lamba phage as an example.
Lytic: phage life cycle that culminates with host cell bursting, releasing virions
Virulent phages: phages that lyse their host during the reproductive cycle i.e. T4 Phage

(1) Early mRNA synthesis (2) Synthesis of proteins that (3) Enable T4 to take over host cell (4) Phage
DNA replication (5) Late mRNA synthesis (6) encode capsid proteins and other proteins needed for
phage assembly

Lysogenic: Nonlytic relationship b/t phage-host; integration of phage genome into host DNA

Defined terms:
a. Prophage integrated phage genome (lysogeny)
b. Temperate phage phages able to establish lysogeny
c. Induction An event in the life cycle of some viruses (e.g., temperate bacteriophage) that results in
the provirus initiating synthesis of mature virions and entering the lytic cycle.

Explain why stress triggers induction events

Triggered by drop in levels of lambda repressor caused by exposure to UV light and chemicals that
cause DNA damage
a. Catalyzed by excisionase: binds integrase which enables integrase to reverse integration process

DNA/RNA
Describe these key experiments that elucidated the central dogma of molecular biology in terms of
objective, methods, results & conclusions:
a. Gliffith (1928), Avery (1944)
i. Griffith: Transformation; Dead S (infectious) + Live R strain injected Mouse dies (live S strain
isolated); conclusion: hereditary element can be transformed from dead to live cells

ii. Avery: Protein not transforming agent; R & S culture PRO/lipid removed transforming
element had DNA chemistry (destroyed by DNAse)
b. Heshey & Chase (1952)
i. DNA injected by viruses; bacteriophage infected cells had radioisotope labeled DNA (phosphorus) +
protein (sulfur) agitation/centrifugation revealed only P isotope still present in cells
c. Fraenkel-Conrat (1957)
i. NA (RNA), not protein coat is infective agent; Tobacco infected with hybrid: TMV protein coat and
HRV RNA observed lesions characteristic of HRV
d. Watson & Crick and Meselson & Stahl (1958)
i. Double helix 2 strands (parental & complementary)
ii. Meselson & Stahl radioisotope labeled DNA revealed semi-conservative replication in DNA
Identify the sugar in nucleic acids and features associated with I ', 2', 3' and 5' positions

Explain difference In strength of G-C pairing versus A-T.

A. GC capable of 3 H bonding
Contrast the structure(s) and function(s) of DNA & RNA including mRNA, tRNA & rRNA

Describe how these enzymes: gyrase, DNA polymerase, helicase and ligase, contribute to DNA replication
A.
B.
C.
D.

Gyrase: Remove supercoil

DNA Polymerase: polymerizes dNTP in 5 3
Helicase: Unwinds double-helix
Ligase: Fuses okazaki fragments

Describe polypetide synthesis using the terms transcription and translation.

RNA polymerase (a large multi-subunit enzyme) is responsible for the synthesis of RNA
The core enzyme (2, , ' subunits) catalyzes RNA synthesis
The sigma subunit () helps the core enzyme bind DNA at the appropriate site

 sequences centered at -35 and -10 BP before the transcription starting point are important in directing
RNA polymerase to the promoter
Prokaryotic mRNA can code for one polypeptide (monogenic) or many polypeptides (polygenic)
Leader sequences consist of 25 to 150 bases at the 5 end of the mRNA, and precede the initiation
codon
Spacer regions separate the segments that code for individual polypeptides in polygenic mRNAs
Trailer regions are found at the 3 end of the mRNA after the last termination codon
Identify where sigma factors bind to activate transcription and explain their role stress response and
antibiotic resistance.
A. Binds to RNA polymerase @ specific promoter sequence; each

factor binds to unique sequence

Sketch a biphasic growth curve using the example of diauxic growth observed when E. coli is grown on
glucose and lactose as sole carbon sources.

 Discuss transcription regulation in this lac operon example using these terms: cAMP, promoter,
repressor, polygenic and inducer.
Discuss how eukaryotes and bacteria generate genotype and phenotype diversity.

Explain degeneracy and wobble in the genetic code and how point mutations and frame-shift mutations in
DNA alter polypeptide sequences or, in the case of silent mutations, don't.
Define wild type, prototrophs and auxotrophs to explain how the Ames test works and how horizontal gene
transfer (MGT) was quantified Lederberg and Zinder (1951).
a. Auxotroph requires an organic growth factor
b. Prototroph can synthesize all necessary growth factors

Using the terms pili, cell-to-cell contact, prophage, lytic phase and phage- like particles, contrast
conjugation, transformation, specialized transduction, generalized transduction and gene transfer agents.

Conjugation

Define plasmids, episomes and conjugative plasmids.

Plasmid

Small replicons, double-stranded, usually circular DNA molecules; have their own origin of
replication; can exist as single copies or as multiple copies

Episome

Plasmids that can exist either with or without integrating into chromosome

Conjugation

The form of gene transfer and recombination in procaryotes that requires direct cell-to-cell
contact

Conjugative
Plasmids

Have genes for pili (ex F factor in E. coli); can transfer copies of themselves to other bacteria during
conjugation

Elimination of plasmid; spontaneous or induced by treatments that inhibit plasmid replication but not
host cell reproduction

Curing
F Factor

F plasmid; the fertility factor; a plasmid that carries genes for bacterial conjugation and makes its E.
coli host the gene donor during conjugation

Generalized
Transduction

The transfer of any part of a prokaryotic genome when the DNA fragment is packaged within a virus
capsid by mistake

HGT

The process by which genes are transferred from one mature, independent organ- ism to another

Prototroph

A microorganism that requires the same nutrients as most of the members of its species

Specialized
transduction

A transduction process in which only a specific set of bacterial or archaeal genes is carried to a
recipient cell by a temperate virus

Transduction

The transfer of genes between bacterial or archaeal cells by viruses.

Transformation
Transposition

Mode of gene transfer in prokaryotes in which a piece of free DNA is taken up by a cell and stably
maintained
The movement of a piece of DNA around a cell's genome. transposon A mobile genetic element that
carries the genes required for transposition

Explain relationship between methylation of cytosine in viral DNA replication and restriction enzymes.
Contains hydroxymethyl-cytosine (HMC) instead of cytosine
o Synthesized by two phage encoded enzymes
Protects phage DNA from host restriction endonucleases
Restriction defends against viral infection
Contrast restriction enzymes and CRISPR as bacterial defenses against phages.

Insertion Sequences
a. Short DNA sequences (600 3,000 bp)
b. Carry transposease

Transposons
a. Known for carrying antibiotic resistance
b. Include conjugative

CRISPR (clustered regularly interspaced short palindromic repeats)

a. Bacteria and Archaea not only produce restriction endonucleases (Sections 8.6 and 11.1) that
function to destroy incoming foreign DNA, they also have an RNA-based defense program to
destroy invading DNA from viral infection and sometimes conjugation. This type of prokaryotic
immune system helps preserve genome stability and is called the CRISPR system, which stands
for clustered regularly interspaced short palindromic repeats
b. DNA repeats spacing between DNA complementary to foreign DNA
c. Processed to generate crRNAs

The CRISPR region is transcribed as a whole into a long RNA molecule that is then cleaved in
the middle of each of the repeated sequences by the nuclease activity of Cas proteins. This
converts the long RNA molecule into spacer segments of small RNAs called CRISPR RNAs
(crRNAs)

2 Methods of Microbial Genetic Diversity

Mutation alteration in existing DNA sequence
DNA transfer (horizontal gene transfer) acquisition of DNA from another source
Discuss, citing Corvaglia et al. (2010), the importance of type Ill-like restriction enzymes in the virulence of S.
aureus through acquisition of multiple antibiotic resistance.

Explain how the roles of RecA and LexA, uvrD in the SOS response.

LexA: repressor; in absence of DNA damage LexA binds to operator

RecA: inducer; DNA damage activates; cleave LexA

UvrD: helicase; removes thymine dimer

Compare structure and organization of chromosomes and genes in archaea, bacteria and eukaryotes using
the terms intron, start amino acids, size (bp), circular/linear, haploid/diploid.

Explain the function of 5' cap and poly A tail in eukaryotic primary transcripts.

Protect from exonuclease activity in Eukaryotes

Capping is the addition of a methylated guanine nucleotide at the 5-phosphate end of the mRNA
a. The cap nucleotide is added in reverse orientation relative to the rest of the mRNA molecule and is
needed to initiate translation.

Poly A tail
a. The tail recognition sequence, AAUAAA, is located close to the 3 end of the primary transcript.
b. The poly(A) tail stabilizes mRNA and must be removed before the mRNA can be degraded.

Discuss how alternative splicing, insertion sequences, methylation and RNA interference contribute to
complexity of organisms.

 Alternative splicing: # of introns; increases w/ complexity of organism

Insertion sequences: Carry transposease (transposons); most bacteria have few; increase variability of
organisms (antibiotic resistance)
RNA interference
Explain why obligate mutualist bacteria generally have small genomes and few insertion sequences.

No selective pressure to undergo evolutionary beneficial adaptations

Describe in general terms nonspecific responses to infection.

Innate Immune System (PAMPs)

a. Cells: Macrophages + Neutrophils + NK cells
b. Proteins: Complement (opsonization; c3b) + Interferons
c. Systemic: Inflammatory + Temperature
i. Cytokines: Proinflammatory (IL-1 + TNF- + IL-6)

Discuss why these PAMPs illicit a general immune response by binding PRRs and identify the class of
microbes associated with them: LPS, teichoic acid, peptidoglycan, chitosan, CpG DNA (methylation
patterns), SS DNA and DS DNA.

PAMPs:

Class of
Microbes

PAMP

PRR (Patern
Recognition
Receptor)

Result

Gram -

LPS

TLR-2/4

Phagocyte activation + inflam

Gram +

Teichoic Acid

CD14TLR-4

Phagocyte + inflam

Gram -/+

Peptidoglycan

TLR-2; NOD1/2

Phagocyte activation + inflam; Antimicrobial

peptide production + proinflamm cytokines

Fungal cell
wall

Chitosan

TLR-6

Phagocyte activation + inflam

Bacteria

CpG DNA
(methylation
patterns

TLR-9

Phagocyte activation + inflam

Virus

ss DNA

TLR-7

Phagocyte activation + inflam

Virus

ds DNA

TLR-3

Phagocyte activation + inflam

Describe how these PAMPs stimulate systemic and local responses by stimulating the release of
endogenous pyrogens, particularly IL-I and TNF-a, or, for viral associated PAMPs, interferon.

Discuss evidence that interferon production interferes with viral replication.

Contrast the three pathways of complement activation and explain the central role of C3.

Explain these effector roles of complement:

Inflammation (C3a & C5a)

Lysis (membrane attack complex

Opsonization.

Present clonal selection theory to describe how the acquired immune system improves with repeated
exposure, using the terms proliferation, differentiation, memory cells, somatic hypermutation and class
switching.

Proposed by Niels Jerne (1955)

All mature B cells have the genetic information to respond to any single antigen

Specificity idiotype and antigen

Population of mature B cells is a library of cell lines, each cell line responds to a single antigen

Binding of antigen by a cell line results in proliferation

a. Plasma cells
b. Memory cells

Describe the roles RAGI/RAG2, AID and Xboxl in primary and secondary immune responses.

RAGI/2 recombination of B/T cells from same parent genome (bone marrow) to produce specific
antibody isotype (IgG, IgM, IgA, IgD, IgE)

XboxI switch recombination IgM (isotype)

Clonal selection. During B-cell differentiation, V(D)J recombination generates a primary antibody
repertoire that is displayed on the surfaces of immature B cells. Cells that fail to bind foreign antigens
or that bind to self-antigens die. Cells expressing antibodies that bind to foreign antigens then undergo
selective expansion and further differentiation via somatic hypermutation of the antibody variable
regions. Iterative rounds of mutation and selection lead to the clonal expansion of an activated B-cell
population expressing antibodies with the highest affinity for foreign antigen. Activated B cells
eventually differentiate into plasma cells, which synthesize secreted antibodies, or into memory B cells.

Explain the role of MHC in antigen presentation as a link between innate and acquired responses.

PAMPs activate neutrophils, M and DC

a. DC cells ingest, mature and home to lymph nodes
b. APC process and present on MHC II to CD4+T cells
i. MHC II Expressed on APCs Present endogenous antigens to T-helper (CD4+) cells

B cells activated
a. Crosslinking w/ antigen

Complement products lowers threshold C3dg CR2

(CD21)
a. C3B C3dg (inactivated derivative of opsonization
protein)
b. Multivalent antigens

Immune synapse w/ T-helper cells (CD4+) , cytokines and

PAMPs induce: proliferation + somatic hypermutation +
class switching

Memory B and T cells generated, which confer long-term immunity

Describe the role of macrophages, neutrophils, dendritic cells and in the immunes system.

Macrophage: phagocytosis

Neutophils: phagocytosis

Dendritic: antigen presentation

Describe two mechanisms of activating natural killer cells.

Nonspecific activation signal on target cell

Absence of MHC I (endogenous) marker on target cell for NK cells

Cell-mediated CD16 binds to Fc region of Ig (+) NK cells

Differentiate MHC class I and Il in terms of endogenous'exogenous peptides and CDS & CD4 positive cells.

Rule of 8

MHC I = CD 8+ = endogenous = T killer

MHC II = CD4+ = exogenous = T-helper

Differentiate the two arms of acquired immunity, humoral and cellular, in tenns of cells (B and helper and
cytotoxic T) involved.

Define antigens, epitopes and haptens. Sketch the basic structure of antibodies including variable and
conserved regions and light and heavy chains.

Identify the structure and function of the five isotypes of antibodies: lgG, IgA, IgM, IgD and lgE.

Macro

Identify those found on mature B cells, where IgM functions as the B cell receptor, and produced following
activation.

IgD

Differentiate active and passive imtiation of specific Immune response, with natural and artificial examples.

Identify the isotypes (IgG and lgA) involved in natural, passive immunity acquis'tion by the fetus and
newborns. Describe how HIV evades the immune system.

List Koch's Postulates.

Pathogen present in every case of disease

Isolate pathogen and grow in pure culture

Isolated pathogen causes disease when inoculated into lab animal

Pathogen isolated from inoculated animal identical to original organism

Differentiate the terms disease and pathogen.

Contrast mutualism, commensualism and parasitism.

Define virulence or pathogen and lethal dose 50

Differentiate opportunistic and nosocomial infections.

Differentiate vectors and fomites.

Vectors living organism transmits pathogen

Fomites inanimate object that transmits pathogen

Explain the relationship between virulence, mode of transmission and reservoirs.

Virulence extent of pathogenicity

Discuss role of probiotics in prevention of disease including the role of attachment and competition for
space in establishment of infection.

Define these pathogens in these genera as either facultative intracellular, obligate intracellular, or
extracellular: Yersinia, Chlamydia, Vibrio and Pseudomonas.

Yersinia Facultative IC

Chlamydia Obligate IC

Vibrio - EC

Psuedomonas - EC

Compare exotoxins and endotoxins in terms of chemical nature, host responses, gram positive/negative,
heat stability and toxicity.

Contrast these exotoxin types: superantigens, type AB and cytotoxins.

Superantigens
Cause T cells to release cytokines
e.g., toxic shock syndrome
AB
diptheria
neurotoxins
target nerve tissue
e.g., botulinum toxin
enterotoxins
target intestinal mucosa
e.g., cholera toxin
cytotoxins

 target general tissues

Include hemolysin and membrane disruptors
Identify MHC and TCR bound by superantigens.

Contrast A and B proteins in AB-types.

A = active; effector

B = mediates attachment

Explain how pore-forming and phospholipases cause cytotoxicity.

Pore-forming
a. bind cholesterol
b. e.g., hemolysins, leukocidins

Phospholipases
a. destroy membrane integrity
b. e.g., gangrene toxin

Explain how siderophores and lgA proteases are virulence factors and help bacteria evade the immune
system.

Collagenase
a. Hydrolyzes collagen

IgA protease
a. Destroys secreted antibodies

sidephores
a. Take essential nutrient, iron, from host enzymes

Capsules
a. not antigenic
b. e.g., Streptococcus pyogenes

N. gonorrhea approaches
a. genetic variation of surface antigens
b. production of IgA proteases

Interfere with antibody-mediated opsonization

a. produce proteins that bind Fc portions of immunoglobins
b. Can bind macrophages as well as normal antibody response
c. e.g., Streptococcus pyogenes

Describe how type Ill secretion systems are acquired as pathogenicity islands and contribute to virulence.

Present evidence that capsules and fimbriae are virulence factors for S. pneumonia and E. coli respectively.