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Virologie > Virus genomes and viral replication strategies > Flashcards

Virus genomes and viral replication strategies Flashcards

1
Q

Viral life cycle - definitions

A

A cell is:
– susceptible for a virus if the virus can penetrate
– permissive for a virus if viral genome replication is possible (this does not necessarily mean that the virus can infect this cells under natural conditions)

Production of infectious virus particels in a cell defines a productive infection

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2
Q

Virus-cell interaction

A

Type of infection
 lytic
 persistent
 latent
 transforming
 abortive

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3
Q

Virus-cell interaction -> lytic

A

Host cell dies. The production of virus- descendents can be observed easily

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4
Q

Virus-cell interaction -> persistent

A
  • Continous production of viruses since
     cells survive the infection
     limited infection, with newly forming cells replacing cells dying from infection
  • Balance between virus and host
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5
Q

Virus-cell interaction -> latent

A
  • Few virus-proteins/RNAs are expressed
  • No infectious virus is produced
  • Well orchestrated molecular controls are necessary to maintain latent status
  • Often, a specific set of latency genes/RNAs is expressed
  • External stimuli drive reactivation
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6
Q

Virus-cell interaction -> transforming

A
  • As a result of infection with specific DNA- or RNA- viruses, some cells show increased cell division rates and a change in their „behavior“
  • Carcinogenesis is multifactorial
  • Viruses are involved in approx. 20% of human cancers.
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7
Q

Virus-cell interaction -> abortive

A
  • Although viruses can normally infect various cell types, as long as they present the proper receptor, the replication-efficiency may vary dramatically
  • Results in:
     reduced proliferation or
     poor virus/infection ratio
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8
Q

What is a virus?
-> Characterics of viruses

A
  • Very small, infectious, obligate intracellular parasites
  • Viral genome consists of DNA or RNA
  • In a suitable host cell the viral genome is replicated and determines the synthesis through virus-own or cellular components
  • New viruses are generated with new synthesized components within the host cell „de novo“
    -The viruses, which are synthesized in this replicative cycle are the vehicels for the transmission of the viral genom into the new host cell or organism, in which the uncoating of the virion and the next replicative cycle starts
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9
Q

Parts of a Virus

A
  • Capsid (Protein)
  • Envelope (lipids and proteins) -> facultative
  • Nucleic acid (RNA or DNA)
  • replicative proteins/accessory proteins -> facultative

Virus = cell free, protected nucleic acid

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10
Q

Reasons for Formation of Virus Particle

A
  • Closed capsid to protect genome
    for the time of transfer to new host (cell)
  • allows specific Entry of defined host (cells)
  • allows Assembly of specific genome
  • allows Budding at specific sites
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11
Q

Viral replication - basic principles

A

1) Virus entry
2) Uncoating
3) genome replication
4) Protein synthesis
5) Virus-assembly and maturation
6) Virus release

Viral protein synthesis only by host cell machinery

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12
Q

Virus structure

A
  • Enveloped (lipid-bilayer)
  • Entry/membrane fusion: Exit/„Budding“ through membrane (Cell membrane or ER or Golgi)
  • Non-enveloped
    Entry?
    Exit by cell lysis? Not always
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13
Q

Amplification Schemes of Bacteria and Viruses

A

Bacteria
- Lag-phase: Adaptation to culture conditions
- logarythmic phase: Amplification by division
- Stationary phase: Slow growth/
End of divisions due to limited nutrients or toxic metabolites

Virus
- Eclipse: Infektious particle invade and dissociate
- Burst: Release of hugh numbers of progeny per cell (1000-10000)
- Stationary phase: End of viral replication due to death of host cell, or limited
host factors

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14
Q

Tropism

A

human Hepatitis B Virus: infects only liver cells of humans and chimps
HIV: infects only CD4-positive cells of humans and chimps

Determined by:
- Receptors on cell surface (virus binding)
- Cell type-specific promoter-enhancer-elements (only DNA viruses)
- Host factors for entry, gene expression, assembly, transport
- Entry route in organism, kind of inoculation

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15
Q

Virus genomes code for gene products as well as informations for:

A
  • particle generation and genome packaging
  • genome replication
  • regulation of replication cycle
  • antagonists of cellular defence mechanisms - spread to other cells/organisms
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16
Q

Virus genomes do not code for:

A
  • protein synthesis machinery
  • enzymes of energy metabolism
  • factors of membrane biogenesis
  • telomers, centromers
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17
Q

Virus genomes display high variability

A
  • DNA or RNA
  • DNA or RNA with covalently bound protein
  • single strand: minus, plus or ambisense orientation
  • double strand
  • linear
  • cirkular
  • segmented
  • double strand DNA with gap

Nature of genome determines replication pathway

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18
Q

RNA virus genomes

A

Relicts from „RNA-world“?
- RNA primary form of replicating organic material?
- autokatalytic RNAs (ribozymes, e.g. Hepatitis delta virus)

Essential requirement:

Copy of genome without loss of information
Strategies:
- Virus encoded RNA-dependent RNA polymerase or reverse transcriptase
- RNA-elements control replication and transcription in cis
- de novo synthesis of RNA (initiation without „primer“; RNA promoter)
- Mechanisms for „priming“ (e.g. protein primer)

Generation of translatable RNAs (mRNA)
Strategies:
- Mechanismens for „capping“ and polyadenylation
- „cap snatching“
- IRES Elements

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19
Q

Classification of viruses: Genome

A

DNA-Viruses
- single stranded
- Double stranded

RNA Viruses
- positive stranded (=mRNA)
- negative stranded
- double stranded
- segmented or non segmented genome

Both
with or without envelope

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20
Q

Size of viral genome

A

Circoviridae 1,7-2,3 kb ssDNA
Coronaviridae 31 kb ssRNA
Mimivirus 1,2 mb dsDNA (isolated from an ameba)

Comparison: obligate intracellular bacterium Mycoplasma genitalium: genome size 582 970 bases (encodes 485 proteins)

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21
Q

Limiting factors for genome size

A
  • time required for genome replication
  • capsid size
  • RNA-stability
  • precision of replicase
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22
Q

Classification by Replication Strategy -> Pathways from genome to mRNA

A
  • serves as mRNA -> (+)-strand RNA viruses
  • Viral RdRp transcribes mRNA -> (-)-strand RNA viruses, dsRNA viruses
  • Viral RT transcribes genome into dsDNA and cellular DdRp transcribes mRNA -> Retroviruses
  • cellular DdRp transcribes mRNA -> ds DNA viruses, with exception of Poxviruses
  • Viral DdRp transcribes mRNA -> Poxviruses
  • Replication of genomes into ds DNA and transcription by cellular DdRp -> ss DNA viruses
23
Q

Replication strategy determines place of replication

A

Exception Poxviruses:
Replication in cytoplasm
(virus encoded replication enzymes)

RNA viruses depend on RdRp, has to be encoded by the virus, replication in cytoplasm

DNA viruses use cellular DNA-polymerases
replication in nucleus

Exception: Orthomyxo viruses, Bornavirus
Replicate RNA in nucleus,
make use of „Splicing“-machinery Retroviruses: Integration into cell genome

24
Q

Difference RNA dependent RNA Polymerase and DNA dependent DNA polymerase

A
  • RNA dependent RNA Polymerases (RdRps) prime at specific site without a primer
  • Most DNA-dep. DNA Polymerases (DdDps) prime via elongation of a primer

Exception to the rule:
Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers.

25
Q

Replication of DNA Viruses
-> The 5’ end problem
-> Two mechanisms of ds DNA synthesis

A

Replication fork -> RNA primers
- Papillomaviruses
- Polyomaviruses
- Herpesviruses
- Retroviral proviruses

Strand displacement (primer) -> never RNA primed
- Adenoviruses (protein)
- Parvoviruses (DNA hairpin)
- Poxviruses (DNA hairpin)

26
Q

DNA Viruses -> Double stranded (linear, circular)

A

circular: Polyomaviruses

linear: Adeno-, Herpes-, Poxviruses

27
Q

Circular ds DNA: Polyomavirus

A
  • replication starts at origin; similar to eukaryotic genome replication
  • genome is complexed with histones (mini chromosome)
  • temporally and quantitatively controlled gene expression
    E: early; L: late
  • model for oncogenesis („T-Antigen“) and transcription control („Enhancer“)
28
Q

SV40: Initiation of Replication

A

DNA replication highly similar to eukaryotic cell

  1. T-Antigen (T-Ag) binds as 2 hexamers in the presence of ATP; strand melting of A/T rich regions.
  2. In the presence of the heterotrimeric “replication protein A” (RPA; binds ssDNA) unwinding of DNA occurs (T-Ag-helicase) under hydrolysis of ATP.
  3. DNA Pol a/primase binds to each single strand and synthesizes short pieces of RNA serving as primers for DNA synthesis. In this process the interaction between Pola/Primase and T-Ag (as chaperon) is important. Pola/Primase are then replaced by replication factor C and “Pold complex”.
29
Q

SV40 elongation

A

Topo I, II and T-Ag unwind supercoil DNA.

Leading strand is synthesized by a complex of pold, replication factor C (RF-C), and proliferating cell nuclear antigen (PCNA).

Lagging strand: pola/primase synthesizes Okazaki fragments which are completed by pold, RF-C, PCNA. RPA keeps DNA in ss state. RNase H and FenI remove RNA primers, pold fills gaps and DNA ligase I ligates the fragments.

30
Q

ds DNA: Adenovirus

A
  • no lipid envelope
  • protein „primer“ (TP) for genome replication
  • virus encoded replication machinery including DdDp - transcription via cellular
    DdRp Pol II und III
  • „Splicing“
  • all mRNAs with identical „leader“ sequence
  • temporally and quantitatively controlled gene expression
    E: early; L: late

Frequently used vector in gene therapy

31
Q

3 main goals of early adenovirus gene expression

A
  1. To induce the host cell to enter the S phase of the cell cycle. E1A, E1B, and E4 gene products play roles in this process.
  2. To set up viral systems that protect the infected cell from various antiviral defenses of the host. The E1, E3 and VA RNA genes contribute to these defenses (PKR).
  3. To synthesize viral gene products needed for viral DNA replication and later for the synthesis of viral structural proteins.
32
Q

E1 functions, E1B

A

Two forms, generated by different translational start codons (different reading frames). Longer form of E1B binds to and inactivates p53 – initiation of S-phase, transformation of the cell in cooperation with E1A

33
Q

E2 functions

A

Three products, generated by alternative splicing. 72 kDa form (E2A) binds to single stranded DNA (AdDBP); 80 kDa form (E2B1) corresponds to pTP that is processed to the 55kDa TP; a 140 kDa form (E2B2) represents the Adenopolymerase.

34
Q

E3 functions

A

Diverse small proteins, not essential for life cycle, but modulate Adenovirus infection. E3-gp19K inhibits glycosylation of MHC class I proteins and thus inhibits the presentation of antigenic peptides to CTLs (persistence). Other E3 gene products inhibit TNF-alpha mediated cell-lysis early during infection. E3-11.6K (death protein) induces cell death to release progeny virus.

35
Q

E4 functions

A

7 proteins, generated by alternative splicing. The proteins modulate transcription, replication, splicing of L-transcripts. ORF6 is oncogenic by binding to p53.

36
Q

L functions

A

The mRNAs of the 11 structural proteins II-XII are spliced from a common pre-mRNA. They form the core, pentons and hexons.

37
Q

Adenovirus -> Initiation of replication

A
  1. Adenovirus terminal protein (TP=55 kD; grey) is covalently bound to 5’-end of Adenoviral origin (at 5 ́terminus of linear Adenovirus genome).
  2. A complex consisting of pre-TP (pTP=80 kD; red) and Adenopolymerase (Pol) as well as other transcription factors bind to end of DNA. Binding of adenoviral ssDNA binding protein DBP leads to strand melting at the origin of the dsDNA.
    Template sequence: 3 ́-GTAGTA…-5 ́
  3. DNA synthesis initiates at a serine residue of pTP by synthesis of the tri-nucleotide CAT;
    template are nucleotides 4-6. pTP-CAT then sildes back to position 1 on the template and starts with elongation (= jump of primer). Common mechanism for protein primed DNA synthesis (also observed for phages like φ 29)!

A complex consisting of pre-TP (pTP=80 kD) and Adenopolymerase (Pol) as well as other transcription factors bind to end of DNA.
Binding of adenoviral ssDNA binding protein DBP leads to strand melting at the origin of the dsDNA.
Template sequence: 3 ́-GTAGTA…-5 ́
DNA synthesis initiates at a serine residue of pTP by synthesis of the tri-nucleotide CAT; template are nucleotides 4-6.
pTP-CAT then sildes back to position 1 on the
template and starts with elongation (= jump of primer). Common mechanism for protein primed DNA synthesis (also observed for phages like φ 29)!

38
Q

Leading strand (no lagging strand!)

A

Ad pol elongates the strand primed by pTP. Ad DBP displaces complementary strand and maintains single stranded state. This strand forms by self complementary ends (inverted repeats) a short double strand, which allows priming.

39
Q

ds DNA: Herpesviruses

A
  • 126 kB ds DNA, linear
  • Immediate early, early, late genes (differential gene regulation)
  • ca. 84 ORFs
  • Latency in neurons; reactivation by „stress“
  • Replication in nucleus; concatamers
40
Q

DNA Viruses single stranded (ss)

A

Circo-, Parvoviruses

41
Q

ss DNA Viruses: Parvovirus (B19, AAV)

A

Causes Erythema infectiosum or fifth disease (mainly during childhood)
German: Ringelröteln
Infects reticulocytes (red blood cell precursors)
- Linear genome, 5 kb, + or – strand
with 5 ́ and 3 ́ „hairpins“; the latter serves as „Primer“
- 5 ́ end covalently bound to viral protein (protein is removed for replication)
- some parvoviruses depend on helperviruses e.g. Adeno Associated Virus
- circular genome, 1.7-2.2 kb
- only 2 ORFs: capsid and replicative protein
- depend in replication strictly on host
- disease in swine and birds
- Linear genome, 5 kb, + or – strand
with 5 ́and 3 ́ „hairpins“; the latter serves as „Primer“
- 5 ́ end covalently bound to viral protein
- some parvoviruses depend on helperviruses e.g. Adeno Associated Virus

42
Q

Parvovirus (B19, AAV) mechanism

A

1) Formation of hairpin structure
2) Initiation of DNA synthesis at 3’ OH end
3) Unfolding of hairpin structure and elongation
4) Cleavage by endonuclease at trs-site
Unfolding of hairpin structure, initiation of DNA synthesis at cleavage site and elongation
5) Formation of hairpin structure
6) Separation of strands and initiation of DNA synthesis

43
Q

DNA Viruses Double stranded with gap

A

Hepadna Viruses

44
Q

Double stranded with gap (gapped) Hepadna Viruses (HBV)

A
  • ds DNA genome 3,4 kb
  • very narrow host range
  • partially single stranded (not for Avi Hepadna), part of (+) strand is missing (gap)
  • RT bound to 5 ́end of DNA (-)-strand
  • (+)-strand of DNA contains 18 base RNA
    with Cap at 5 ́end
  • host enzyme completes (+)-strand
    to ccc (covalently closed circular) form
  • cellular Pol II transcribes mRNAs
  • this is used by HBV RT to synthesize in the capsid a partiell ds DNA
45
Q

RNA Viruses -> Polarity of genomic RNA determines mechanism of replication

A

(+)-strand RNA: mRNA polarity
(-)-strand RNA: complementary to mRNA
Ambisense RNA: one RNA serves as sense as well as antisense RNA

46
Q

Polarity of genomic viral RNA

A
  • Positive strand: Genome = mRNA (e.g. Poliovirus)
  • Negative strand: antigene = mRNA (e.g. influenza virus)
  • Ambisense: antigenome and genome = mRNA
47
Q

(+)-strand-RNA Viruses

A

Genome:
- serves as mRNA
- is translated immediately after infection

Replication:
- by viral RdRP
- in cytoplasm
-„Capping“ by viral enzymes

Enlargement of coding capacity:
- Polyprotein
- subgenomic mRNA ́s
- ribosomal „frameshifting“
- nonsense suppression

48
Q

(+)-Strand-RNA Virus: Poliovirus

A
  • RNA encodes polyprotein
  • proteolytic processing
  • protein as „primer“
  • no 5 ́CAP but IRES structure for initiation of translation
49
Q

Segmented (+)-strand-RNA Viruses Reoviruses: Blue Tongue Virus Rotaviren -> Genome

A
  • 11 ds RNA segments
  • each segment is a template for a monocistronic mRNA
  • RdRp is packaged into the capsid
  • templates remain in capsid
  • cotranscriptional export of mRNA from capsid
50
Q

(-)-Strand-RNA Viruses: Orthomyxo-, Paramyxo-, Rhabdoviruses -> Genome (segmented or not)

A
  • serves as template for mRNA-synthesis
  • always in complex with proteins = ribonucleoprotein (RNP) complex
  • not infectious without these proteins
  • viral RdRP always packaged in virion
51
Q

(-)-Strand-RNA Viruses -> Rhabdovirus: Vesicular Stomatitis Virus (VSV)

A

Genom not segmented
- serves as template for mRNA-synthesis
- always in complex with proteins
- replication in cytoplasm

Viral RdRP (L)
- transcribes antigenome/genome
- transcribes 5 monocistronic mRNAs
- capping, polyadenylation

52
Q

(-)-Strand-RNA Viruses -> Orthomyxovirus: Influenzavirus

A

Genome segmented
- 8 segments
- code for 1-2 proteins (each)
- splicing
- RNA-replication in the nucleus
- reassortment

Viral RdRP
- transcribes antigenome/ genome and mRNAs
- Cap snatching: 5 ́ends of cellular pre-mRNAs are cleaved off in the nucleus and serve as primers for viral mRNA synthesis
- 3 subunits

53
Q

ambisense-(-)-Strand-RNA Viruses -> Arena-, Bunyaviridae (Hantavirus)

A

Segmented genomes:
- in 2 genera of Bunyaviridae one genome segment is ambisense
- Arenaviridae: both genome segments are ambisense

54
Q

(+)-Strand-RNA Viruses with DNA Intermediate Retroviridae

A

Characteristics:
- diploid: 2 (+)-Strand RNAs packaged
- each is complexed with a tRNA (primer)
- does not serve as mRNA
- serves as template for DNA-synthesis
- reverse Transcriptase
- viral integrase: integrates viral DNA into host cell genome
- cellular Pol II transcribes mRNAs from integrated viral genome
- splicing, frameshifting

Virologie (11 decks)

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