Week 3 Module 1

Question 1

Do viruses have several modes of genome replication?

Answer 1

-Yes since viruses can have different genome structures,
there are several modes of genome replication.

-For viruses with dsDNA genomes, replication occurs through semi-conservative replication.

-For viruses with ss (+) strand genomes and ssDNA viruses, a replicative form – a ds DNA version of the genome must be made after infection; Will be used for replication and transcription.

-The mechanism of replication and transcription depends on
genome structure.

Question 2

How does the Phage T4- dsDNA virus do replication?

Answer 2

-T4 phage encodes its own DNA Polymerase, some primases and
helicases
-genome replicated through semi-conservative replication
-‘single-genome’ units produced and then several units recombine
to form a concatamer
-during maturation, headful packaging occurs: endonuclease cuts one DNA length just long enough to fill one head

Question 3

How does the Phage T4- dsDNA virus do transcription and translation?

Answer 3

-host cell replication, transcription and translation shut down quickly
-transcription produces both monocistronic and polycistronic
transcripts
-three sets of genes: early, middle and late
-translation follows order of transcription

Question 4

Early proteins?

Answer 4

-early proteins include an anti-sigma factor: binds to host
RNA Pol (and its σ); Prevents transcription of host genes
which results in transcription of viral early genes

Question 5

Middle proteins?

Answer 5

-middle protein includes factors that direct host RNA Pol to
viral middle gene promoters

Question 6

Late proteins?

Answer 6

-late proteins include new T4 sigma that directs host RNA Pol to late gene promoters
-also, late proteins include phage components necessary for
Packaging viral genome

Question 7

How does Phage ΦX174 – circular ssDNA ‘+’ Strand replicate?

Answer 7

-after infection, host enzymes convert circular ssDNA for into
circular dsDNA which is replicative form
-first more several copies of replicative form of genome are made by semi-conservative replication of a circular genome (Genetics course)
-purpose of this is to create a template for transcription
-then many copies of ‘+’ strand are made; These are copies of
genome for packaging
-occurs through rolling circle replication

Question 8

How does Phage ΦX174 do transcription and translation?

Answer 8

-genome is about 5.4 kb and has overlapping genes
-transcription occurs using ‘-’ DNA strand as template
-some parts of genome read more than once
-for overlapping genes, transcription initiated at more than one place to produce distinct mRNA with distinct ORFs

-also, one protein A* made by another initiation of translation
at a later start codon in same frame as A i.e. A* is shorter version
of A

Question 9

What are the consequences of Animal Infection by viruses?

Answer 9

When animal cell infected by virus there are at least four possible consequences which include: Virulent infection, Latent infection, budding, transformation

Question 10

Virulent infection

Answer 10

-virulent infection (lysis of host) most common

Question 11

Latent infection

Answer 11

– viral DNA maintained in host but
new virions not produced and no lysis

Question 12

Budding

Answer 12

– release of new virions from host cell is slow and host cell may not be killed
-host cell continues to produce virions over time; persistent infection
-in some cases, host cells carrying viral genome can continue to divide

Question 13

Transformation

Answer 13

– virus converts normal animal cell
into tumour cell

Question 14

Latent infection is similar to?

Answer 14

-latent infection, similar to lysogeny in bacteria, virus is quiescent (ex. Mononucleosis, stress induced)

Question 15

Can a latent infection revert back to lysis?

Answer 15

-Latent infection can revert back to lysis( example: chicken pox infection and then showing up as shingles later in life)

Question 16

What are Poxviruses?

Answer 16

-A dsDNA viruses that infects Many Species

-large group of viruses that can cause diseases in: birds, Insects, reptiles, marsupials, and mammals

-human diseases/conditions include: smallpox (variola virus),
chicken pox and shingles, (Varicella Zoster virus), monkeypox (monkeypox virus or MPXV)

Question 17

What is the genome information on monkeypox virus?

Answer 17

-~197 kb, ~190 ORFs (about 190 proteins)
-about 1/3rd of genome gives proteins that evade/combat host
immune system e.g. making viral mRNA look like host mRNA

Question 18

How does replication happen with Poxviruses?

Answer 18

Replication:
-occurs in cytoplasm (unusual) in factories – compartments
surrounded by ER membrane; can get bigger over time and
Sometimes fuse
-poxviruses encode replication enzymes e.g. DNA Pol, helicase-
primase, SSBs (single stranded binding proteins) etc.

Question 19

How does transcription & translation happen with Poxviruses?

Answer 19

Transcription & Translation
-also occurs in factories
-virus encodes RNA Pol for transcription
-use host cell ribosomes and some proteins for translation;
viral proteins reprogram/co-opt host proteins

Question 20

What are Coronaviruses?

Answer 20

-cause about 15% of common colds, SARS (SARS-CoV), and
Middle East respiratory syndrome (MERS-CoV)
-attacks respiratory system
-History: SARS cases first recorded in China in 2002
-went from China to Hong Kong to Singapore to Canada
-first time epidemic spread through air travel
-now, Covid-19!
-glycoprotein spikes give virion shape as ‘crown’ (latin = corona)
-largest RNA genome for virus it is 30 kb (huge for an RNA virus)
-Positive sense single stranded RNA genome of 29,891 nucleotides (1) (sequence of genome is same as mRNA)

Question 21

What happens during transcription & translation of Coronaviruses?

Answer 21

‘+’ strand is polycistronic mRNA in part
-ribosomal frame shifting also occurs to get different proteins from same sequence in genome.
-polyprotein made and cleaved by proteases to give individual
proteins including replicase
-replicase – makes RNA using RNA as a template (i.e. RNA-dependent RNA polymerase or RdRp)
-Virus must make replicase because no cellular protein has RdRp
activity

Question 22

What is the function of replicase?

Answer 22

-replicase does two things:
1. Makes copies of RNA genome
2. Transcribes RNA to give an mRNA

Question 23

Why do Coronaviruses need an RdRp?

Answer 23

-Coronavirus genome is RNA, it needs to use its RNA genome and new genomes need to be made, one of the enzyme that the virus provides that the host cell can’t provide is a replicase (an RNA dependent RNA polymerase). In normal transcription DNA is template to make RNA.

Question 24

What is Ribosomal frame shifting in coronaviruses?

Answer 24

Ribosomal frame shifting:
One mRNA molecule is present and translation starts at the same
start codon for both ORFs. During translation the ribosome
Pauses.

-Pause causes shift upstream or downstream (-1 in case of corona
virus) in certain percentage of cases (< 100%)
-amino acids put in with ‘shift’ will differ compared to no shift
i.e. different C-terminal portions of proteins produced

Question 25

Why could a ribosome pause during translation and cause ribosomal frameshifting?

Answer 25

-Certain codons, are rare have few matching tRNAs, anticodon would be rare too. Ribosome can pause because it’s waiting for tRNA. When it pauses in certain percentage of cases it can slip (move forward by one base or backwards by one base). If it moves backwards one base it will give you a different protein (i.e the -1 frameshift)

Question 26

What are some other characteristics of coronaviruses?

Answer 26

-Possible that host cells carrying
viruses will fuse; Get multinucleate host cell making virions that can
spread between cells but are hidden from antibodies.

-Virus mimics host cell mRNA by having a 5’ cap and poly A tail

-With coronaviruses cells can fuse so the virus doesn’t have to exit cell and be exposed to antibodies

Question 27

Overview of Retroviruses Transcription & translation of retrovirus

Answer 27

Retroviruses – ssRNA ‘+’ Strand that use DNA Intermediates

-includes HIV, human T-cell leukemia virus (HTLV)
-retroviruses have two copies of RNA genome that dimerize

Transcription & Translation
-the enzyme reverse transcriptase (RT) is packaged into mature
virions so present from the start with new infection
-RT has 3 activities:
i) reverse transcription- makes DNA using RNA template
ii) ribonuclease – in this case degrades RNA in
RNA:DNA hybrid
iii) polymerase – makes DNA using DNA template

-Reverse transcriptase can make a DNA copy using RNA template, same enzyme can get rid of RNA part and then make another DNA strand using a DNA template (since it is a polymerase)