Giant Viruses and Nidovirales

Question 1

What indicates that giant viruses (mimi and mama viruses) are indeed viruses?

Answer 1

• capsid protein (double jelly-roll fold)
• ikosahedral structure
• eklipse phase („disappearance“) as part of life cycle
• absence of genes for ribosomes (but for aminoacyl tRNA synthetases), energy production and energy conversion
• phylogeny

Question 2

What is sputnik?

Answer 2

a virophage
- 50 nm
- 18.3 kb circular ds DNA
- depends in its replication on mamavirus (helper virus)
- its 21 genes are a mixture from viruses infecting Archaea, Bacteria and Eukarya - tree genes derived from Acanthamoeba polyphaga Mamavirus
- archaeal integrase
- packaging ATPase related to bacteriophages/ DNA viruses
Attractive hypotesis based on presence of integrase protein: Gene transfer by virophages
Viruses as a genetic tool box between kingdoms of life?
A lot of recombinations found between host and virus or in between different viruses!

Question 3

In which groups is the scheme of life categorised after discovery of giant viruses?

Answer 3

Capsid-encoding-organisms:

• viruses of archea
• viruses of bacteria
• viruses of eukarya

Ribosome-encoding organisms:

• bacteria
• archea
• eukarya

All living organisms probably contain 34 ribosomal genes and viruses are „gene-taxis“ between the living organisms and drive evolution

Question 4

What are the families and genera of nidovirales and their characteristics?

Answer 4

Single positive stranded RNA genome
Spike structures common in all nidovirales

Arteriviridae:
13-16 kb RNA genome e.g. Equine Arteritis Virus

Roniviridae:
Okavirus

Coronaviridae:
26-32 kb RNA genome
- Torovirus
- Coronavirus
e.g. - SARS Coronavirus
- Human CV 229E
- Mouse Hepatitis Virus

Question 5

What are the three groups of corona virus? Name an example for each one.

Answer 5

• Alphacoronaviruses: HCoV 229E- Common cold virus
• Betacoronaviruses: SARS-CoV 2
• Gammacoronavirus: Mers-CoV

Question 6

What are the Naturalien intermediate hosts for corona pathogens?

Answer 6

Natural host: bats (SARS-cov 2) and mouse - no severe effect but can infect other hosts
Intermediate hosts: Cow, alpaca, civet cat, camel, pangoline (?Sars-CoV 2) - no severe effects but can infect humans
in humans mild to severe infections

Bats are resorvares for viruses

Question 7

What are the Feature of the virus particle of coronaviridae?

Answer 7

-100 to 200 nm diameter
-lipid envelope
-envelope proteins:
•S (Spike): receptor binding and membrane fusion
•E (small envelope protein): critical for budding
•HE (Hemaglutinin-acetyl-esterase) not in Sars-CoV
•M (membrane protein): interacts with N protein - contact to nucleocapsid
-Nucleocapsid: genome + N protein = more like RNP of negative strand RNA viruses
-no ikosahedral internal structure in the virion

Question 8

Describe the life cycle of coronavirus

Answer 8

• receptor binding via spike protein
• fusion and uncoating
• assembly of polymerase complex and double membrane vesicles (DMV) for transcription and replication
• translation
• budding
• Exocitosis

Question 9

Describe the structure of the spike protein and name two therapy strategies targeting the spike protein.

Answer 9

Trimeric structure: S1 subunit (globular receptor binding domain) with receptor binding motifs at the three ends
S2 subunit (stalk fusion domain) 
Intercellular tail 

Therapy strategies

• there are two critical proteolytic steps happening in the processing of the prossesing of the s protein, therapies targeting the protease could interfere with the proper function of the Spike - no entering of the virus into the host cell
• soluble receptor mimicing peptides could be used, that bind stronger to the receptor binding motif then the ACE2 (angiotensin-converting enzyme 2) receptor (protection of medical staff before vaccine development)

Question 10

Describe the genome features of coronavirus and the general replication process

Answer 10

Genome
- (+) strand RNA genome with 5 ́cap and 3 ́poly A
- 5 ́ and 3 ́untranslated regions (UTRs)
- 9-14 open reading frames (ORFs)
- ORF1 encodes a polyprotein which is proteolytically processed
- translation of ORF1a stops at leaky stop codon; read through in 25% of cases
‘slippery’ heptanucleotide sequence and pseudoknot; ribosome makes −1 frameshift.
(Exact balance: deregulation severely hampers virus replication)

Replication:

• first step: translation of incoming genome; assembly of replicase
• synthesis of a complete (-)-strand, complementary to the (+)-strand genome - followed by the synthesis of many (+)-strand genomes on (-)-strand template

Question 11

Describe the special mechanism of the transcription of coronavirus

Answer 11

A nested set of 3‘ co-terminal mRNAs with identical 5‘ end sequence is generated
Mechanism:

Discontinous transcription (leader primed) mechanism:
1. negative strand synthesis starts at 3 ́end of genome
2. stopatinternalgenomicsequence,thetranscription-regulatingsequence(TRS)
transfer of this (-)-strand RNA to the TRS at the 5 ́end of the (+)-genome RNA
via base pairing between TRS and leader TRS sequence
4. restart of RNA synthesis and completion of (-)-strand synthesis;
5 ́ends of all negative strands identical = leader sequence
5. subgenomic (sg) negative strand RNA serves as template for synthesis of mRNA

Question 12

What are the functions of the 16 NSP (none structural proteins) in Covid?

Answer 12

NSP1: Host mRNA degradation, cell cycle arrest, translation inhibition, inhibition of IFN signaling
NSP2: unknown
NSP3: polyprotein processing, Part of DMV pores
NSP4: DMV formation ?
NSP5: Main protease (Mpro/3CLpro) polyprotein processing 
NSP6: DMV formation ?
NSP7: single strand RNA binding
NSP8: Primase 
NSP9:Part of replicase complex
NSP10: Part of replicase complex 
NSP11: unknown 
NSP12: RdRp
NSP13: helicase
NSP14: 3’-5’ endonuclease activity → proof reading functio in complex with NSP10
NSP15: endonuclease 
NSP16: RNA cap formation