Microbio 244 - Viruses and other acellular infectious agents (2)

Genome strategies and viral life cycles

What is the core principle of genomic strategies in viral agents

Every virus needs its genome converted into mRNA so it can be translated by host ribosomes, how it does this determines its whole replication strategy.

Why is viral phylogeny so difficult?

Viruses don’t have a marker gene like cellular organisms do (conserved genes like small subunit rRNA). Viral genomes are small and modular, and horizontal genome transfer is common. This means that viral phylogeny takes place through a combination of genome type, hallmark proteins and comparative genomics.

What is the ICTV?

The International Committee on Taxonomy of Viruses system is the formal authority on the taxonomy of viruses. Modern Viral taxonomy increasingly relies on comparative genomics. Important genomic approaches include genome type, gene-sharing networks, hallmark protein comparison (capsid formation) and capsid structure.

Realm → Phylum → Class → Order → Family → Genus

What is the Baltimore system?

It is a system that does not group viruses based on phylogenetic information, but rather according to genome type and the route to mRNA. It is a useful organisation as ever viral cycle must reach translateable mRNA.

Describe positive-strand and negative-strand genomes

Positive strand genomes are in the same direction as mRNA and can be directly translated.

Negative strand genomes are oriented in the opposite direction to mRNA and cannot be directly translated, they must first be copied into positive strand mRNA by RNA-dependent RNA polymerase.

What are the seven types of viral genome?

1. dsDNA

2. ssDNA

3. dsRNA

4. +ssRNA

5. -ssRNA

6. Retrovirus

7. Reverse Transcribing DNA virus

A T4 Bacteriophage is an example of a dsDNA virus, describe its characteristics, early events, genome replication, packaging and release.

Characteristics:

Virulent phage = lytic cycle

uses long tail fibres for reversible attachment to membrane, short tail fibres for irreversible attachment to receptor site.

Tail sheath contraction drives tubule which deposits viral DNA into the cell.

Phage protein gp5 lysozyme degrades the peptidoglycan layer.

Genome replication:

Host RNA polymerase transcribes T4 genes immediately after entry.

early proteins are produced that redirect host metabolism to phage production

host mRNA is degraded, freeing up those pathways for use by the phage.

DNA replication:

T4 encodes for its own DNA dependent DNA polymerase.

replication initiates from multiple origins, allowing rapid genome copying.

Hydroxymethylcytosine (HMC) replaces cytosine in T4 DNA

Genome packaging and release:

T4 DNA is made up of concatemers that is packaged using a packasome and a terminase. Holin forms holes in the plasma membrane and Endolysin passes through these holes to degrade the peptidoglycan layer. These combined results in cell lysis. → releasing infectious progeny.

What is a Lambda virus?

A lambda virus is an example of a dsDNA virus that can follow a lytic cycle or a lysogenic cyce. It is regulated by proteins cI (Lambda repressor), cII , cIII, integrase and excisionase.

High cII levels favour lysogeny → cII promotes the production of cI(lambda repressor) and integrase. CI represses the genes required for the lytic cycle.

Environmental factors can cause damage to the host DNA, leading to the release of the host protein RecA. Activated RecA leads to the self-cleavage of cII, triggering the expression of the genes for the lytic cycle.

Compare Herpesvirus latency to Bacteriophage lysogeny

Explain how ssDNA cannot directly support DNA to RNA flow

ssDNA must first have a dsDNA intermediate, in replicate form. This serves as a template off which mRNA transcription and genome replication are based. Host DNA polymerase often used to synthesize the complementary dsDNA strand.

name 3 dsDNA virus examples and describe them briefly

1. ϕX174

Infects E.coli

Circular +ssDNA phage (positive strand) - same sequence as mRNA

Blocks peptidoglycan synthesis, leading to cell lysis

2. PF1

Filamentous Pseudomonas

Does not lyse host.

Virions are assembled and secreted.

Host continues to grow, but more slowly.

3. Parvovirus

Small, non-enveloped animal virus.

no replication enzymes encoded, relies heavily on host biosynthesis.

Lyses host cell.

What is the difference between +ssRNA and dsRNA and -ssRNA?

+ssRNA (positive strand) is in the same sequence as the viral mRNA and therefore does not need to be transcribed; but can rather be directly translated by the host ribosome to produce viral mRNA.

-ssRNA is in the complementary direction to the viral mRNA and thus, requires RNA dependent RNA polymerase (acting as a replicase) to transcribe the mRNA before it can be translated.

This is required for dsRNA as well, which remains safely within the capsid while transcription takes place.

What is the process for +RNA strands upon entry into the host cell?

+RNA is immediately useful to the host and after entry and uncoating, is immediately translated into mRNA. Translation usually results in mRNA, RdRp and other non-structural proteins. The RdRp makes -RNA, this is used as a viral template with which to produce more +RNA genomes and viral mRNA. Replication is often associated with membrane-associated Replication factories.

What happens with the entry of the SARS-CoV-2 virus?

SARS-CoV-2 has a large, linear +RNA genome (30kB) that encodes for non-structural proteins, structural proteins and accessory proteins.

A spike protein binds to an ACE2 receptor on the host epithelial cells and TMPRSS2 protease cleaves the spike and exposes the domain that promotes membrane fusion. The +RNA genome is released into the cytoplasm and can be translated immediately due to its 5’ cap. ORF1a and ORF1b code for large polyproteins that are cleaved by viral proteases.

Summary:

spike protein binds to ACE2 receptor → TMPRSS2 protease cleaves spike and promotes membrane fusion → +RNA genome is released into cytoplasm and is translated immediately → ORF1a and ORF1b code for large poly proteins that are cleaved by viral proteases.

What is ribosomal Frameshifting?

Because viruses have smaller genomes, they cannot encode for as many proteins. How they get around this problem is through ribosomal frame shifting. Because proteins are constructed based off the sequence of nucleotides read, shifting the frame/codon by even one can give you a completely new protein. Therefore, viruses use ribosomal frame shifting to create many amounts of long codon chains to code for different kinds of proteins. Frame shifting isn’t 100% efficient, but this means that viruses can control the amount of proteins produced.

How are NSP 1,4,6, 12 and 14 used in the creation of replication factories and release of SARS-CoV-2?

Ribosomal frameshifting is used to produce proteins from the overlapping of reading frames.

NSP 1 inhibits host translation, sequestering the resources for viral translation.

NSP 4 and 6 promote the formation of double-membrane vesicles from ER membrane. RNA template and replication enzymes are concentrated in these enzymes and they serve as replication factories.

NSP 12 acts as RdRp, while NSP 14 acts as proof-reading. The RNA template is used to generate full length +RNA genomes with subp-genomic RNAs (these are used to interfere with a hosts antiviral response).

The RNA is packaged as a full viral genome in a nucleocapsid and leaves the host cell once mature by exocytosis.

What is the influenza life cycle?

The influenza virus is a segmented, negative-strand virus.

Entry:

A receptor mediates endocytosis, low pH of the endosome triggers conformational changes and the viral envelope fuses with the endosomal membrane.

The Nucleocapsid enters the nucleus and RdRp obtains the 5’ cap from the host cell mRNA(Cap-snatching)

PA cleaves a short fragment of the host mRNA to use as a primer for viral mRNA synthesis.

Genome replication:

A full length +RNA genome is synthesized from the -strand genome .

Templates for synthesis of new - strand genome segments are also synthesized.

Assembly and release:

Structural proteins are synthesized via the ER/Golgi pathway and are inserted into the plasma membrane.

Viral genomes are packaged into assembling virions. They acquire envelopes and are released from the plasma membrane via budding.

In short:

Endocytosis → Nucleus → Cap-snatching → genome synthesis → Budding

Why are segmented viral genomes advantageous?

Segmented genomes allow genetic mixing of relative viruses that infect the same host cell. This allows for the generation of new influenza strains through reassortment, where entire intact gene segments from either parent is mixed and randomly packed together.

How do retroviruses use reverse transcription as a strategy to bypass viral RNA transcription?

Instead of using their + RNA strands directly, reverse transcription with the reverse transcriptase enzyme is used. The viral genomic + RNA is converted into dsDNA before being integrated into the host chromosome as a provirus. This means that host RNA polymerase transcribes viral mRNA as new genomic RNA.

This strategy makes integration and long-term persistence central to the life-cycle.

What is the preferred drug-target for HIV and why is it susceptible?

HIV is integrated as a provirus and is transcribed by the host RNA polymerase. Viral RNAs are spliced to produce different viral mRNAs. Viral proteins and genomic RNA assemble as the host membrane. The virions bud but are initially immature, viral protease cleaves Gag proteins and this causes capsid rearrangement and subsequent virion maturation. Viral Proteases are essential for bud maturation and are thus a prime target for drug action.

How does the Hepatitus B virus use reverse transcription even though it is a DNA virus?

Differs from a regular retrovirus as its genome contains DNA, but uses RNA as an intermediate. HBV has a circular, dsDNA genome with a nicked but complete strand and an incomplete, gapped strand. Viral Polymerase P is attached to the genome.

Host repair enzymes fill in the gap of the incomplete genome, creating covalently closed circular DNA (cccDNA).

Host RNA polymerase transcribed the cccDNA to viral RNA, including pregenomic RNA.

Reverse transcription converts the pregenomic RNA into -strand DNA. Most RNA is degraded, what remains primes synthesis of the incomplete positive DNA strand. This regenerates the characteristic, gapped dsDNA viral genome.

Compare the logic between the transcription of retroviruses and HBV

Compare the genome types replication strategies

What are the 5 strategies viruses use to compress information even though they have such a small genome?

1. Frame shifting

2. Polyproteins

3. read through

4. subgenomic RNA

5. overlapping ORFs (open reading frames) with segmented genomes.

What are the 5 factors targeted by antiviral drugs?

1. Entry

2. Integrase

3. Polymerase

4. Protease

5. Release