Viral Vectors 101: What is a Virus?

Question 1

How does the textbook describe viruses?

Answer 1

Viruses are simple — a genome packaged in a protein shell

Question 2

How do viruses survive?

Answer 2

Viruses survive by hijacking the machinery of the cells they infect. Once they enter a cell, they treat their viral genome as a “payload” that is delivered to the cell, which then uses its own machinery to replicate the viral genome and proteins.

Question 3

What are meant by enveloped and non-enveloped viruses?

Answer 3

While all viruses have a genome inside a protein shell known as a capsid, some also have a lipid bilayer surrounding the capsid, called an envelope. Viruses without an envelope are referred to as
naked or non-enveloped, while viruses with an envelope are called enveloped viruses.

Question 4

How are individual viral particles referred to as?

Answer 4

“Virus” refers to a single type of virus, not the individual viral particles. A single viral particle is called a virion.

Question 5

Are viruses single or double stranded, DNA or RNA?

Answer 5

When it comes to replication, viruses tend to march to the beat of their own drum.

They have a wide variety of possible genome types that can be either double-stranded (ds) or single-stranded
(ss) (dsDNA, ssDNA, dsRNA, positive sense ssRNA, or negative sense ssRNA) and varying methods of replication.

In fact, an entire classification scheme, known as the Baltimore replication classes, has been developed around how they replicate

Question 6

How may viruses with DNA genomes replicate?

Answer 6

Viruses with DNA genomes either replicate themselves following central dogma (gDNA => RNA => proteins) or require an intermediary dsDNA replication step between DNA and mRNA.

Question 7

Describe the arrow plot shown for the the 7 classes of virus replication

Answer 7

I dsDNA: dsDNA genome is transcribed into mRNA, which is translated into proteins while the DNA is replicated and the virus is assembled.

II ssDNA: ssDNA genome forms dsDNA and is transcribed into mRNA, which is translated into proteins while the ssDNA is replicated and the virus is assembled.

III dsRNA: dsRNA genome is transcribed into mRNA via RNA-dependent RNA polymerase (RdRp), which is translated into proteins while the mRNA is also replicated via RdRp transcription and the virus is assembled (also with RdRp?). RdRp then assisted in forming the dsRNA in the capsid.

IV +ssRNA: +ssRNA genome creates proteins including RdRp. This RdRp is then also used to to make an anti-genome (-ssRNA). RdRp is then used again to form another +ssRNA genome and it is assembled using proteins formed before into virions.

V: -ssRNA: From the -ssRNA genome, RdRp is used to form both an anti-genome (+ssRNA) and mRNA. RdRp is used to form an -ssRNA and proteins are translated from the mRNA and the virion is assembled.

VI RNA viruses that reverse transcribe: +ssRNA genome reverse transcribes (using reverse transcriptase) to form cDNA. This cDNA is then integrated into the infected host cell via integrase (proviral DNA). From here mRNA is transcribed from the proviral DNA and proteins are translated. Full length transcription of the proviral DNA also produces a +ssRNA genome and virions are assembled.

VII DNA viruses that reverse transcribe: an ssDNA or dsDNA genome forms dsDNA (cccDNA: a special DNA structure that arises during the propagation of some viruses in the cell nucleus and may remain permanently there. It is a double-stranded DNA that originates in a linear form that is ligated by means of DNA ligase to a covalently closed ring.) From here mRNA is transcribed and proteins are translated including p protein. pregenomic RNA (pgRNA) is also transcribed from the dsDNA and these are assembled into ss/dsDNA (rcDNA: relaxed circular DNA) in a capsid.

Question 8

Describe what happens more generally when a virion infects a cell

Answer 8

After a virion infects a cell, the single particle creates many copies of itself, sometimes up to millions of new copies, which then are released from the cell like baby spiders from an egg sac.

Question 9

What are viral vectors?

Answer 9

Viral vectors are viruses that have been genetically modified to either limit or completely eliminate their replicative ability. The remaining particle, which retains the protein coat (and envelope), has a “gutted” genome with proteins central to the replication process removed. This means it can deliver a genetic payload to a cell but cannot create new particles.

Question 10

What bonus does the gutting of the genome of a virus have for researchers?

Answer 10

As a bonus, this increases the carrying capacity of the viral vector, increasing both the safety and the usefulness of the tool. . In fact, most gutted genomes have as much genome removed as possible, just to increase the viral vector’s carrying capacity.

Question 11

How is it ensured that viral vectors are only allowed to reproduce when experimentally necessary?

Answer 11

In order to allow the viral vectors to reproduce when experimentally necessary, genes for the required proteins are packaged in separate plasmids and expressed via cells cultured with the vectors This allows the viral vectors to reproduce only in experimentally controlled conditions, making them far safer to work with than virions.

Question 12

What are the four types of viral vectors called?

Answer 12

The four types of viral vectors (which many researchers will refer to as viruses even when they are referring to the viral vectors) are named after the viruses they’re derived from: AAVs, lentivirus, gamma retrovirus, and adenovirus

Question 13

What are retroviruses?

Answer 13

Retroviruses are single-stranded RNA viruses that use reverse transcriptase to synthesise complementary DNA copies of their genome upon infecting a host cell. This viral DNA (also called proviral
DNA) is then inserted into the host’s genome and is used to make more virus.

Question 14

What is the most studied and used gamma-retrovirus?

Answer 14

The most studied (and used in the lab) type of gamma-retrovirus is Murine Leukemia Virus (MLV).

Question 15

Naturally isolated gamma-retroviruses have been associated with cancer
in mammals, why might this be?

Answer 15

It is thought to be due to their
propensity to integrate into proto-oncogenes and disrupt their expression. To add insult to injury, retroviruses generally have moderate to high immunogenicity, meaning they induce an immune response in the host.

Question 16

Why use gamma-retroviruses in the lab?

Answer 16

So why use a gamma-retrovirus in the lab? Well, they are essentially evolutionarily optimised gene delivery devices. Their survival depends on their ability to deliver and permanently integrate a genetic cargo into a cell. These viruses have a cargo capacity of ~8 kb, ample room for most experimental needs, and can transduce (infect) a wide range of cell types with a high efficiency

Question 17

What limitations are there to the types of cells which can be infected by gamma-retroviruses?

Answer 17

However, gamma-retrovirus infection is restricted to dividing cells only, so is not suitable for infection of quiescent
cells or infrequently cycling cells but is fine for experiments with immortalized cell line cultures.

Question 18

What is a lentivirus?

Answer 18

Lentivirus is a type of retrovirus and shares many common features with gamma-retrovirus including genome architecture, host integration, and its association with disease

Question 19

What is the most well studied lentivirus?

Answer 19

The most well-studied lentivirus is HIV, which many lentiviral tools have been derived from.

Question 20

How does the carrying capacity and the versatility in cell type differ between the gamma-retrovirus and the lentivirus?

Answer 20

Lentiviruses have slightly larger packaging capacities than gamma-retrovirus, coming in around 9 kb. Unlike gamma,
lentivirus can infect both dividing and non-dividing cells.

Question 21

Why can lentiviruses infect non-dividing cells?

Answer 21

Lentiviral particles enter the nucleus
through nuclear pores and thus do not require breakdown of the nuclear envelope during cell division to enter the nucleus as gamma-retroviruses do.

Question 22

What is an AAV?

Answer 22

Adeno-associated virus (AAV) is a small, single-stranded DNA parvovirus.

Question 23

Can AAV infect humans and mammals?

Answer 23

AAV can infect humans and some other mammals but is not currently known to cause any diseases.

Question 24

How does an AAV replicate?

Answer 24

Recombinant AAV largely does not integrate, and the addition of a helper virus or plasmid allows it to replicate episomally, essentially remaining an extrachromosomal part of the host genome that can replicate and divide along with the endogenous DNA.

Question 25

Comment on the immunogenicity of AAV

Answer 25

AAV also has low immunogenicity, meaning it does not induce the same high immune response as retroviruses.

Question 26

Comment on the range of cell types which can be infected by AAV

Answer 26

This virus can also transduce dividing and nondividing cells, making it conducive to targeting all cell types.

Question 27

Outside of the lab where is AAV often used?

Answer 27

AAV has many excellent features for human gene therapy and is currently being used in clinical settings.

Question 28

When is AAV often used in the lab?

Answer 28

It is ideal for whole animal administration, such as in mouse experiments.

Question 29

What can assist in the specificity of AAV in experiments?

Answer 29

AAV also has multiple serotypes (12 naturally occurring and more
synthetic capsids are routinely being developed) to choose from, which can target the virus to specific cell types of interest.

Question 30

What is a constraint of AAV as compared to lentivirus and gamma-retrovirus?

Answer 30

The cargo capacity of AAV is ~4.7 kb, almost half that of lenti and gamma-retro viruses. This is large enough for many applications, but in some cases multiple AAVs (co-infection), an alternative virus, or other strategies may need to be considered if size constraints are an issue.

Question 31

To what extent are elements of gamma retro-viruses and lentiviruses interchangeable?

Answer 31

Since lentivirus and gamma-retrovirus are so closely related, their required components are very similar. While plasmids containing virus-specific elements such as the long terminal
repeats (LTRs) or structural proteins are not interchangeable between the two systems, other, more general, viral components such as a heterologous envelope or a post-transcriptional regulatory element can be used for either type of retrovirus.

Question 32

Describe the structure of an adenovirus

Answer 32

Adenovirus has a linear, double stranded DNA genome which is non-enveloped

Question 33

What species can be infected by adenovirus?

Answer 33

It can infect humans, many mammals, and even the avian population.

Question 34

Can adenoviruses be pathogenic?

Answer 34

Some adenoviruses are known to cause respiratory disease across species, as well as cancer in hamsters, although no cancer connection has been identified in humans to date.

Question 35

Comment on the immunogenicity of adenovirus

Answer 35

Adenovirus generally has high immunogenicity, but some strains have been engineered to induce a lower inflammatory response within hosts.

Question 36

Relative to the other viruses discussed, what two characteristics does the adenovirus excel at / maximise?

Answer 36

Relative to the other viruses discussed in this article, adenovirus has the highest maximum titer, a plus for production and infection.

These viruses are roomy: some adenoviruses pack up to ~ 35 kb of space for cargo!

Question 37

What cells can be infected by adenovirus? Does it integrate into the genome?

Answer 37

This virus can infect dividing and non-dividing cells and doesn’t require genomic integration to express its cargo; in fact its viral life cycle does not include genomic integration.

Question 38

Comment of the expression of adenovirus as compared to AAV

Answer 38

While the expression of adenovirus cargo is high, it’s generally not as long-lasting as AAV (up to several weeks vs. AAV’s several years).

Adenovirus is an effective option if you need high, transient expression of a genetic cargo or have a very large cargo.

Question 39

In nature, viruses infect host cells via what?

Answer 39

In nature, viruses infect cells and organisms (which we refer to as hosts) via viral particles, the functional infection unit

Question 40

How does the particle access the cell?

Answer 40

Each particle has a viral envelope, which fuses with the target cell’s membrane, allowing the viral particle access into the cell. Once inside
the host cell, the virus unleashes its cargo — its own genome.

Question 41

How can the viral envelope be utilised in a lab?

Answer 41

The viral envelope and its proteins determine what type of cell the virus will target and fuse with. In nature, this determines which organisms and cells they infect. In the lab, this means viruses can be engineered to be cell type-specific

Question 42

In what regard is the genome insufficient for the virus to replicate and spread? What is done to remedy this?

Answer 42

Its genome, however, isn’t enough to ensure the virus can replicate and spread. Instead, viruses replicate by hijacking the host’s system to produce more virus and continue the infection cycle.

Question 43

Viruses are essentially DNA (or RNA) delivery vectors, so what makes them better than the alternatives? (3)

Answer 43

(1) They have a high transduction efficiency and can effectively infect a wide range of cell types that are typically “difficult to transfect,”
such as primary cells. They can also be directly injected into hard-to-reach organs, such as the brain.

(2) Some viruses have a low immunogenicity, which makes them ideal for applications with whole organisms.

(3) Viral delivery is typically straightforward across applications, while electroporation of a human, for example, isn’t a reasonable option.

Question 44

What extra measures have to be taken since main viruses used are derived from those which can infect humans?

Answer 44

Since viruses used in labs are derived from viruses that infect people in nature, extra safety measures, like replication-incompetent viral particles, are required.

Question 45

What is the relationship between lentivirus and retroviruses?

Answer 45

Lentiviruses are a genus of the retroviral family that can infect both non-dividing and dividing cells.

Question 46

How do lentiviruses infect non-dividing cells?

Answer 46

They accomplish this through nuclear localisation signals, which can traffic their viral particles inside the nucleus, without the need for envelope disassembly.

Question 47

When are adenovirus and AAVs typically appropriate as opposed to retroviruses?

Answer 47

Adenoviruses infect non-dividing cells but they do not permanently integrate into the host genome (non-integrating) as lentiviruses and retroviruses do. They are generally used to achieve transient, high expression of the relevant cargo without the fear of random genomic integration causing mutagenesis in the host.

Question 48

What are AAVs biggest advantage over adenovirus?

Answer 48

AAVs primary advantage over adenovirus is their low immunogenicity, which means they do not stimulate a strong immune response when introduced into an organism. This feature makes
them an attractive tool for human subject delivery, as immunogenicity was previously a large barrier for gene therapy.

Question 49

Describe three ways in which viruses can be used to manipulate gene expression

Answer 49

shRNAs are short RNA sequences which target complementary mRNA sequences for degradation. Viral introduction of shRNA plasmids can induce gene-specific silencing temporarily, long-term, or even inducibly, depending on the vector and virus.

Viral CRISPR-Cas9 tools have been engineered to introduce frameshift mutations in genes of interest, turning expression off permanently.

Viral vectors can also be used to express proteins. Viral expression cassettes typically contain a promoter (inducible or constitutive), your gene of interest, and often a selectable
marker. This vector can express your construct in target cells transiently or constitutively depending on the virus used.

Question 50

What is meant by sgRNA?

Answer 50

Guide RNA (gRNA) or single guide RNA (sgRNA) is a short sequence of RNA that functions as a guide for the Cas9-endonuclease or other Cas-proteins that cut the double-stranded DNA and thereby can be used for gene editing.

Question 51

Why deliver these systems virally instead of by another method?

Answer 51

Viral delivery is attractive due to the ease of transduction in difficult-to-transfect cell types ranging from primary cells to embryonic stem cells.

Viral integration of a construct is also reliably higher than traditional transfection and random genomic integration of a plasmid.

Viral transduction is also generally less toxic to cells than transfections, preserving cell viability.

Question 52

How can you test thousands of elements simultaneously in your system rather than just one at a time?

Answer 52

Screening libraries, large collections of
biologically independent variables, are becoming increasingly popular tools with many applications. These libraries include sgRNAs targeting the entire genome, overexpression of all human
transcription factors, barcoded libraries to trace cell lineage, and so much more! Libraries allow you to test thousands of elements simultaneously in your system instead of just one at a time.

Question 53

Many of these libraries are packaged and delivered with viral vectors instead of a traditional transfection method. Why is this?

Answer 53

This is because viral systems have a high infection frequency and the viral titer (the number of viral particles capable of infecting a host cell) can be tuned so that the copies of library cargo delivered are controllable.

For many screening applications, it is essential to deliver a single copy of your library cargo to isolate the effects of each variable being manipulated. Thus, the adjustable nature of viral libraries makes them the ideal vector for the job.

Question 54

Briefly describe two therapeutic applications for viruses

Answer 54

Vaccine development
Attenuated and inactivated viruses pioneered vaccine development well over a hundred years ago.

Clinical applications
Viruses in vaccines helped pave the way for their use in other clinical applications. In the last decade, viral applications in cancer treatment and gene therapy have also been in the limelight.

Question 55

What was the first oncolytic viral therapy approved by the FDA?

Answer 55

The FDA approved the first oncolytic viral therapy in 2015 to treat melanoma, and many more are in the pipeline.

Question 56

How has vaccine development moved away from the delivery of the actual virus?

Answer 56

Vaccine development is moving away from delivery of the actual virus to which immunity is desired. Instead,
components of the virus are now delivered in other forms (mRNA, DNA, etc.) to raise immunity.

Question 57

What is being explored as the delivery mechanism for these new vaccines? What benefits may they provide? (3)

Answer 57

Viral vectors are being explored as the delivery mechanism for increasingly popular nucleic acid based vaccines.

These vectors provide several
advantages over traditional vaccines, including
1) Cellular responses in addition to antibody response
2) Very high immunogenicity
3) Longlasting immune response even after a single dose.

Question 58

What viruses in particular are being looked at for gene therapy and what advantages do they bring?

Answer 58

Viruses, specifically AAV, are also being exploited as delivery mechanisms for gene therapy.

The tractability of AAV, low immunogenicity, and cell-specific targeting features make them an ideal delivery system for human patients.

Question 59

What plasmids are often used when delivering viral vectors?

Answer 59

The components of these vectors
are split across several plasmids (often three), for safety purposes. The primary plasmid with the delivered cargo (often a gene) is called the transfer plasmid, while the other plasmids with packaging or replication components are called helper plasmids.

Question 60

Describe what should be included in the vector in the region to be expressed

Answer 60

Genetic cargo, this is often cDNA,
shRNA, or something similar, and is contained on the transfer plasmid. If expression is the goal, a promoter should flank this component as well. On both sides of this promoter-gene combo are two long terminal repeats (LTRs). These LTRs define the limit of what will be packaged. The 5’ LTR
and 3’ LTR, respectively, act as RNA polymerase II promoter and terminator regions and are slightly different in sequence

Question 61

The genetic cargo requires viral packaging to infiltrate and integrate into the host genome. Describe this

Answer 61

First, it needs to be virally enveloped which is achieved by expression of the vesicular stomatitis virus G glycoprotein (VSVG). VSVG is a broad tropism envelope protein that can also be
pseudotyped to alter infectivity.

Next, the virus must assemble and have appropriate factors displayed on the outside of the particle to interact with the host. This is the role of Gag proteins: they contain the matrix, capsid, and nucleocapsid viral components needed for viral packaging, assembly, and display.

Pol, the last component, contains the reverse transcriptase and integrase
components to convert the cargo from RNA to DNA and permanently insert the cargo into the host genome.