Microbiology and Immunology

Viral vectors in gene therapy WORLD OF MICROBIOLOGY AND IMMUNOLOGY

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is transcribed by the host own machinery. The resulting tran-

scripts are either used to synthesize proteins or produce new

viral particles. These new viruses are released by budding, usu-

ally without killing the host cell. Both HIVand HTLVviruses

belong to this class of viruses.

Virus genetics are studied by either investigating genome

mutationsor exchange of genetic material during the life cycle

of the virus. The frequency and types of genetic variations in the

virus are influenced by the nature of the viral genome and its

structure. Especially important are the type of the nucleic acid

that influence the potential for the viral genome to integrate in

the host, and the segmentation that influence exchange of

genetic information through assortment and recombination.

Mutations in the virus genome could either occur spon-

taneously or be induced by physical and chemical means.

Spontaneous mutations that arise naturally as a result of viral

replication are either due to a defect in the genome replication

machinery or to the incorporation of an analogous base instead

of the normal one. Induced virus mutantsare obtained by either

using chemical mutants like nitrous oxide that acts directly on

bases and modify them or by incorporating already modified

bases in the virus genome by adding these bases as substrates

during virus replication. Physical agents such as ultra-violet

light and x rays can also be used in inducing mutations.

Genotypically, the induced mutations are usually point muta-

tions, deletions, and rarely insertions. The phenotypeof the

induced mutants is usually varied. Some mutants are condi-

tional lethal mutants. These could differ from the wild type

virus by being sensitive to high or low temperature. A low tem-

perature mutant would for example grow at 88°F (31°C) but

not at 100°F (38°C), while the wild type will grow at both tem-

peratures. A mutant could also be obtained that grows better at

elevated temperatures than the wild type virus. These mutants

are called hot mutants and may be more dangerous for the host

because fever, which usually slows the growth of wild type

virus, is ineffective in controlling them. Other mutants that are

usually generated are those that show drug resistance, enzyme

deficiency, or an altered pathogenicity or host range. Some of

these mutants cause milder symptoms compared to the parental

virulent virus and usually have potential in vaccinedevelop-

ment as exemplified by some types of influenzavaccines.

Besides mutation, new genetic variants of viruses also

arise through exchange of genetic material by recombination

and reassortment. Classical recombination involves the break-

ing of covalent bonds within the virus nucleic acid and

exchange of some DNA segments followed by rejoining of the

DNA break. This type of recombination is almost exclusively

reserved to DNA viruses and retroviruses. RNA viruses that do

not have a DNA phase rarely use this mechanism.

Recombination usually enables a virus to pick up genetic

material from similar viruses and even from unrelated viruses

and the eukaryotic host cells. Exchange of genetic material

with the host is especially common with retroviruses.

Reassortment is a non-classical kind of recombination that

occurs if two variants of a segmented virus infect the same

cell. The resulting progeny virions may get some segments

from one parent and some from the other. All known seg-

mented virus that infect humans are RNA viruses. The process

of reassortment is very efficient in the exchange of genetic

material and is used in the generation of viral vaccines espe-

cially in the case of influenza live vaccines. The ability of

viruses to exchange genetic information through recombina-

tion is the basis for virus-based vectors in recombinant DNA

technology and hold great promises in the development of

genetherapy. Viruses are attractive as vectors in gene therapy

because they can be targeted to specific tissues in the organs

that the virus usually infect and because viruses do not need

special chemical reagents called transfectants that are used to

target a plasmid vector to the genome of the host.

Genetic variants generated through mutations, recom-

bination or reassortment could interact with each other if

they infected the same host cell and prevent the appearance

of any phenotype. This phenomenon, where each mutant

provide the missing function of the other while both are still

genotypically mutant, is known as complementation. It is

used as an efficient tool to determine if mutations are in

unique or in different genes and to reveal the minimum num-

ber of genes affecting a function. Temperature sensitive

mutants that have the same mutation in the same gene will

for example not be able to complementeach other. It is

important to distinguish complementation from multiplicity

reactivation where a higher dose of inactivated mutants will

be reactivated and infect a cell because these inactivated

viruses cooperate in a poorly understood process. This reac-

tivation probably involves both a complementation step that

allows defective viruses to replicate and a recombination

step resulting in new genotypes and sometimes regeneration

of the wild type. The viruses that need complementation to

achieve an infectious cycle are usually referred to as defec-

tive mutants and the complementing virus is the helper virus.

In some cases, the defective virus may interfere with and

reduce the infectivity of the helper virus by competing with

it for some factors that are involved in the viral life cycle.

These defective viruses called “defective interfering” are

sometimes involved in modulating natural infections.

Different wild type viruses that infect the same cell may

exchange coat components without any exchange of genetic

material. This phenomenon, known as phenotypic mixing is

usually restricted to related viruses and may change both the

morphology of the packaged virus and the tropism or tissue

specificity of these infectious agents.

See alsoViral vectors in gene therapy; Virology; Virus repli-

cation; Viruses and responses to viral infection

VIRAL INFECTIONS•seeVIRUSES AND RESPONSES

TO VIRAL INFECTION

VViral vectors in gene therapyIRAL VECTORS IN GENE THERAPY

Genetherapy is the introduction of a gene into cells to reverse

a functional defect caused by a defect in a host genome (the

set of genes present in an organism).

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