XMRV Not Laboratory Contaminant -Strong Argument

[Guest guest]

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Many thanks to Dr Speedy

Professor Racaniello: Why XMRV is not a

laboratory contaminant at: http://bit.ly/dRhPZu

~jvr

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http://bit.ly/hh2KQn

virology blog

ABOUT VIRUSES AND VIRAL DISEASE

Retroviral integration

and the XMRV provirus

4 January 2011

By Racaniello

A virology Professor unravels viruses

Questions? virology@...

A strong argument that the novel human retrovirus

XMRV is not a laboratory contaminant is the the finding

that viral DNA is integrated [http://bit.ly/eTDb5T] in

chromosomal DNA of prostate tumors.

Why does this result constitute

such strong proof of viral infection?

Establishment of an integrated copy of the viral genome –

the provirus – is a critical step in the life cycle of retroviruses.

Proviral DNA is transcribed by cellular RNA polymerase II to

produce the viral RNA genome and the mRNAs required to

complete the replication cycle. Without proviral DNA, retroviral

replication cannot proceed.

To produce proviral DNA, the retroviral RNA genome is

converted to a double-stranded DNA by the viral enzyme

reverse transcriptase.

This step occurs in the cytoplasm.

Specific and efficient insertion of the viral DNA into the host

cell DNA is catalyzed by a viral enzyme called integrase.

This enzyme recognizes and nicks the two ends of viral DNA,

and the new 3'ends are then joined covalently to the host

DNA at staggered nicks made by integrase.

The image below shows some of the characteristic features

of retroviral integration.

A the top is the unintegrated linear DNA of avian

sarcoma/leukosis virus produced by reverse transcription.

Upon completion of integration, two base pairs (AA•TT) are

lost from both termini, and a 6-bp target site in host DNA

(pink) is duplicated on either side of the proviral DNA.

This target site varies in length from 4 to 6 bp among

different retroviruses. The proviral DNA (middle) ends with

the conserved 5'-T G…C A-3' sequence.

The provirus serves as a template for the production of the

viral RNA genome (bottom).

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Photo: http://bit.ly/hh2KQn

a bigger version can be found at: http://bit.ly/ePtaUU

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To identify XMRV proviral DNA, genomic DNA was isolated from

prostate tumors, and DNA was amplified using a primer that

annealed in the viral env gene, near the right-hand LTR.

Nucleotide sequence analyses of amplified DNAs from 14

different patients showed the expected viral CA sequence

followed by human DNA.

However, the other cardinal sign of retroviral integration –

duplication of host DNA sequences flanking the integration

site – could not be confirmed, because only the right-hand

integration site was studied.

The isolation of the entire proviral DNA, including both flanking

integration sites, from patients with prostate cancer or chronic

fatigue syndrome would be additional evidence that XMRV is a

virus that infects humans.

Kim, S., Kim, N., Dong, B., Boren, D., Lee, S., Das Gupta, J.,

Gaughan, C., Klein, E., Lee, C., Silverman, R., & Chow, S.

(2008). Integration Site Preference of Xenotropic Murine

Leukemia Virus-Related Virus, a New Human Retrovirus

Associated with Prostate Cancer Journal of Virology, 82 (20),

9964-9977 DOI: 10.1128/JVI.01299-08 [http://bit.ly/ffRhsS]