The development of recombinant DNA methods by Cohen and Boyer in 1973, together with the discovery of reverse transcriptase by Temin and Baltimore in 1970, made it possible to introduce a mutation at any location in a viral genome. The essential reagent is an infectious DNA clone, a double-stranded DNA copy of the viral genome carried in a bacterial plasmid. These DNAs (or RNAs produced from them) can be introduced into cells by transfection1 to produce infectious virus.
Infectious clones of viral genomes were initially produced in the late 1970s and early 1980s. The first was made in 1978 by inserting a DNA copy of the RNA genome of the bacteriophage QB, made with reverse transcriptase, into a plasmid vector. Infectious virus was produced when the cloned viral DNA was inserted into E. coli. In 1980, infectious cloned retroviral DNA was produced by inserting the integrated viral DNA from the cellular genome into a plasmid vector. The next year, a DNA copy of the RNA genome of poliovirus was produced by reverse transcription and inserted into a plasmid vector. When the cloned copy of the viral genome was introduced into mammalian cells, infectious virus was produced.
Since these early findings, infectious DNAs of members of nearly every virus family have been reported. Some have been more difficult to produce than others. For example, to recover infectious influenza virus from cloned DNA, an expression system is used in which cloned DNA copies of the eight RNA segments are flanked by two promoters. Upon introduction of the eight plasmids into cultured cells, two types of RNAs are produced: mRNAs for the synthesis of viral proteins, and viral RNAs for replication and incorporation into virions. The production of infectious DNAs of (-) strand RNA viruses was counterintuitive. The (-) strand genomic RNA of these viruses is not infectious because it cannot be translated or copied into mRNA in the cell. In the first attempts, full-length (-) strands, produced by in vitro transcription of cloned DNA, were introduced into cells that produce the proteins required for mRNA synthesis. However, no infectious virus was recovered. The solution, found first with rabies virus, was to transfect full-length (+) strand RNA into cells that produce the viral nucleocapsid protein, phosphoprotein, and polymerase. In these cells, the (+) strand RNA is copied into (-) strand RNAs which then initiate an infectious cycle.
The double-stranded RNA genome of reoviruses is not infectious because it cannot be translated. To produce an infectious clone, DNA copies of the genome segments are placed in plasmids under the control of a T7 RNA polymerase promoter. When all 10 plasmids are introduced into cells that synthesize T7 RNA polymerase, viral mRNAs are produced which initiate an infectious cycle.
The complete genomes of many DNA viruses, including polyomaviruses, papillomaviruses, and adenoviruses, are sufficiently small to be carried in plasmid vectors. However, conventional plasmid vectors cannot accommodate the larger DNA genomes of herpesviruses and poxviruses; therefore cosmids and bacterial artificial chromosomes vectors, which can accept larger inserts, have been used. Such vectors have also been used to carry DNA copies of the largest RNA genomes, those of members of the Nidovirales. Poxvirus DNA is not infectious, because cellular DNA-dependent RNA polymerase cannot recognize the viral promoters. Viral DNA-dependent RNA polymerase and transcription proteins must therefore be provided.
The infectious viral DNA clone is a double-edged sword. It enables manipulation of the viral genome at will, allowing unprecedented genetic analysis and the use of viruses as vectors for gene therapy. But nearly any virus can now be recovered from the nucleotide sequence – effectively making it impossible to ever truly eradicate a virus from the globe.
1The introduction of DNA or RNA into cells with the object of obtaining infectious virus is called transfection (transformation-infection). This phrase was originally coined to describe production of bacteriophage lambda after transformation of cells with viral DNA. Transfection is now incorrectly used to describe the introduction of any DNA into cells. This usage has come about to avoid confusing DNA-mediated transformation with the process of oncogenic transformation.
Taniguchi T, Palmieri M, & Weissmann C (1978). A Qbeta DNA-containing hybrid plasmid giving rise to Qbeta phage formation in the bacterial host [proceedings] Annales de microbiologie, 129 B (4), 535-6 PMID: 754572
Lowy DR, Rands E, Chattopadhyay SK, Garon CF, & Hager GL (1980). Molecular cloning of infectious integrated murine leukemia virus DNA from infected mouse cells. Proceedings of the National Academy of Sciences of the United States of America, 77 (1), 614-8 PMID: 6244569
Racaniello, V., & Baltimore, D. (1981). Cloned poliovirus complementary DNA is infectious in mammalian cells Science, 214 (4523), 916-919 DOI: 10.1126/science.6272391
Schnell MJ, Mebatsion T, & Conzelmann KK (1994). Infectious rabies viruses from cloned cDNA. The EMBO journal, 13 (18), 4195-203 PMID: 7925265
KOBAYASHI, T., ANTAR, A., BOEHME, K., DANTHI, P., EBY, E., GUGLIELMI, K., HOLM, G., JOHNSON, E., MAGINNIS, M., & NAIK, S. (2007). A Plasmid-Based Reverse Genetics System for Animal Double-Stranded RNA Viruses Cell Host & Microbe, 1 (2), 147-157 DOI: 10.1016/j.chom.2007.03.003
25 thoughts on “Infectious DNA clones”
I am just beginning to anticipate needing to produce infectious clones of reovirus and rotavirus, and I had not even begun to look at the available literature. The Kobayashi reference is going to help immensely, I predict!
(Just found your blog and I have enjoyed reading it…)
What I did not mention in the post was that many labs tried for years
to produce infectious DNA of reovirus. All kinds of reasons for why it
didn't work were set forth. In the end, it turned out that the
sequences were wrong. Kobayashi et al sequenced many clones until they
got it right, and the result was infectivity. So make sure your
sequences are correct….sequence multiple independent clones for each
dsRNA segment and arrive at a consensus. Good luck, and glad you enjoy
That's bad an infectious clone. Well DNA sometimes cause bad.
That's bad an infectious clone. Well DNA sometimes cause bad.
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Looks like you’ve done your research very well.
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NextFect 2012 was created by organic chemists and validated by cellular
biologists for maximum Universal transfection performance. It is a novel agent
that is highly effective in a universal fashion.
â€¢ Universal Nucleic acid Delivery (exceptional for DNA and RNA)
rapid protocol with fewer steps for consistent & reproducible results
Compatible with a broad range of cell types
â€¢ Streamlined protocol
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A little late to the party, but here: http://www.ncbi.nlm.nih.gov/pubmed/21798351
So basically this is how they created HTLV-3 (HIV), cloning MVV & BIV with the possibility of EIAV? Everything makes sense. It was so easy.
I too am a Little late here but I am fasinated about the origins of HTLV-3, WOW, does this all start to make perfect sense! So basically HTLV-3 could be the ancestor of many SIVÂ´s not the other way around! This is scary stuff!
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Idiot: this was disproved about as long ago as the idea was first floated. The first known isolate of virus dates back to 1959 – which is way before anyone could do anything like whole genome cloning.
Vincent, I was just getting around to this part of history – and I see you already wrote it! I shall refer out, then. Thanks!
HIV or HTLV-3(4) are man made lab viruses I would hazard a guess the HTLV-1 is BLV. There is absolutely no empirical evidence to remotely suggest HIV came from Africa. Many 100,000Â´s (millions) were dying of HIV/AIDS, which went unnoticed & then in 1979 (2 cases of KS in NYC in 77 (cluster)) in jumped the Atlantic & landed in New YorkÂ´s gay district. This has never been explained how a heterosexual viruses landed in a minority sector. HIV/AIDS came out of no where. ItÂ´s certainty not a SIV, if it was not a cattle virus it could come from any where. Note, KSHV (HHV-8), is very close to a DNA, owl monkey virus (central America). There were stealth viruses in the Hepatitis B vaccinations on 1979.81! NOTHING HAS EVER BEEN DISPROVED! I would not trust any scientist!
Also I should have noted that in African HIV/AIDS, KS & PCP donÂ´t exist. Chris Jenkins “AIDS/HIV Facts & Fiction, Africa) Chris Jenkins, is a Harvard educated, & one of the top authors on this subject. Not a conpiracist. He denies that HIV derived from a lab, unlike myself.
Moreover, a single isolated case can NOT be HIV, it has to be a cluster as HIV is an acquired illness not a primary. Primary (CIDÂ´s) do exist abeit very rare, the isolated HIV cases you cite are either PIDÂ´s or CVIDÂ´s certainly not HIV, now who is the idiot?
That’s the nice thing about conspiracy theorists: they can just ignore all evidence that doesn’t fit with their obsessions. Good bye.
You should listen to him about HIV not coming from a lab.
Actually Jennings, states that HIV was created by accident, Matt Essex, although HIV could have been created any where. It is Visna (MVV) but it is something(s) else as well. The genetic make up of the virus is closest to Visna but the synchronisation harmology is out. HIV, as Jennings, states came out of the U.S. not Africa! Where is your proof that HIV, didnÂ´t come from a lab? Viruses, donÂ´t come out of no where.
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So are deproteinated DNA viral genomes infectious if they are able to be transported into the nucleus?, Also, why are dsRNA viral genomes non-infectious?
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