Retroviral reverse transcription

Proceedings of the National Academy of Sciences, 1990

Retroviral transduction of cellular nucleic acid sequences requires illegitimate RNA or DNA recombination. To test a model that postulates transduction via efficient illegitimate recombination during reverse transcription of viral and cellular RNAs, we have measured the ability of Harvey sarcoma viruses (HaSVs) with artificial 3' termini to recover a retroviral 3' terminus from helper Moloney virus (MoV) by illegitimate and homologous recombination. For this purpose, mouse NIH 3T3 cells were transformed with Harvey proviruses and then superinfected with MoV. The proviruses lacked the 3' long terminal repeat and an untranscribed region of the 5' long terminal repeat to prevent virus regeneration from input provirus. Only 0-11 focus-forming units of HaSV were generated upon MoV superinfection of 3 x 106 cells transformed by Harvey proviruses with MoV-unrelated termini. This low frequency is consistent with illegitimate DNA recombination via random Moloney provirus integration 3' of the transforming viral ras gene in the 10'-kilobase mouse genome. When portions of murine viral envelope (env) genes were attached 3' of ras, 102-105 focus-forming units of HaSV were generated, depending on the extent of homology with env of MoV. These recombinants all contained HaSV-specific sequences 5' and MoV-specific sequences 3' of the common env homology. They were probably generated by recombination during reverse transcription rather than by recombination among either input or secondary proviruses, since (i) the yield of recombinants was reduced by a factor of 10 when the env sequence was flanked by splice signals and (ii) HaSV RNAs without retroviral 3' Abbreviations: HaSV, Harvey sarcoma virus; MoV, Moloney virus; LTR, long terminal repeat; ffu, focus-forming unit(s); pfu, plaqueforming unit(s).

Proceedings of the National Academy of Sciences, 1990

After mixed infection, up to half of related retroviruses are recombinants. During infection, retroviral RNA genomes are first converted to complementary DNA (cDNA) and then to double-stranded DNA. Thus recombination could occur during reverse transcription, by RNA template switching, or after reverse transcription, by breakage and reunion of DNA. It has not been possible to distinguish between these two potential mechanisms of recombination because both single-stranded cDNA and double-stranded proviral DNA exist in infected cells during the eclipse period. Therefore we have analyzed for recombinant molecules among cDNA products transcribed in vitro from RNA of disrupted vinons. Since recombinants from aflelic parents can only be distinguished from parental genomes by point mutations, we have examined the cDNAs from virions with distinct genetic structures for recombinant-specific size and sequence markers. The parents share a common internal allele that allows homology-directed recombination, but each contains specific flanking sequences. One parent is a synthetically altered Harvey murine sarcoma virus RNA that lacks a retroviral 3' terminus but carries a Moloney murine retrovirus-derived envelope gene (env) fragment 3' of its transforming ras gene. The other parent is intact Moloney virus. Using a Harvey-specific 5' primer and a Moloney-specific 3' primer, we have found recombinant cDNAs with the polymerase chain reaction, proving directly that retroviruses can recombine during reverse transcription unassisted by cellular enzymes, probably by template switching during cDNA synthesis. The recombinants that were obtained in vitro were identical with those obtained in parallel experiments in vivo.

Nature Reviews Microbiology, 2007

Retroviruses are a unique family of RNA viruses that use virally encoded reverse transcriptase (RT) to replicate genomic RNA through a full-length viral DNA intermediate. These viruses have been shown to be present in the genomes of many vertebrates, including fish, rodents, birds, cats, ungulants, non-human primates and humans. Infection by retroviruses causes a wide variety of pathologies, most commonly cancers, such as leukaemias, sarcomas and mammary carcinomas, but retroviral infection can also cause immunodeficiencies, anaemias, arthritis and pneumonia 1. The retroviridae are classified into seven different genera that are named α through to ε retroviruses, as well as the lentiviruses and spumaviruses (see the International Committee on Taxonomy of Viruses database). Historically, retroviruses have been the source of many key discoveries in biology during the twentieth century, including cell transformation, viral and cellular oncogenes, RT and viral transduction, which paved the way to cDNA cloning and design of retroviral vectors for gene therapy 1. This Review will focus on the mechanisms used by retroviruses to ensure the correct viral protein synthesis within the cytoplasm of the host infected cell with particular emphasis on αand γ-retroviruses (whose prototypes are the avian leukosis virus (ALV) and murine leukaemia virus (MLV), respectively) and primate lentiviruses (HIV-1, HIV-2 and simian immunodeficiency virus (SIV)). An overview of the retroviral life cycle. Retroviruses are enveloped RNA viruses that encapsidate two copies of the same capped and polyadenylated (positive sense) RNA molecule that ranges from 8,000 to 11,000

Proceedings of the National Academy of Sciences, 1988

We have studied whether the origin of retroviral onc genes, by transduction of sequences from cellular proto-onc genes, involves DNA or RNA recombination. By using altered Harvey sarcoma proviruses as models for transduction intermediates, we have investigated the mechanism of regeneration of transforming virus from truncated proviruses with only a single 5' long terminal repeat (LTR) but with a complete 5'-LTR-ras transforming gene. The Harvey proviruses were specifically altered to discriminate between virus regeneration by RNA template switching during reverse transcription, as has

Cancer Research

Retroviruses (without transforming genes) are thought to cause leukemias and other cancers in animals and humans because they were originally isolated from those diseases and because experimental infec tions of newborns may induce leukemias with probabilities of 0 to 90%. According to this hypothesis viral cancers should be contagious, polyclonal, and preventable by immunization. However, retroviruses are rather widespread in healthy animals and humans where they typically cause latent infections and antiviral immunity. The leukemia risk of such infections is less than 0.1% and thus about as low as that of virus-free controls. Indeed retroviruses are not sufficient to initiate transformation (a) because of the low percentage of symptomatic virus carriers and the complete lack of transforming function in vitro-,(b) because of the striking discrepancies between the long latent periods of 0.5 to 10 years for carcinogenesis and the short eclipse of days to weeks for virus replication and direct pathogenic and immunogenic effects; (c) because there is no gene with a late transforming function, since all genes are essential for replication; (d) because host genes, which do not inhibit virus, inhibit tumorigenesis up to 100% if intact and determine the nature of the tumor if defective; and above all (e) because of the monoclonal origin of viral leukemias, defined by viral integration sites that are different in each tumor. On these bases the probability that a virus-infected cell will become transformed is estimated to be about 10 ", The viruses are also

Nature Reviews Cancer, 2012

Retroviruses are the original source of oncogenes. The discovery and characterization of these genes were made possible by the introduction of quantitative cell biological and molecular techniques for the study of tumor viruses. Key features of all retroviral oncogenes were first identified in src, the oncogene of Rous sarcoma virus. These include non-involvement in viral replication, coding for a single protein, and cellular origin. The myc, ras and erbB oncogenes quickly followed src, and these together with pi3k are now recognized as critical driving forces in human cancer.

Retrovirology, 2009

The discovery of HIV-1 as the cause of AIDS was one of the major scientific achievements during the last century. Here the events leading to this discovery are reviewed with particular attention to priority and actual contributions by those involved. Since I would argue that discovering HIV was dependent on the previous discovery of the first human retrovirus HTLV-I, the history of this discovery is also re-examined.

Proceedings of the National Academy of Sciences of the United States of America, 1981

Journal of virology, 1990

Reverse transcription of the retroviral RNA genome begins with tRNA-primed synthesis of a minus-strand DNA, which subsequently acts as the template for the synthesis of plus-strand DNA. This plus-strand DNA is initiated at a unique location and makes use of a purine-rich RNA oligonucleotide derived by RNase H action on the viral RNA. To determine the variables that are relevant to successful specific initiation of plus-strand DNA synthesis, we have used nucleic acid sequences from the genome of Rous sarcoma virus along with three different sources of RNase H: avian myeloblastosis virus DNA polymerase, murine leukemia virus DNA polymerase, and the RNase H of Escherichia coli. Our findings include evidence that specificity is controlled not only by the nucleic acid sequences but also by the RNase H. For example, while the avian reverse transcriptase efficiently and specifically initiates on the sequences of the avian retrovirus, the murine reverse transcriptase initiates specifically ...

Journal of Virology, 1993

There is a copy of a short terminal repeat segment, r, at each end of the retroviral RNA genome. During reverse transcription, r is copied from the genomic RNA to form the R component of the long terminal repeat in viral DNA. Although our current model for reverse transcription suggests that the 5' r is copied, it is not known whether the 5' copy, the 3' copy, or part of each r in the genomic RNA serves as the template for the R region in the progeny viral DNA. To assess the relative contribution of the 5' and 3' r templates, we examined the effect of mutations located at the center of the 5' or 3' r of spleen necrosis virus and determined the sequence of the R region in the progeny proviruses after a single round of retroviral replication. In approximately 90% of the proviruses, the 5' r marker was copied, whereas 10% of the proviruses had derived the R marker from the 3' r.