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Retroviral Reverse Transcription Retroviral DNA Integration Is A Retroviral Reverse Transcription Hepadnaviral Reverse Transcription Discovery A DNA Virus with Reverse Impact Transcriptase? The Pathways of Reverse Transcription Pathway of Reverse Transcription General Properties and Structure of Perspectives Retroviral Reverse Transcriptases There Are Many Other Examples of References Reverse Transcription Retroviral DNA Integration Is a Unique Process Integrase-Catalyzed Steps in the Integration Process Integrase Structure and Mechanism Reverse Transcription and Integration “One can’t believe impossible things,” Retroviral Reverse Transcription said Alice. Discovery “I dare say you haven’t had much In 1970, back-to-back reports in the scientifi c journal Nature from the laborato- practice,” said the Queen. “Why, sometimes ries of Howard Temin and David Baltimore provided the fi rst concrete evidence I’ve believed as many as six impossible things for the existence of an RNA-directed DNA polymerase activity in retrovirus before breakfast.” particles. The pathways to this discovery were quite different in the two labo- LEWIS CARROLL Alice in Wonderland ratories. In Temin’s case, the discovery came about through attempts to under- stand how this group of RNA-containing viruses could permanently alter the heredity of cells, as they do in the process of oncogenic transformation. Temin proposed that retroviral RNA genomes become integrated into the host cell’s chromatin in a DNA form, an idea supported by the observation that purifi ed cellular DNA polymerases can use RNA as a template under certain reaction conditions in vitro. Furthermore, studies of bacterial viruses such as bacterio- phage lambda had established a precedent for viral DNA integration into host DNA (Box 7.1). However, with the technology available at the time, it was a diffi cult hypothesis to test, and attempts by Temin and others to demonstrate the existence of such a phenomenon in infected cells were generally met with skepticism. Baltimore’s entrée into the problem of reverse transcription came from his interest in virion-associated polymerases, in particular one that he had just discovered to be present in vesicular stomatitis virions, a virus with a (−) strand RNA genome. It occurred to Baltimore and Temin independently that retrovirus particles might also contain the sought-after RNA-dependent DNA polymerase. As subsequent experiments showed, this was indeed the case, and the retrovirus particles themselves yielded up the enzyme activity that had earlier eluded Temin. In 1975, Temin and Baltimore were awarded the Nobel Prize in physiology or medicine for their independent discoveries of retroviral reverse transcriptase (RT). 205 206 CHAPTER 7 BOX DISCUSSION 7.1 Bacteriophage lambda, a paradigm for the joining of viral and host DNAs In 1962, Allan Campbell proposed an ele- chromosome (called attB) would produce was favored by a number of investigators. gant, but at the time revolutionary, model an integrated viral genome, with a link- However, shortly after Campbell’s elabo- for site-specifi c integration of DNA of the age map that was a circular permutation ration of his model, circular molecules bacteriophage lambda into chromosome of that of the linear phage genome, as had of lambda phage DNA were detected of its host, Escherichia coli. The model was been observed (see part B of fi gure). in infected cells, and the linear DNA deduced from the fact that different linkage Induction of the integrated provirus extracted from purifi ed virus particles maps could be constructed for viral genomes and subsequent viral DNA replication was found to possess short complemen- at different stages in its life cycle. One link- would proceed by a reversal of this site- tary single-strand extensions, “cohesive age map, that of the prophage, was obtained specifi c integration reaction, a process ends,” that could promote circle forma- from the study of lysogenic bacteria. A differ- called excision. The model explained how tion. Other predictions of the model were ent linkage map was obtained by measuring abnormal excision (arrows a and b) could also validated in several laboratories, and recombination frequencies in the phage yield give rise to the observed rare, specialized viral and cellular proteins that catalyzed from infected cells (see part A of fi gure). transducing phages that lacked certain integration and excision were identifi ed. Campbell proposed that these unique viral genes and carried either the gal or bio Lambda DNA integration remains an features could be explained by a model genes of the host. These were the fi rst cel- important paradigm for understanding the for integration in which the incoming, lular “cloned” DNAs to be identifi ed. molecular mechanisms of DNA recombi- linear double-stranded DNA phage Although this model seems obvious nation and the parameters that infl uence genome must fi rst circularize. Subsequent today, it was not obvious in the 1960s. An the joining of viral and host DNAs. recombination between a specifi c, internal alternative model in which the linear viral sequence in the phage genome (called attP) DNA was attached by a partial binding or Campbell, A. M. 1962. Episomes. Adv. Genet. and a specifi c sequence in the bacterial “synapse” with the bacterial chromosome 11:101–145. Impact could also be a “retrograde” fl ow of information from RNA The immediate impact of the discovery of RT was to amend to DNA, and the name retroviruses eventually came to the then-accepted central dogma of molecular biology, replace the earlier designation of RNA tumor viruses. In that the transfer of genetic information is unidirectional: the years following this revision of dogma, many addi- DNA → RNA → protein. It was now apparent that there tional reverse transcription reactions have been discovered. Reverse Transcription and Integration 207 Furthermore, as Temin hypothesized, study of RT has transcription system is poised for action as soon as the virus contributed to our understanding of cancer. As described enters the cell. Indeed, small amounts of viral DNA can be in Volume II, Chapter 7, the study of oncogenic retrovi- detected in purifi ed human immunodefi ciency virus type ruses has provided a framework for current concepts of 1 particles, presumably having been synthesized using sub- the genetic basis of this disease. Study of reverse transcrip- strates picked up from the infected cell during budding. tion (and integration) has also allowed us to understand Retroviral reverse transcription intermediates have also the persistence of retroviral infections and aspects of the been analyzed in totally reconstituted reactions with puri- pathogenesis of acquired immunodefi ciency syndrome fi ed enzymes and model RNA templates. (AIDS) that is caused by the human immunodefi ciency Retroviral RT is the only protein required to accom- virus. Finally, RT itself, fi rst purifi ed from virions and now plish all the diverse steps in the pathway described below. produced in bacteria, has become an indispensable tool in However, as the reactions that take place inside cells are molecular biology, for example allowing experimentalists signifi cantly more effi cient than those observed in either to capture cellular messenger RNAs (mRNAs) as comple- endogenous or reconstituted systems, it is unlikely that all mentary DNAs (cDNAs), which can then be amplifi ed, the essential features have been reproduced. cloned, and expressed by well-established methodologies. For such reasons, we devote an entire chapter to these Essential Components very important reactions. Genomic RNA. Retrovirus particles contain two cop- ies of the RNA genome held together by multiple regions of The Pathways of Reverse Transcription base pairing. (See Box 7.2 for labeling conventions.) This Signifi cant insight into the mechanism of reverse tran- RNA sediments in a 70S complex, as expected for a dimer scription can be obtained by comparing the amino acid of 35S genomes. Partial denaturation and electron micro- sequences of RTs with those of other enzymes that catalyze scopic analyses of the 70S complex indicate that the most similar reactions. For example, RTs share (with the RNA stable pairing is via sequences located near the 5′ ends of and DNA polymerases of both prokaryotes and eukary- the two genomes (Fig. 7.1). Retroviral genomes can be otes) certain sequence motifs in regions known to include thought of as being annealed head to head, an arrange- critical active-site residues (see Fig. 6.4). It is hypothesized, ment that may discourage the encapsidation of multiples therefore, that these enzymes employ similar catalytic larger than two (i.e., concatemers). The 70S RNA complex mechanisms for nucleic acid polymerization reactions. also includes two molecules of a specifi c cellular transfer Like DNA polymerases, viral RTs cannot initiate DNA syn- RNA (tRNA) that serves as a primer for the initiation of thesis de novo, but require a specifi c primer. In this chapter, reverse transcription (discussed below). we provide a detailed description of priming and reverse Despite the fact that two genomes are encapsidated, transcription for retroviral and hepadnaviral enzymes. But only one integrated copy of the viral DNA typically is it should be noted that even as arcane and distinct from detected after infection with single virion. Therefore, ret- each other as these two systems may appear, they do not roviral virions are said to be pseudodiploid. Why should exhaust the repertoire for reverse transcription reactions such a feature have been selected during evolution? One that have evolved in nature. A wide variety of primers, as popular notion is that the availability of two RNA templates well as sites and modes of initiation, are used by the RTs of can help retroviruses survive extensive damage to their other retroelements. genomes. At least parts of both genomes can, and typically Much of what has been learned about reverse transcrip- are, used as templates during the reverse transcription pro- tion in retroviruses comes from the analysis of intermedi- cess, accounting for the high rates of genetic recombination ates in the reaction pathway that have been identifi ed in in these viruses.
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