Mini-Paper: A Fo c u s o n t h e R e s e a rc h T H E D I S CO V E R Y O F R E V E R S E T R A N S C R I P TA S E D. Baltimore. 1970. RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature 226:1209–1211. H. Temin and S. Mizutani. 1970. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature 226:1211–1213.
Context By the 1960s, several lines of evidence indicated that the replication of RNA tumor viruses, like the tumorigenic virus ﬁrst described by Peyton Rous in 1911, included a DNA intermediate. First, researchers observed that DNA synthesis inhibitors block replication of these viruses, but only if these inhibitors are added to cells early after their infection. Second, researchers observed that actinomycin D, a drug that inhibits the DNA-dependent RNA polymerase responsible for transcription in mammalian cells, also prevents the formation of new virus particles. Third, several experiments showed that cells infected by RNA tumor viruses contain DNA that hybridizes with, or binds to, viral RNA. Based on these observations, Baltimore and Temin independently postulated that the replication of RNA tumor viruses proceeds through a DNA intermediate. In other words, the RNA genome is converted into DNA, which later is converted back into RNA genomes. This model would explain the observations previously described. This model also would require the existence of a RNA-dependent DNA polymerase, or an enzyme that could convert RNA into DNA. Such an enzyme had never been observed in any organism. Furthermore, such an enzyme clearly would violate the central dogma of biology: that DNA is converted to RNA, which is converted to protein.
Experiments To determine if RNA tumor viruses contained RNA-dependent DNA polymerases, the researchers asked two fairly straightforward questions. First, do the viruses produce DNA? Second, what is the template from which this DNA is derived? To address the ﬁrst question, both groups started with puriﬁed preparations of two RNA tumor viruses: Rauscher mouse leukemia virus
the disease also is limited. Thus, a very detailed understanding of how inﬂuenza virus replicates was essential for the development of this effective antiviral medication. Inﬂuenza viruses, however, may develop resistance to this drug. By October 2009, the WHO had reported 39 oseltamivir-resistant isolates of H1N1 inﬂuenza virus. The emergence of these drug-resistant strains of inﬂuenza has created concern and a need for rapid detection of resistance. Following a signiﬁcant increase in Tamiﬂu resistance during the 2007–2008 ﬂu season, researchers have been closely monitoring resistance rates. The method they use to screen for increased oseltamivir resistance is called the neuraminidase inhibition assay. This assay allows scientists to quantitate viral neuraminidase activity
262 Chapter 8 Viral Replication Strategies
(R-MLV) or Rous sarcoma virus (RSV). Refer to Chapter 5 to review how viruses can be isolated, puriﬁed, and quantiﬁed. Next, they added these viruses to a standard DNA polymerase assay. The ingredients of this assay included the four deoxyribonucleotide triphosphates (dATP, dTTP, dCTP, and dGTP), the precursors of DNA. Additionally, one of the nucleotides, thymidine, was labeled with 3H, a radioactive form of hydrogen. Following an incubation, trichloroacetic acid was added to the reaction and the entire reaction then was ﬁltered through a ﬁne ﬁlter. Previous research had shown that DNA was acidinsoluble. In other words, any DNA produced in the reaction would precipitate out of solution when the acid was added and would be retained on the ﬁlter. By simply measuring the amount of radioactivity retained on the ﬁlter, the researchers could determine if any DNA had been produced. The reaction did, indeed, result in the formation of an acid-insoluble product. To conﬁrm that this product was DNA, Baltimore then treated the completed reaction with deoxyribonuclease, an enzyme that destroys DNA, ribonuclease, an enzyme that destroys RNA, and micrococcal nuclease, a relatively non-speciﬁc nuclease that destroys both RNA and DNA. Deoxyribonuclease and micrococcal nuclease, but not ribonuclease, digested the radioactive product. These two results support the conclusion that puriﬁed preparations of R-MLV could produce DNA. To address the second question, Baltimore and Temin both investigated the sensitivity of the virus to ribonuclease (Figure B8.6). In the absence of ribonuclease, the amount of DNA produced increased over time (line 1). A similar increase was observed when ribonuclease was added after the virus and deoxynucleotides were allowed to incubate together for several minutes (line 2). When ribonuclease was included initially in the reaction mixture, however, the amount of DNA produced decreased markedly (line 3). When the virus was pre-incubated with ribonuclease before being added to the polymerase assay reaction, the amount of DNA produced decreased even more dramatically (line 4). These results support the conclusion that RNA is the template for DNA production.
as a means of detecting resistance to the drug. Resistant strains continue to have neuraminidase (NA) activity even in the presence of the drug. Oseltamivir is mixed with varying dilutions of the virus being tested and then a substrate for the neuraminidase enzyme is added to the mixture. When the substrate is degraded, chemiluminescence or light levels are detected by a luminometer. This chemiluminescence, therefore, allows you to “see” levels of neuraminidase activity. An understanding of the process of evolution indicates that widespread use of Tamiﬂu increases the probability of Tamiﬂuresistant inﬂuenza, as those strains will be selected for in the population. Surveillance of Tamiﬂu resistance will be a key strategy in monitoring and limiting resistance.
Impact The identiﬁcation of this enzyme that converts RNA to DNA, now referred to as reverse transcriptase (RT), had an immediate and profound eﬀect on the ﬁelds of virology and, more generally,
incorporated (c.p.m. × 10–3)
biology itself. Indeed, as the editors of Nature noted in a comment preceding these two articles, “This discovery, if upheld, will have important implications . . . for the general understanding of genetic transcription.” The discovery has been upheld. Today, two classes of viruses, the retroviruses and the Class VII double-stranded DNA viruses, have been shown to utilize RT. Eukaryal cells, too, utilize forms of this enzyme both for the maintenance of the ends of linear chromosomes (see Section 7.2) and the movement of certain transposable elements. Reverse transcriptase also has become an indispensable tool for the molecular biologist. As we saw in Chapter 5, RT is used to convert the RNA of HIV into DNA as part of a standard viral load test. In addition, it is used routinely in the laboratory to convert messenger RNA into DNA. Not only did the discovery of reverse transcriptase change the way biologists viewed the ﬂow of information within cells, but it also changed the way we can do experiments within the laboratory.
Questions for Discussion 3 5 4
Figure B8.6. Incorporation of radiolabeled dTTP by purified virions Virus particles were incubated with deoxynucleotides, one of which was radiolabeled. At speciﬁed times, the amount of acid-insoluble radioactivity was measured. As seen in line 1, the amount of incorporated radioactivity increased over time. The amount of incorporated radioactivity was greatly diminished if the virus was pre-incubated with ribonuclease (line 4), indicating that the template for DNA production was the viral RNA. (From D. Baltimore. Nature 226:1209–1211, 1970. Figure 1. Reproduced with permission.)
1. Given the information presented here, would you hypothesize that retroviruses bring RT into the cell or produce it after infecting a cell? 2. Do you think that RT would be a viable target for antiretroviral drugs? Give reasons why or why not. 3. We mentioned that earlier experiments showed that cells infected by RNA tumor viruses contained DNA that hybridizes with viral RNA. One explanation for this finding is that the viral RNA is converted to DNA within the infected cell. Provide another possible explanation for this initial finding.
Summary Section 8.1. How do viruses recognize appropriate cells? Most viruses interact with appropriate host cells through a speciﬁc interaction between the viral attachment protein and a cellular receptor. N For enveloped viruses, this attachment protein generally exists as a spike embedded in the envelope. N For non-enveloped viruses, the capsid may contain a spike protein or, alternatively, a less deﬁned component of the capsid may interact with the host cell. N For bacteriophages, the tail ﬁbers generally serve as the attachment proteins.
This interaction, in many cases, determines the host range of a virus and which tissues within a host a virus may infect. N Studies with the bacteriophage T2 have demonstrated that altering the tail ﬁbers of the phage can alter the host speciﬁcity of the phage. N Studies with the mammalian virus mouse hepatitis virus have demonstrated that strains of mice are more or less susceptible to this virus depending on which form of a surface protein they express. N Information about viral attachment proteins and receptors can be gleaned from two related techniques: SDS-PAGE and Western blotting.
Chapter 8: Mini-paper