BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 30. VIRUSES

30.15. Darwinian Evolution of Phage RNA in vitro

The purification of phage Qβ RNA and Qβ replicase from nuclease contaminants enabled Sol Spiegelman to study Evolutionary Processes outside of a living Cell. One of the central questions was: what happens to RNA molecules when the sole Selection pressure placed upon them is the requirement to replicate as rapidly as possible? Phage Qβ RNA, Qβ replicase, and ribonucleoside triphosphates were incubated for 20 minutes. This incubation period favors the selection of mutant RNA molecules that replicate rapidly. A sample of this incubation mixture was then transferred and diluted into a fresh batch of standard reaction mixture containing Qβ replicase and ribonucleoside triphosphates. After 75 such serial transfers, the resulting RNA products were analyzed. The incubation time was progressively shortened because the RNA molecules replicated at an increasingly fast rate throughout the experiment. Most remarkably, after 75 transfers ("generations"), the length of the RNA molecules was only 12% of the original phage Qβ RNA. NUCLEOTIDES nonessential for Replication were lost as shorter molecules replicated faster. The primary constraint imposed by the experimental conditions was the retention in the mutant molecules of the initiation sequence recognized by Qβ replicase.

30.16. Lysogenic Phages Can Integrate Their DNA into the Host Cell Genome

Some Bacteriophages can follow one of two developmental pathways after infecting a host cell: they can replicate and lyse the infected cell (the lytic pathway), or their DNA can integrate into the host cell DNA without exhibiting replication or lytic activity (the lysogenic pathway). Viruses that do not invariably kill their host Cells are termed temperate. The best-studied temperate virus is phage λ (Fig. 30.27), which we discussed earlier in connection with METABOLISM/31.html">Transcription regulation (Section 28.11). Recall that the λ phage repressor binds to two sets of operator sites, O_L and O_R, and regulates its own synthesis.

Class="center">Fig. 30.27. Electron micrograph of phage

The virion DNA is a linear double-stranded molecule of 48 kb. The 5'-end of each strand features a single-stranded sequence of 12 nucleotides. These sequences are called cohesive (sticky) ends because they are mutually complementary and can base-pair with one another. In fact, they anneal almost immediately upon infection. As a result, the 5'-phosphate of each strand is brought into close proximity with its own 3'-hydroxyl terminus. The host cell DNA ligase seals these nicks, yielding a circular phage DNA molecule (Fig. 30.28).

Fig. 30.28. Conversion of linear phage DNA into a circular form

Replication of this circular phage λ DNA molecule proceeds through the interaction of Proteins encoded by phage with the host cell replication machinery. Alternatively, the circular phage λ DNA can integrate into the bacterial chromosome via a single reciprocal recombination event between specific 15-base-pair DNA regions of phage and E. coli. The attachment site (att site) for phage in the E. coli chromosome is designated attλ and is located between the galE and bioA GENES OF THE galactose and biotin operons. The base sequence of attλ can be represented symbolically as B-B' (where B stands for bacterial). The specific attachment site in phage λ is designated attP and is located near the int (integrase) and xis (excisionase) genes. The base sequence of attP is denoted P-P' (where P stands for phage). The int protein recognizes the P-P' sequence in the phage DNA and the B-B' sequence in the E. coli DNA. A reciprocal strand exchange then takes place: P joins with B', and B joins with P'. This mechanism (Figs. 30.29 and 30.30) was originally proposed by Allan Campbell on The basis of genetic data.

Fig. 30.29. Scheme of reciprocal recombination between phage DNA and E. coli DNA

Fig. 30.30. Integration and excision of phage λ DNA. This is a simplified diagram, as some factors involved in these processes remain unidentified

Phage λ DNA now forms an integral part of the E. coli DNA molecule. This integrated state is referred to as a prophage, and the E. coli cell harboring the prophage is called a lysogenic bacterium. The prophage remains stable in the absence of the xis protein. Transcription of the xis Gene is repressed by the phage λ repressor (Section 28.11). When this repression is lifted, the xis and int proteins jointly catalyze the Cleavage of the B—P' and P—B' junctions, and reciprocal strand exchange occurs once again (Fig. 30.30): P joins with P', and B joins with B', thereby regenerating the circular phage λ DNA molecule and the non-lysogenic E. coli chromosome. The key feature of this recombination system is that the int protein alone is unable to recognize the two novel hybrid sequences at the prophage borders (B-P' and P-B'), rendering them stable. Thus, phage insertion requires only the int protein, whereas prophage excision requires both the int and xis proteins.

30.17. Retroviruses and Certain DNA Viruses Can Induce Cancer in Susceptible Host Cells

In 1911, Peyton Rous prepared a cell-free filtrate from a Connective Tissue tumor that had arisen spontaneously in a chicken and injected it into normal chickens. Remarkably, the recipients developed highly malignant tumors of the same type, known as sarcomas. Rous also discovered that the tumor-inducing agent in the filtrate—now known as Rous Sarcoma virus (RSV) or avian sarcoma virus (ASV) (Fig. 30.31)—could be propagated through serial passage in chickens. Avian sarcoma virus belongs to the group of RNA tumor viruses (oncogenic RNA viruses). These viruses contain (+) RNA within their virions and replicate via a double-stranded DNA intermediate; consequently, they are termed Retroviruses. Retroviruses are the only RNA viruses capable of causing Cancer. In addition, A number of DNA viruses can induce malignant tumors. Simian virus 40 (SV40) and polyomavirus belong to the papovavirus family (Fig. 30.32) and are the most intensively studied oncogenic DNA viruses. These RNA and DNA viruses are of particular interest because they contain only four or five genes. Since tumor induction involves just one or two viral genes, researchers hold high hopes of elucidating their MECHANISM OF ACTION.

Fig. 30.31. Electron micrograph of an avian sarcoma virus (Rous sarcoma virus), a retrovirus. Heavily stained virions are visible near The surface of an infected chicken cell

Fig. 30.32. Electron micrograph of the oncogenic DNA virus SV40

Tissue culture systems have been developed to study cancer at THE MOLECULAR LEVEL. When an oncogenic virus infects suitable animal cells, they become permanently transformed, meaning they acquire cancer-like characteristics. Transformed cells differ from normal ones in their growth patterns and cell Structure/108.html">Surface Properties (Table 30.3). The most striking change is that transformed cells grow continuously and haphazardly, regardless of neighboring cells. Furthermore, transformed cells contain virus-specific DNA integrated into the host cell genome. This explains why transformation is a heritable phenotypic change. Cultures derived from transformed colonies permanently retain the abnormal properties of transformed cells. In addition, some transformed cells obtained from tissue culture will form cancerous tumors when introduced in sufficient numbers into a suitable host.

Table 30.3. Alterations in cell properties upon transformation by DNA or RNA tumor viruses

Growth Characteristics

Form tumors when introduced into susceptible animals

Grow to a much higher density; growth becomes unoriented in space, and cells detach from the growth surface

Protease activators are secreted into the medium, increasing cell invasiveness

Reduced requirement for serum growth factors

Surface Properties

Appearance of new virus-specific Antigens

Loss of Fibronectin, an outer cell surface protein

Decreased ganglioside content

Appearance of fetal antigens on The Cell surface

Increased rate of nutrient transport

Enhanced agglutinability by plant Lectins

Markers of Tumor Virus Presence

Presence of viral DNA sequences

Presence of virus-specific mRNAs

Detection of virus-specific antigens