BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 30. VIRUSES

30.18. SV40 and Polyoma Viruses Can Cause Productive Infection or Host-Cell Transformation

SV40 and polyoma Viruses contain a small circular double-stranded DNA molecule enclosed within an icosahedral shell. In certain Cells (termed permissive host cells), The Development of these viruses follows a lytic cycle, leading to The production of numerous new virions (Fig. 30.33).

Class="center">Fig. 30.33. Electron micrograph of a nuclear membrane fragment from an SV40-infected Cell, showing nuclear pores and numerous virions

These viruses kill permissive cells during a productive infection. In other cell types (nonpermissive host cells), certain stages of viral Gene Expression are blocked for reasons that remain unclear, and no progeny virus is produced. However, a small fraction of these cells—on the order of one in 10⁵—becomes transformed As a result of the integration of viral DNA into the host cell genome.

To date, the complete nucleotide sequence of the 5243 Base Pairs of SV40 DNA has been elucidated, and many aspects of its Replication and METABOLISM/31.html">Transcription are under intensive investigation. Half of the DNA is transcribed early in infection, while the other half is transcribed late, concurrently with viral DNA Synthesis (Fig. 30.34).

Fig. 30.34. Genetic Map of SV40 DNA, containing 5243 base pairs. The early region (transcribed counterclockwise) is shown in yellow, the late region (transcribed clockwise) in blue, and THE ORIGIN OF replication (ori) in red

Table 30.4. Proteins encoded by SV40

The origin of replication is located in the same region as the start sites for early and late transcription. The early region is transcribed counterclockwise and encodes the T antigen (protein A), which is required to initiate DNA replication. Another immunologically distinct protein, the small t antigen, is also encoded by the early region. The synthesis of T-antigen mRNA involves the excision of an intervening sequence (intron) from the primary transcript.

Thus, SV40 utilizes the splicing machinery of the host Cell Nucleus. The base sequence at the origin of replication is also noteworthy. It contains two 13-base-pair sequences possessing twofold rotational Symmetry, situated adjacent to an AT-rich region:

The T antigen binds to this segment.

Papovaviruses are a group of Introduction/6.html">DNA-containing viruses. The name is derived from the names of three group members: papillomaviruses, polyomaviruses, and the vacuolating virus (SV40).

Transcription of the late region proceeds clockwise from the origin of replication (Fig. 30.34), resulting in the synthesis of three capsid proteins: VP1, VP2, and VP3. Here too, intervening sequences are removed from the primary transcript. These three mRNAs appear to be generated via different splicing pathways. The N-terminal Amino Acid Sequence of VP3 overlaps 70% of the C-terminal sequence of VP2. Furthermore, an overlapping stretch of 22 NUCLEOTIDES is read in one reading frame during the synthesis of VP2 and VP3, and in a different frame during the synthesis of VP1. Thus, the limited Genetic information of SV40 is utilized with maximal efficiency. Another example of genetic economy is that SV40 does not synthesize its own proteins for DNA packaging; newly synthesized DNA associates with host-cell Histones (Fig. 30.35). This supercoiled complex is then packaged into the capsid with the assistance of proteins VP1, VP2, and VP3. Ultimately, progeny virions are released upon lysis of the host cell, which dies in the process.

Fig. 30.35. Electron micrograph of assembling tumor-causing SV40 virus particles associated with a cellular chromosome

In nonpermissive cells, the early region of the SV40 genome is expressed, whereas the late region is neither replicated nor transcribed. A small proportion of these cells become transformed through the Integration of the SV40 genome into cellular DNA. Unlike the integration of phage λ DNA, the integration of SV40 DNA does not appear to involve specific homologous sites on either the viral or cellular DNA. Both viral Antigens (T and t) are required for transformation and, in all likelihood, for the Maintenance of the transformed state. Introducing such transformed cells into susceptible animals leads to the rapid formation of tumors. The primary objective of current research on SV40 is to determine how the expression of the integrated viral early region renders a cell cancerous.

30.19. Retroviruses Contain Reverse Transcriptase, Which Synthesizes Double-Stranded DNA Using (+) RNA as a Template

Another class of Oncogenic Viruses—Retroviruses—contains a (+) RNA genome enclosed within an icosahedral shell. This spherical nucleoprotein core is surrounded by a membrane composed of virus-encoded glycoprotein molecules embedded in a lipid bilayer derived from the host cell Plasma Membrane. Retroviruses typically have a diameter of 1000 Å (see Fig. 30.31).

In 1964, Howard Temin observed that the infection by RNA tumor viruses, such as avian Sarcoma virus, is blocked by DNA synthesis inhibitors. Inhibitors such as amethopterin, 5-fluorodeoxyuridine, and cytosine arabinoside are effective within the first twenty hours following viral inoculation. This discovery led to the hypothesis that DNA synthesis is required for the replication of RNA tumor viruses. Furthermore, the production of progeny virus particles is suppressed by actinomycin D. This antibiotic is known to inhibit template-directed RNA Synthesis (Section 25.18). Thus arose the hypothesis that DNA Transcription is necessary for the reproduction of RNA tumor viruses. These unexpected findings led Temin to suggest that a DNA provirus serves as an intermediate in the replication and oncogenic action of these viruses:

Temin's hypothesis that genetic information could flow from RNA to DNA was initially met with skepticism by most researchers. It required the existence of a hitherto unknown enzyme capable of synthesizing DNA from an RNA template (RNA-dependent DNA polymerase). In 1970, Temin and Baltimore independently discovered such an enzyme, known as Reverse Transcriptase, in the virions of certain RNA tumor viruses. All subsequently studied viruses of this group were found to contain reverse transcriptase, which is why they are called retroviruses (from reverse transcriptase).

The life cycle of a typical retrovirus begins with the binding of virions to specific receptors on the host cell surface and entry into The Cell. In the Cytosol, the viral (+) RNA is uncoated. Then, the reverse transcriptase contained within the viral particle synthesizes the (-) DNA strand. The same enzyme degrades the genomic RNA strand within the RNA–DNA hybrid. Next, reverse transcriptase synthesizes the (+) DNA strand using the (-) strand as a template. Thus, reverse transcriptase catalyzes three sequential reactions: RNA-dependent DNA synthesis, RNA Hydrolysis, and DNA-dependent DNA synthesis (Fig. 30.36).

Fig. 30.36. DNA synthesis on an RNA template by reverse transcriptase. The primer is not shown

Like other DNA polymerases, reverse transcriptase synthesizes DNA in the 5' → 3' direction and is unable to initiate chains de novo. Where does the primer for viral DNA synthesis come from? The initiation process is remarkably economical: the (+) RNA of the viral genome contains a non-covalently linked Transfer RNA (in avian sarcoma virus, this is a Tryptophan tRNA) that was captured from the host cell during the previous infection cycle. The 3'-OH group of this tRNA, whose bases are paired with the genomic RNA, acts as a primer for DNA synthesis. How is the replication of the full-length (+) RNA strand achieved?

Recall that the replication of any linear DNA presents a specific problem in filling in the 5' ends (Section 30.6). Retroviruses have found a very ingenious solution to this problem. Their genomes consist not of a single molecule, but of two (+) RNA molecules (Fig. 30.37). These molecules are joined together by Hydrogen Bonds near their 5' ends. In addition, the (+) RNA contains the same sequence at both the 5' and 3' ends. This terminal redundancy appears to be essential for the replication process, much like in phage T4 (Section 30.6).

Fig. 30.37. Schematic diagram of the avian sarcoma virus genome. Two identical (+) RNA molecules are non-covalently linked. The 5' ends bear caps, and the 3' ends carry poly(A) tails. A tRNA molecule serving as a primer is attached via base pairing to each RNA molecule

30.20. Retroviral DNA Is Transcribed Only If It Is Integrated into the Host Cell Genome

Double-stranded viral DNA circularizes and enters The Nucleus. Transcription of retroviral DNA occurs only after it has integrated into the host cell DNA. Thus, integration is an obligatory step in the retroviral life cycle. In contrast, for oncogenic DNA viruses, integration and productive infection are alternative pathways. Another difference is that the frequency of retroviral DNA integration is very high, as would be expected given its key role in productive infection.

The avian sarcoma virus genome, which is 10 kb long, contains four genes (Fig. 30.38). Three of these—gag, pol, and env—are required for productive infection. The gag gene encodes a 76 kDa polyprotein that is cleaved into four proteins forming the viral core. The pol gene encodes reverse transcriptase, which consists of $\alpha$ and $\beta$ subunits. The 65 kDa $\alpha$ subunit is a proteolytic fragment (derived from a 90 kDa precursor) of the $\beta$ chain. The env gene encodes the viral envelope glycoprotein, which is necessary for viral attachment to the host cell surface. The fourth gene, src (from sarcoma), is not required for viral reproduction, but is essential for cell transformation, as discussed below.

Fig. 30.38. Genetic map of the avian sarcoma virus genome. The Genome is 10 kb in length. The letter T denotes terminal repeat sequences

Various viral mRNAs are presumably generated by splicing from the 10 kb primary transcript. They are then transported to the cytosol and translated there. Genomic RNA and viral proteins migrate to The Plasma Membrane and become incorporated into it. Subsequently, a portion of the modified membrane buds off to form new Viral Particles. Thus, unlike infection by oncogenic DNA viruses, productive retroviral infection is not lytic. Retroviruses generally do not kill their host cells. Retroviral DNA remains in the infected cell's genome and continues to be expressed. Moreover, the integrated viral DNA replicates along with cellular DNA, ensuring that daughter cells inherit the viral genome.