EUKARYOTIC VIRUSES are of many different types with a remarkable variety of properties. Beyond the interest in viruses as infectious agents, they also have provided crucial model systems for understanding cell biology and genetics and are important tools for recombinant DNA and transgenic technologies, including gene therapy.

A. Viruses may have either RNA OR DNA GENOMES (Table 1.3). Their genomes may consist of a single nucleic acid molecule or a collection of several. Generally virus genomes are very compact (i.e., there is little RNA or DNA that does not encode proteins). Typically viruses encase their genomes in a capsid made up of one or several proteins (Fig. 1.40). Sometimes this "nucleocapsid" is surrounded by a membrane "envelope" (taken from the host cell plasma membrane). Many viruses also have integral membrane proteins sticking out of the envelope which bind to "receptor" proteins on the host cell membrane.

B. One type of RNA virus of particular importance (including Human Immunodeficiency Virus, HIV, the causative agent of AIDS) are the RETROVIRUSES, pp. 99-101, 103. The retroviral RNA genome is converted to DNA upon entering a host cell, Fig. 3.15. This "backward or retro" flow of information from RNA to DNA is catalyzed by a type of DNA polymerase called REVERSE TRANSCRIPTASE (RT). The double stranded DNA copy of the retroviral single stranded RNA is made by a complex series of steps using different primer molecules and an RNA degrading activity of the reverse transcriptase called RNase H (Fig. 5.47, you need not know this information). Once the retroviral genome is converted to double stranded DNA, it can integrate into the host chromosomal DNA (like a lysogenic DNA virus in prokaryotes). The integrated form of the virus is called a PROVIRUS. Unlike lysogenic viruses, a retrovirus must go through the integrated proviral state to replicate. Also unlike the prophage of lysogenic viruses, the provirus often remains active, producing new viral RNA genomes and viral mRNAs using the hostís RNA polymerase II. All functional retroviruses contain at least 3 basic genes, although each of these genes produces several protein molecules due to proteolytic processing of a large protein precursor (Fig. 15.17). Viral proteins and genomic RNAs assemble the viral nucleocapsid and bud new virus off the host cell by exocitosis which migrates to infect other cells in the host animal.

C. Some retroviruses (RNA tumor viruses) cause cancer by one of two mechanisms:

1. A virus may contain an additional gene, called an oncogene, which induces the cancerous growth phenotype in the host cell (e.g., Rous sarcoma virus of chickens contains an oncogene called v-src, for viral sarcoma(-causing) gene, Fig. 15.19). It has been shown that RNA tumor viruses picked up their oncogenes by incorporating normal host genes into their genomes, p. 614-615 and Fig. 15.20. The host form of the oncogene is called a proto-oncogene. Proto-oncogenes do not cause cancer in their normal form; one or more changes in the viral form of the gene convert it to an oncogene (e.g., Fig. 15.21). However, as we will see, proto-oncogenes in the host cell can mutate and become oncogenic without any involvement of a virus.

2. Proviruses integrate fairly randomly throughout the host cell genome (but, like l in E. coli, only one provirus per cell). Rarely (but it only takes once) the provirus integrates close to a proto-oncogene and the integration of viral sequences (like enhancers) nearby genetically alters the proto-oncogene to become oncogenic. In this way an RNA tumor virus without its own oncogene can cause cancer, but much more slowly and more rarely.

3. Very little human cancer is caused by RNA tumor viruses of either type. This is not the way in which HIV causes AIDS. Most human cancers are not caused by viruses, but those that are viral are most often caused by DNA viruses.

D. Human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). HIV is a retrovirus which contains an envelope protein that specifically binds to a cell surface receptor on and infects a type of human white blood cell called a T helper cell. The T helper cell is an essential component of the immune system needed to ward off infection with virtually all pathogenic microbes including viruses like HIV (many pathogens, by the way, have "immunosuppression" mechanisms, but HIVís is especially effective.) Initial HIV infection generally leads to little or no clinical signs, low levels of virus production, and little change in the patientís T helper cell profile. For reasons that are probably complex and arenít yet fully understood, HIV proviruses can become activated, causing much higher virus levels and, eventually, massive destruction of helper T cells in the disease we know as AIDS. The loss of T cells causes the victim to become immuno-compromised (e.g., the boy in the bubble) and susceptible to a host of infectious agents and cancer. Like most retroviruses, HIV does not itself kill the helper T cells. The mechanism of AIDS pathogenesis is not fully known but may relate to a mechanism by which immune cells can be triggered to undergo programmed cell death. AIDS was initially treated primarily with drugs which bind to and inhibit the HIV RT. However, drug-resistant forms of HIV usually emerge due to the fact that retroviral replication by RT has a much higher error rate than normal DNA polymerases. Recently drugs have been designed which inhibit the viral protease required to cleave the initial viral protein products to smaller functional proteins. Treatment with a combined regimen of RT and protease inhibitors has shown considerable promise.

E. DNA tumor viruses. A variety of DNA viruses cause cancer in humans and other animals (Table 15.2). Viruses like SV40 (simian virus40) and polyoma, and adenovirus (Figs. 15.13, 14, and 16) generally grow in a lytic mode and kill their host cells rather than causing cancer. However, in cells that do not support lytic growth (usually from species related to, but outside of, the normal host range), all or part of the viral DNA is integrated into the host genome (at random) which can lead to transformation of the cell to a cancerous state (Fig. 15.13). Cancer is caused by viral proteins (T-antigen in SV40 and E1A and E1B in adenovirus) which bind to and inactivate the host tumor suppressor gene products Rb and p53 which normally suppress the cell cycle and thereby regulate cell growth. (Many animal viruses can only replicate when the cell is in S phase, so they express gene products which shift the host cells out of a resting state into the cell cycle.) Papillomaviruses (Fig. 15.15) usually induce benign cell proliferation, again by blocking Rb and p53, but they can also cause cancer directly or in combination with additional mutations in the host genome. The mechanisms by which hepatitis B virus and herpesviruses (e.g., Epstein Barr virus) cause cancer are not yet well understood.

How do we know?

1. That HIV causes AIDS? Obviously one cannot inject HIV into humans as a test, however:

a. Virtually all AIDS patients can be shown to have been exposed to HIV.

b. The appearance of AIDS in new populations parallels (with the expected delay time) the appearance of HIV. Furthermore, somewhat different types of AIDS correlate with variant HIV strains. AIDS cases appeared in hemophelia victims who received blood products containing HIV, and the death rate increased dramatically, but only in those who became HIV+. (Now that blood supplies are screened for HIV, this is no longer a problem. Furthermore, the necessary factors often can now be made by recombinant DNA techniques. Finally, three lab workers whose bloodstreams were accidentally exposed to a molecularly cloned HIV virus have since developed AIDS-like symptoms. None of the three had any of the common risk factors for getting AIDS.

c. A very similar virus, SIV, has been shown to cause an AIDS-like illness in monkeys.

d. HIV specifically attaches to and infects the cell types which decline in AIDS patients.

For these and other reasons, virtually no major AIDS researcher doubts that HIV causes AIDS.


Molecular Basis of Cancer (Chapter 15)

A. Cancer is the unregulated growth of cells in a multicellular organism. There are many types of cancer (Table 15.1), but they can generally be grouped into carcinomas (cancer of epithelial cells), sarcomas (cancer of connective tissues) and leukemias and lymphomas (cancer of hematopoietic or blood-forming cells). Cancer is due to heritable changes to the genome (e.g., mutations) and most cancers involve several stages and therefore several different genetic alterations (Fig. 15.4 and 15.5). These stages may include initial overproliferation of a group of cells which then develop into a benign (locally contained) tumor. Strictly speaking, cancer begins when the tumor becomes malignant (invading surrounding cells and tissues) and eventually tumor cells metastasize (invade other organs) by spreading through the bloodstream. One or more genetic alterations may occur at each stage and, since most spontaneous mutations occur during DNA replication, the more rapidly the tumor cells grow, the more likely they are to accumulate further mutations that lead to tumor progression. The study of cancer is called oncology and cancerous changes in cell behavior are often called oncogenic.

B. Much of what we know about cancer cells derives from studies of cultured cells. Oncogenic changes to tissue culture cells are recognized by: cells gaining the ability to cause tumors when transplanted into (immunocompromised) animals, cells escaping the restraints of contact or density-dependent inhibition and forming foci (Fig. 15.8 and 15.12), cells becoming able to grow with fewer or no added growth factors, changes in cell shape (Fig. 15.10), and changes in many proteins within and secreted by the cell. Note that one or a few genetic alterations indirectly affect a wide variety of cell traits (in genetic terms, the mutations have highly pleiotropic phenotypes). Not surprisingly, oncogenic mutations usually occur in critical control and signalling genes.

C. The existence of RNA tumor viruses in animals provided the first clue that tumors co uld be caused by improper regulation or expression of host proto-oncogenes. In other words, about 1-200 of our 50-100,000 normal genes can occasionally be mutated into forms which cause cells to grow out of control. This can occur at random or due to mutagenic agents without any involvement of viruses. Many types of mutations of different proto-oncogenes have been shown to be involved in cancer (Table 15.4).

D. Oncogenes are the mutant form of normal proto-oncogenes which impair normal regulation of cell growth. Generally, mutation of a proto-oncogene to become an oncogene is a "gain of function" mutation and is dominant (only one allele needs to be changed to exhibit the phenotype). Many types of oncogenic mutations have been observed including virus integration (ALV integration near c-myc), point mutation (ras, Fig. 15.23), chromosomal translocations (c-myc and abl, Fig. 15.24-25) and gene amplification (N-myc in neuroblastoma). Oncogenic changes have been found to occur in hormones, hormone/growth factor receptors, transcription factors and proteins which couple receptors to cell responses (Fig. 15.26-15.29). Some, but by no means all, oncogenic mutations in human cancers can be detected by transfecting isolated human tumor DNA into cultured (mouse) cells and isolating that piece of human DNA that causes the cells to form foci (Fig. 15.22).

E. Other oncogenic mutations occur in tumor suppressor genes, which are genes that normally function to stop unwanted cell growth (Table 15.3). Such mutations are generally "loss of function" mutations and so they are usually recessive (in and of themselves). The first well-characterized tumor suppressor gene was Rb (retinoblastoma). Genetic analysis of a familial tendency to have multiple retinoblastomas showed that affected offspring receive one damaged or deleted Rb gene. During normal growth of their retinas, mutations occasionally occurred which made the Rb defect homozygous and led to tumors (Fig. 15.33). Non-inherited retinoblastomas can occur by the chance mutation of both normal Rb genes, but this, of course, is much rarer. As we know (Fig. 14.19 or 15.36), Rb is a critical cell cycle regulatory molecule which couples growth factor stimulation and cyclin D accumulation to passage through the G1-S restriction point. Another critical tumor suppressor gene is p53, which arrests the cell cycle in the presence of damaged DNA (Fig. 14.20) and leads to programmed cell death of cells whose DNA is irreversibly damaged (Fig. 15.37). As discussed above, several DNA tumor viruses cause cancer through their effects on p53 and Rb (Fig. 15.35).

F. Most cancer is due to the accumulation of somatic mutations (i.e., no effect in the germ line, so not inherited), and usually several such mutations are required to lead to full blown cancerous growth. Colon cancer has been particularly well studied. While a variety of patterns can occur, many colon cancers appear to derive from a series of mutations in a ras oncogene and three tumor suppressor genes (e.g., APC, DCC, and p53, Fig. 15.38).

How do we know?

1. That viral oncogenes in RNA tumor viruses are derived from normal host cell genes? The initial experiment that proved that RSV v-src has a homologue in normal chicken DNA, a proto-oncogene, called c-src is described on pp. 614-615. More convincing experiments were later done by Southern blotting in the same lab. [Chickens, like almost all animals, have remnants of ancient proviruses called endogenous proviruses in their genomes, so when a Southern blot is hybridized to labeled cDNA to RSV, several different bands show up. However, generally only one or two of those bands hybridize to RSV which contains v-src, but not to ALV which doesn't. These bands are due to the c-src gene. Furthermore, if one does blots with many different chicken lines, the endogenous virus bands are of often of different size, due to the fact that they arose fairly recently in evolution (or have since recombined), but the c-src band(s) will generally be the same (except for an RFLP) in all chicken lines since it is an original part of the avian (and vertebrate) genome. The final confirmation of the proto-oncogene hypothesis came with the recombinant DNA cloning of the chicken c-src gene and of many other proto-oncogenes.

2. How retroviruses cause cancer in chickens? As described previously (p. 19 of notes), Peyton Rous discovered Rous sarcoma virus of chickens in 1910 and Temin showed that it went through a DNA intermediate, the provirus. Peter Vogt isolated a mutant version of RSV which did not cause sarcomas. He and others showed this virus was missing most of one gene, subsequently called the v-src gene. Therefore, src must be the gene that allows RSV to cause cancer. Subsequently, it was shown that v-src is a protein tyrosine kinase which causes aberrant signals for cell growth. There are also naturally occurring variants of RSV called ALV, avian leukemia virus, that do not contain src. Just like Vogt's mutant version of RSV, ALV does not cause sarcomas, but, as implied by its name, it does cause leukemia (after long times of infection). Several groups (including one here at MSU) about the same time showed how this occurs. They did Southern blots of DNA from ALV-infected leukemic cells. By hybridizing the blot to radioactive cDNA copies of ALV RNA, they showed that the provirus was integrated into the host genome in a particular region. The same size DNA fragment would hybridize to a gene called the c-myc gene, a gene originally isolated because it was related to the oncogene in avian myelocytomatosis virus, AMV. Thus, ALV normally causes leukemia by integrating its provirus adjacent to and activating the c-myc gene of normal chicken lymphoid cells.

3. That mutations at proto-oncogenes can be involved in human cancer? Experiments in the labs of Robert Weinberg and Geoff Cooper (author of your text) demonstrated that genomic DNA from a human bladder carcinoma was found to transform mouse tissue culture cells to a cancerous form (uncontrolled growth, Fig. 15.22). It was then possible to use recombinant DNA techniques to isolate the responsible human gene. In this case, it was found to be the H-ras oncogene. When the DNA sequence of the responsible H-ras gene from this and other human tumors was determined, it was found to differ from the normal human H-ras proto-oncogene by one of a few specific mutations (Fig. 15.23). Subsequent recombinant DNA tests showed that one of these key mutations was required to deregulate H-ras expression and contribute to cancer. Subsequently this and similar assays identified other mutant oncogenes involved in human cancer. However, this approach only works to identify some kinds of the multiple mutations required for human cancer to progress to full blown malignancy. (Fig. 15. 38).

Food for thought questions, Cell Cycle, Viruses and Cancer:

1. The cdc2 gene was first identified by a mutation obtained in the yeast, S. pombe. Mammals have a closely homologous gene to cdc2. Could the role of cdc2 be studied by making "knock-out" mice lacking a functional cdc2 gene and, if so, how?

2. During the early cleavage stages of embryonic growth in animals, cell growth is minimal but mitosis is very rapid. What synthetic activities must be especially high during this period?

3. Proto-oncogenes can do damage to an organism by mutating to the cancerous (oncogenic) state and often causing lethal disease. Why have they not been eliminated from the genome by the process of evolution which selects against

4. Almost all virologists agree that HIV is the causative agent of AIDS; however a small group of people feel this remains unproven. What factors about the biology of HIV and AIDS might make it difficult to provide absolute scientific resolution of this issue?