Naturally Occurring Retroviruses (RNA Tumor Viruses). II

This is the second of two papers describing the biology of the naturally occurring RNA tumor viruses (oncoviruses). It will appear in two parts. In the first paper [Cancer Investigation 1(1):67-83 (1983)] the general properties of this class of viruses and the biology of the retroviruses of the “lower” vertebrates was discussed. In this paper the oncoviruses of the “higher” animals are described. Part one deals with cat retroviruses.     

The multifaceted retrovirus

I have attempted to illustrate the many different properties of retroviruses and their presence in a wide variety of animal species including humans. Since the turn of this century, progress in the field of retrovirology has been noteworthy and many new and important scientific observations have been made (Table 7). Along the way, certain dogmas were replaced with new tenets. The recent recognition of retroviruses associated with human cancer and immunodeficiency places them into consideration as potential agents responsible for other human diseases such as autoimmunity and multiple sclerosis. Not only can retroviruses be oncogenic or cytopathic agents but they can also exist highly conserved as endogenous genes in the chromosomal DNA of many different species and not cause disease. In fact, this latter group appears to be predominant, suggesting their role in normal developmental processes and as progenitors of the pathogenic types. The virus-like genomes recognized in Drosophila and other lower animal species could be examples of this fact and may represent important biological entities throughout nature. The genetic material of retroviruses resembles transposons and may reflect the ability of these viruses to be passed within the host and to affect the evolutionary pathway. They could, as transposable elements, be transmitted as well to many different animal species. By their ability to move within the genetic machinery of the cell, these viruses could influence development in animals through promotion, enhancement, or suppression of specific cellular genes. This idea has been proposed for the noninfectious type A particles that have been observed to show these effects in cultured cells. One important observation is that the effect of retroviruses on cells has a varied pattern which may be emphasized by one group (e.g., vacuolization by foamy virus) or shared by other groups (e.g.., syncytial cell formation by type C and type D oncovirinae, spumavirinae, and lentivirinae) (Table 6). Moreover, the heterogeneity of the lentiviruses and the transduction of normal cellular genes by many of the oncogenic viruses indicate the changes that can occur as retroviruses infect and replicate within the cell. The overview is very informative. Virus-cell interaction can lead to biological expressions that depend on the phenotype of the cell and the viral genetic structure. Throughout its existence in nature the retrovirus has been evolving, conserving certain features while developing new ones with different properties; it clearly emerges as a multifaceted agent.     

Conventional and immunocolloidal gold electron microscopy of eight simian retroviruses closely related to human T-cell leukemia virus type I

Simian retroviruses closely related to human T-cell leukemia virus type I (HTLV-I) were isolated from 8 species, examined by both conventional and thin section immunocolloidal gold electron microscopy, and compared with HTLV-I. Mature forms of simian viruses were found in extracellular aggregates and within cytoplasmic vacuoles. They were morphologically similar to each other and to HTLV-I. They consisted of a seemingly smooth envelope and a centrally located nucleoid. Their size varied considerably among species and also within the same species; this is characteristic of this group of retroviruses. No budding particles of simian viruses were observed. Thin section immunocolloidal gold electron microscopy using various human and simian sera showed that simian viruses were antigenically related to each other and to HTLV-I. One drawback of this otherwise very useful technique was the difficulty in identifying virions because of the poor preservation of their fine structure by fixation with glutaraldehyde alone. This was overcome by using materials prepared for conventional electron microscopy, in which virions showed weak but specific reactions with gold particles after deosmification and antigen restoration with sodium metaperiodate.     

Leukemogenicity of clonal isolates of murine leukemia viruses.

The leukemogenicity of three types of cloned, in vitro grown murine retroviruses was studied. Two Moloney virus clones caused leukemia, as did five clones of the B-tropic endogenous virus of BALB/c mice. Neither of two clones of N-tropic BALB/c virus caused leukemia in Fv-1n/n mice, and the viruses were not recoverable from the animals. The ability to induce leukemia therefore appeared to reside in the genome of at least certain nondefective murine retroviruses.     

Type D primate retroviruses: a review.

The prototype virus of the type D retroviruses is the Mason-Pfizer monkey virus (MPMV). MPMV was originally isolated from a breast carcinoma of a female rhesus monkey (an Old World monkey). MPMV is of obvious importance in that it is the only retrovirus thus far isolated from a mammary tumor of a primate and has been shown to have transforming potential for primate cells in vitro. Subsequent to the isolation of MPMV viruses morphologically and immunologically indistinguishable from MPMV have been isolated from normal placenta and lactating mammary glands of other rhesus monkeys in captivity. Recently, viruses morphologically resembling MPMV have been isolated from a langur monkey (another Old World monkey) and from squirrel monkeys (a New World monkey). Based on nucleic acid hybridization studies, the latter 2 viruses represent endogenous viruses in their species of origin, whereas MPMV appears to be a horizontally related to the langur monkey isolate. Studies on the immunological relatedness of the type D retroviruses have demonstrated interspecies cross-reactivities between the major internal and external proteins of the viruses. Furthermore, these viruses also share cross-reactivity of their major external glycoproteins with those of the type C baboon endogenous virus. These interspecies reactivities can also be demonstrated in natural sera from both imported and laboratory-bred monkeys. The demonstration of these interspecies cross-reactivities shared by distantly related primate retroviruses provides a means for detecting determinants that are representative of all primate retroviruses presently known and yet to be isolated and may provide new assays for detection of a human retrovirus.     

Molecular comparison of retroviruses associated with human and simian AIDS

Infectious retrovirus(es) associated with the human (LAV, HTLV-III, ARV) and simian (SAIDS-1) acquired immune deficiency syndrome were compared by electron microscopy, immunofluorescence and immunoblotting techniques and by restriction endonuclease mapping of the viral genomes. The extracellular virus particles had similar type D morphology, but intracytoplasmic type A nucleoids were found only in SAIDS virus infected cells. Although the antigens of the three prototype AIDS viruses were similar, no cross-reactivity with the SAIDS virus was detected. Molecular hybridization and restriction enzyme analysis also revealed that the SAIDS and AIDS viruses were genetically unrelated. However, only minor differences, consistent with strain polymorphism, were found between the three AIDS virus isolates. Thus, the retroviruses associated with AIDS in macaques and humans are unique to each species.     

Gene therapy applications of viral vectors

Viral vectors have frequently been applied in gene therapy with the final goal of treating various diseases in the areas of neurology, neurodegeneration, metabolic disease, and cancer. Vectors have been engineered based on AAV, adenoviruses, alphaviruses, herpes simplex viruses, lentiviruses, and retroviruses. Some vectors are suitable for short-term episomal transgene expression, whereas others are integrated into the host cell genome to provide long-term expression. Additionally, hybrid vectors with favorable features from different viruses have been developed. Therapeutic genes of choice have typically been toxic genes such as thymidine kinase, pro-apoptotic genes like Bax, and immunostimulatory genes (for instance, interleukin-12). A large number of animal studies have demonstrated proof of concept of viral gene therapy. Many types of viral vectors have been employed in more than 700 clinical trials that have been carried out or are currently in progress.     

Inhibition by 2′,3′-dideoxythymidine of retroviral infection of mouse and human cells

Infection of mouse and human cells with type C murine leukemia/sarcoma viruses was inhibited by the nucleoside analogue 2′,3′-dideoxythymidine (ddT). Cell growth and virus synthesis by previously infected cells were not affected by the analogue. Infection of rat cells with murine retroviruses was not inhibited by ddT.     

Effects of ribonuclease and deoxyribonuclease on a murine leukemia virus (Rauscher)

Plasma pellets and femoral bone marrow from BALB/cJ mice infected with the Rauscher leukemia virus were fixed, embedded, and sectioned. The thin sections were incubated in ribonuclease and deoxyribonuclease solutions, stained, and examined in the electron microscope. Specific attention was paid to the action of the nucleases on characteristic virus particles in the plasma preparations and on viruses being produced by the "budding" phenomenon in the femoral bone marrow. Ribonuclease solutions digested the nucleoids of the virus particles in the plasma preparations from mice infected with Rauscher leukemia virus. The nucleoid portion of the "budding" virus particles in bone marrow, and the connecting cytoplasm of the stalks were also digested by ribonuclease solutions. In addition, the outer coat of the "budding" particles was affected in a nonspecific manner. The centers of the "budding" particles in the bone marrow and the nucleoids of viruses in plasma preparations were not digested by deoxyribonuclease solutions. Influenza virus, a known ribonucleic acid (RNA) virus, was used as a control for nuclease activity. The nucleoids of influenza virus particles were digested by ribonuclease but not by deoxyribonuclease solutions. After coriphosphine staining of the plasma virus preparations, the fluorescence was quenched in preparations treated with ribonuclease, but did not appear to be diminished in those treated with deoxyribonuclease. This study suggests that infection of mice with Rauscher leukemia virus produces virus particles in plasma and "budding" particles in bone marrow, both of which contain RNA.     

Analysis of structural polypeptides of the lymphoproliferative disease virus (LPDV) of turkeys

The polypeptide composition of the lymphoproliferative disease virus (LPDV) of turkeys was shown to comprise several polypeptides with apparent molecular weights of 76, 31, 28, 20 and 15 kDa. This polypeptide pattern is distinctly different from the protein profiles of avian leukosis viruses, reticuloendotheliosis virus, or murine leukemia viruses. Moreover, LPD virions contain 2 major structural proteins (p31 and p28), in contrast to only one major internal protein present in most other retroviruses. The 76 kDa protein was established as the major viral envelope glycoprotein. The uniqueness of the LPDV polypeptide pattern is consistent with the lack of genetic relatedness of LPDV genome to other retroviruses, establishing LPDV as a representative of a distinct group of retroviridae.     

Viral vaccines for cancer immunotherapy

The understanding that tumor cells can be recognized and eliminated by the immune system has led to intense interest in the development of cancer vaccines. Viruses are naturally occurring agents that cause human disease but have the potential to prevent disease when attenuated forms or subunits are used as vaccines before exposure. A large number of viruses have been engineered as attenuated vaccines for the expression of tumor antigens, immunomodulatory molecules, and as vehicles for direct destruction of tumor cells or expression of highly specific gene products. This article focuses on the major viruses that are under development as cancer vaccines, including the poxviruses, adenoviruses, adeno-associated viruses, herpesviruses, retroviruses, and lentiviruses. The biology supporting these viruses as vaccines is reviewed and clinical progress is reported.     

Sheep pulmonary adenomatosis: a contagious tumour and its cause

Sheep pulmonary adenomatosis (SPA) is a contagious lung tumour that can be transmitted experimentally. Two viruses have been associated with the disease, a herpesvirus and a retrovirus. All ovine herpesviruses are related antigenically and have been isolated only from SPA tumour tissues. They do not appear to cause the tumour and their association with SPA appears to arise from reactivation of latent virus in the respiratory tract. SPA tumour tissue and lung fluid contain a retrovirus that has properties similar to those of type B and type D retroviruses. Homogenates of tumour that contain this retrovirus can transmit SPA to experimentally inoculated sheep. Various retroviruses have been cultured from such tumours. Only one of these has properties similar to that of the retrovirus detected in the tumour and it can transmit SPA experimentally. The others appear to be isolates of the non-oncogenic ovine lentivirus, maedi-visna virus, and play no part in the aetiology of SPA.     

Augmented immunogenicity of tumor cell membranes produced by surface budding viruses: parameters of optimal immunization.

Membranes prepared from tumor cells infected with surface budding viruses are much more immunogenic than membranes from uninfected tumor cells. Factors affecting immunization with membranes from virus-infected tumor cells were studied. Preparations made with influenza virus were clearly superior to those prepared with vesicular stomatitis virus (VSV). Membranes infected with VSV were maximally immunogenic at a dose equivalent to a 10% cell pack whereas influenza-virus-infected membranes were immunogenic at 1/100th of this dose. Subcutaneous inoculation was better than other routes of administration. Maximum protection against challenge with viable tumor cells was afforded by two inoculations of VSV-infected membranes spaced 3 days apart or a single inoculation with influenza-virus-infected membranes. Administration of membranes in complete Freund's adjuvant either had no effect of induced a slight degree of tumor enhancement. Immunization with influenza-virus-infected membranes significantly reduced tumor size and incidence even at a challenge dose of tumor cells which was 50 times the LD100.     

Viral carcinogenesis: revelation of molecular mechanisms and etiology of human disease

The RNA and DNA tumor viruses have made fundamental contributions to two major areas of cancer research. Viruses were vital, first, to the discovery and analysis of cellular growth control pathways and the synthesis of current concepts of cancer biology and, second, to the recognition of the etiology of some human cancers. Transforming retroviruses carry oncogenes derived from cellular genes that are involved in mitogenic signalling and growth control. DNA tumor viruses encode oncogenes of viral origin that are essential for viral replication and cell transformation; viral oncoproteins complex with cellular proteins to stimulate cell cycle progression and led to the discovery of tumor suppressors. Viral systems support the concept that cancer development occurs by the accumulation of multiple cooperating events. Viruses are now accepted as bona fide etiologic factors of human cancer; these include hepatitis B virus, Epstein-Barr virus, human papillomaviruses, human T-cell leukemia virus type I and hepatitis C virus, plus several candidate human cancer viruses. It is estimated that 15% of all human tumors worldwide are caused by viruses. The infectious nature of viruses distinguishes them from all other cancer-causing factors; tumor viruses establish long-term persistent infections in humans, with cancer an accidental side effect of viral replication strategies. Viruses are usually not complete carcinogens, and the known human cancer viruses display different roles in transformation. Many years may pass between initial infection and tumor appearance and most infected individuals do not develop cancer, although immunocompromised individuals are at elevated risk of viral-associated cancers. Variable factors that influence viral carcinogenesis are reviewed, including possible synergy between viruses and environmental cofactors. The difficulties in establishing an etiologic role for a virus in human cancer are discussed, as well as the different approaches that proved viral links to cancer. Future directions for tumor virus studies are considered.     

Type-C particles in human tissues. II. Electron microscopic study of embryonic cultures infected with a murine leukemia virus

Type-C virus particles are synthesized by cells from a number of human embryonic cultures infected with the Rauscher murine leukemia virus (RV). These particles have the characteristic morphology of the murine leukemia viruses. In some cultures, type-C particles were also observed budding into the cisternae of rough endoplasmic reticulum of a few cells. Virus particles in the vicinity of such cells were of “immature” type. In contrast, particles in the vicinity of those cells which did not synthesize intracisternal type-C particle were of the “mature” type. It is not known if the “mature” intracisternal particles have the morphology of the “mature” extracellular type-C particles.     

Ultrastructural characterization of the retrovirus particles (Sm-MTV) liberated from the mammary tumor cell line (Sm-MT) of a house musk shrew, Suncus murinus (Insectivora)

Detailed ultrastructure of a new type of retrovirus (Sm-MTV) released by cultured cells (Sm-MT) of a spontaneous mammary tumor from a house musk shrew Suncus murinus, Insectivora, is described. The virus particles were revealed as three forms: intracellular; budding; and extracellular. The intracellular type A particles were similar in profile to those associated with mouse mammary tumor cells and tended to form a small cluster of several particles in the cytoplasm. In addition, horseshoe-shaped particles as well as smaller particles in clusters, with doughnut-shaped morphology similar in structure to type A particles, were identified near the clusters of type A particles, although in smaller numbers. The budding particles contained a doughnut-shaped nucleoid, although the nucleoids decreased in size as compared with intracytoplasmic type A particles. The extracellular virions consisted of an envelope and a centrally located nucleoid. In routinely fixed specimens, the former was covered with irregularly distributed fuzzy materials in its surface, and the latter was further composed of a small electron dense core surrounded by an intermediate layer. Tannic acid treatment of cells resulted in the visualization of surface projections on the envelope of virions. Similar projections were also detected exclusively on the plasma membrane where virus budding took place, and not on the normal plasma membrane. The presence of surface projections on the viral envelope was further confirmed by the whole-cell-mounting technique. Together with our previous results of biochemical and immunological investigations, we concluded that Sm-MTV seemed to have closer phylogenetic relatedness with type D viruses of primates than with murine mammary tumor virus.