PARKE-DAVIS LECTURE RECOMBINANT ORIGINS OF LEUKEMOGENIC MURINE VIRUSES
Recombinant Origins of Leukemogenic Murine Viruses Parke-Davis Award Lecture, 1978 Richard A. Lerner, MD
I want to capture for vou some of the recent excitement in the RNA tumor virus field. We have come a long wav in the field in the past 5 to 10 years, with, in mv view, the most significant advances being made in an understanding of the virus-host relationship. And it is, in fact, the virus-host relationship which in broad outline is the subject of this presentation. On Table 1 are listed those points which I plan to derive concerning retroviral genes and their hosts. For most issues, I will be using data from our own laborator and, therefore, most remarks will be confined to murine systems. However, much of this information can be generalized to other species, including primates. There is an enormous number of proviruses in the mouse genome. It is not known how manv there are, but we can get some kind of minimum number bv counting the number of gene copies for known viral isolates. The methodology will not be detailed here. Suffice it to sav that in the mouse at least 0.01 % of the genome is thought to be composed of proviruses.' Remember that this kind of gene counting depends on having a complete infectious virus in hand to make a DNA probe, and highlv divergent proviruses not yet induced in the laboratory will, of course, not have been counted. The concept of a multigene family is a significant departure from our thinking during the 1960s.2 During that time all the natural viruses of the mouse were considered to be Gross' virus and anything else was a laboratorv variant thereof. During this presentation, please keep the multigene familv in mind and mentallv record how the agents important to neoplasia derive from interactions between two or more proviral genes. The second point which I will make is that proviral expression is regulated during embryogenesis and later during differentiation. As late From the Research Institute of Scripps Clinic. La Jolla. California. This is Publication 1545 from the Research Institute of Scripps Clinic, La Jolla. California. and Publication 68 from the Department of Cellular and Developmental Immunology. Address reprint requests to Dr. Richard A. Lerner. Department of Cellular and Developmental Immunologp. Research Institute of Scripps Clinic. 10666 N. Torrey Pines Road. La Jolla. CA 9-2037 10 0002-9440/78/1010-0009$01.00
Vol. 93, No.1 October 1978
LEUKEMOGENIC MURINE VIRUSES
11
Table 1 1. 2.
Retroviral genes, as proviruses, comprise an extensive multigene family. As with host genes, proviral expression is regulated during development and
3.
diferentiation. Members of the retroviral gene family are constructed out of functionally distinct
4.
modules. Viruses with new or altered pathogenicity may be constructed of nonpathogenic members of the multigene family.
as 1972 the prevailing opinion was that expression of proviral genes causes cancer and their expression must be avoided. However, this is decidedly not the case. Some of the major differentiation specific glycoproteins of the mouse are, in fact, encoded by proviruses. The third point is especially important. Perspectively, one usually thinks of a viral genome as a series of genes which act in concert to allow the virus to replicate itself. Thus, there are genes, the products of which allow adsorption to the cell, replication of the genome, packaging of the chromosome, lysis of the cell, and other processes. Basically we usually think of viruses in terms of a gene complex with a sole strategy of self replication. Reassortment of individual units of such a genome, eg, by recombination, allows formation of a particle more or less capable of carrying out its replication strategy. But the biologic uniqueness of any recombinant depends on the diversity of the gene pool from which it can draw. There is a large number of proviral genomes in the mouse, which are composed of related yet functionally distinct genes or modules. Thus, the possibility exists for construction, by recombination, of a vast array of biologically distinct viruses. In other words, most viral families consist of members which biologically, at least, are relatively similar. To be sure, they can be discriminated by finely tuned serologic methods, but recombination among them does not significantly change their biology because they are essentially exchanging highly homologous genes. This is not the case for the retroviruses. Allelic genes in this family are functionally distinct and their exchange leads to the biologic uniqueness which will be discussed later. For our purposes, the fourth point is the most important. So far we have been painting the picture of a large proviral gene family, the members of which are composed of functionally distinct genes or modules. Within the infinite reassortment and rearrangement which occurs by recombination, there is room for construction of biologically distinct viruses. Now we add the fact that in all this rearrangement, viruses are constructed with unique pathogenicity.
12
American Journal of Pathology
LERNER
Cnical Data
In Figure 1 we show the polypeptides of the retroviruses from several species. The purpose of this figure is twofold: a) to illustrate the virus protein components and b) to show that the retroviruses isolated all the way from mouse to primates have the same basic structure. Thus, we recognize the major component by mass, ranging in size between 30,000 and 27,000 daltons; three smaller components with sizes of approximately 12,000, 15,000, and 10,000 daltons; and 70,000-dalton glycoprotein. Text-figure 1 is a map of the retroviral genome. Proceeding in the direction 5' -_ 3', we can identify the gag or group antigen gene. The translation product of this gene is a large polyprotein which is cleaved into smaller proteins which then become part of the virus.' Recently in a collaboration with George Vande Woude's group at the NIH, we were able to obtain the first structural evidence for the order of the proteins in the gag gene. Proceeding from left to right, the gag gene encodes p15, p12, p30, and plO, representing proteins of 15,000, 12,000, 30,000, and 10,000 daltons, respectively. The next gene is pol encoding polymerase or reverse transcriptase, which in the murine viruses is a single polypeptide with a molecular size of 70,000 daltons. Finally we come to the env gene which encodes two proteins: a) a hydrophilic 70,000-dalton glycoprotein which is approximately 45,000 daltons protein and the rest carbohydrate and b) a hydrophobic 15,000-dalton protein called p15E, the "E" designating "envelope" to differentiate it from the 15,000-dalton core protein.' Another important way of looking at the viral genome is in terms of constant and variable regions. From virus to virus, all the proteins, of course, vary somewhat. But our studies have shown that the envelope protein is by far the most variable.6 As shown in Text-figure 2, this protein serves a recognitive function for the virus, and biologically its structural variability is reflected in the differential cell attachment or host range of the virus. The variable env gene is itself divided into constant and variable portions. One can prepare a single antiserum which will react with the
gag
pol
env
TEx-r-FIGcRE 1-Prototype leukemia retroviral genome. No attempt is made to be accurate about gene lengths. The gag gene is located on the 5' end and the common region (c) on the 3' end.
p5
p1O p30
c
LEUKEMOGENIC MURINE VIRUSES
Vol. 93, No.1 October 1978
4 n
1
CONSTANT AND VAR/ABLE C/STRCWS C PrTMI W/IDA /rCAIFC
53
9
P°lO
le
>
AKV-I Gp7
V:
A
A AR
_V eA?
_wp
_
O-bb
C A-Virus product Fgu 5-Tryptic peptides of gp7Os expressed in the AKR mouse. of Akv-1 gene locus (AKV-1). B-Virus product of Akv-2 gene locus (AKV-2). D-Free gp7O of the serum (serum gp7O). C-AKR xenotopic virus (AKR-X).
AKR-247 gpO
_e
AKR-13 gp70
a .
...
AI
D Fpe 6-Tryptic peptides of gp7O from four
env gene
recombinant (MCF) viruses of the AKR mouse.
Recombinant Origins of Leukemogenic Murine Viruses Parke-Davis Award Lecture, 1978 Richard A. Lerner, MD
I want to capture for vou some of the recent excitement in the RNA tumor virus field. We have come a long wav in the field in the past 5 to 10 years, with, in mv view, the most significant advances being made in an understanding of the virus-host relationship. And it is, in fact, the virus-host relationship which in broad outline is the subject of this presentation. On Table 1 are listed those points which I plan to derive concerning retroviral genes and their hosts. For most issues, I will be using data from our own laborator and, therefore, most remarks will be confined to murine systems. However, much of this information can be generalized to other species, including primates. There is an enormous number of proviruses in the mouse genome. It is not known how manv there are, but we can get some kind of minimum number bv counting the number of gene copies for known viral isolates. The methodology will not be detailed here. Suffice it to sav that in the mouse at least 0.01 % of the genome is thought to be composed of proviruses.' Remember that this kind of gene counting depends on having a complete infectious virus in hand to make a DNA probe, and highlv divergent proviruses not yet induced in the laboratory will, of course, not have been counted. The concept of a multigene family is a significant departure from our thinking during the 1960s.2 During that time all the natural viruses of the mouse were considered to be Gross' virus and anything else was a laboratorv variant thereof. During this presentation, please keep the multigene familv in mind and mentallv record how the agents important to neoplasia derive from interactions between two or more proviral genes. The second point which I will make is that proviral expression is regulated during embryogenesis and later during differentiation. As late From the Research Institute of Scripps Clinic. La Jolla. California. This is Publication 1545 from the Research Institute of Scripps Clinic, La Jolla. California. and Publication 68 from the Department of Cellular and Developmental Immunology. Address reprint requests to Dr. Richard A. Lerner. Department of Cellular and Developmental Immunologp. Research Institute of Scripps Clinic. 10666 N. Torrey Pines Road. La Jolla. CA 9-2037 10 0002-9440/78/1010-0009$01.00
Vol. 93, No.1 October 1978
LEUKEMOGENIC MURINE VIRUSES
11
Table 1 1. 2.
Retroviral genes, as proviruses, comprise an extensive multigene family. As with host genes, proviral expression is regulated during development and
3.
diferentiation. Members of the retroviral gene family are constructed out of functionally distinct
4.
modules. Viruses with new or altered pathogenicity may be constructed of nonpathogenic members of the multigene family.
as 1972 the prevailing opinion was that expression of proviral genes causes cancer and their expression must be avoided. However, this is decidedly not the case. Some of the major differentiation specific glycoproteins of the mouse are, in fact, encoded by proviruses. The third point is especially important. Perspectively, one usually thinks of a viral genome as a series of genes which act in concert to allow the virus to replicate itself. Thus, there are genes, the products of which allow adsorption to the cell, replication of the genome, packaging of the chromosome, lysis of the cell, and other processes. Basically we usually think of viruses in terms of a gene complex with a sole strategy of self replication. Reassortment of individual units of such a genome, eg, by recombination, allows formation of a particle more or less capable of carrying out its replication strategy. But the biologic uniqueness of any recombinant depends on the diversity of the gene pool from which it can draw. There is a large number of proviral genomes in the mouse, which are composed of related yet functionally distinct genes or modules. Thus, the possibility exists for construction, by recombination, of a vast array of biologically distinct viruses. In other words, most viral families consist of members which biologically, at least, are relatively similar. To be sure, they can be discriminated by finely tuned serologic methods, but recombination among them does not significantly change their biology because they are essentially exchanging highly homologous genes. This is not the case for the retroviruses. Allelic genes in this family are functionally distinct and their exchange leads to the biologic uniqueness which will be discussed later. For our purposes, the fourth point is the most important. So far we have been painting the picture of a large proviral gene family, the members of which are composed of functionally distinct genes or modules. Within the infinite reassortment and rearrangement which occurs by recombination, there is room for construction of biologically distinct viruses. Now we add the fact that in all this rearrangement, viruses are constructed with unique pathogenicity.
12
American Journal of Pathology
LERNER
Cnical Data
In Figure 1 we show the polypeptides of the retroviruses from several species. The purpose of this figure is twofold: a) to illustrate the virus protein components and b) to show that the retroviruses isolated all the way from mouse to primates have the same basic structure. Thus, we recognize the major component by mass, ranging in size between 30,000 and 27,000 daltons; three smaller components with sizes of approximately 12,000, 15,000, and 10,000 daltons; and 70,000-dalton glycoprotein. Text-figure 1 is a map of the retroviral genome. Proceeding in the direction 5' -_ 3', we can identify the gag or group antigen gene. The translation product of this gene is a large polyprotein which is cleaved into smaller proteins which then become part of the virus.' Recently in a collaboration with George Vande Woude's group at the NIH, we were able to obtain the first structural evidence for the order of the proteins in the gag gene. Proceeding from left to right, the gag gene encodes p15, p12, p30, and plO, representing proteins of 15,000, 12,000, 30,000, and 10,000 daltons, respectively. The next gene is pol encoding polymerase or reverse transcriptase, which in the murine viruses is a single polypeptide with a molecular size of 70,000 daltons. Finally we come to the env gene which encodes two proteins: a) a hydrophilic 70,000-dalton glycoprotein which is approximately 45,000 daltons protein and the rest carbohydrate and b) a hydrophobic 15,000-dalton protein called p15E, the "E" designating "envelope" to differentiate it from the 15,000-dalton core protein.' Another important way of looking at the viral genome is in terms of constant and variable regions. From virus to virus, all the proteins, of course, vary somewhat. But our studies have shown that the envelope protein is by far the most variable.6 As shown in Text-figure 2, this protein serves a recognitive function for the virus, and biologically its structural variability is reflected in the differential cell attachment or host range of the virus. The variable env gene is itself divided into constant and variable portions. One can prepare a single antiserum which will react with the
gag
pol
env
TEx-r-FIGcRE 1-Prototype leukemia retroviral genome. No attempt is made to be accurate about gene lengths. The gag gene is located on the 5' end and the common region (c) on the 3' end.
p5
p1O p30
c
LEUKEMOGENIC MURINE VIRUSES
Vol. 93, No.1 October 1978
4 n
1
CONSTANT AND VAR/ABLE C/STRCWS C PrTMI W/IDA /rCAIFC
53
9
P°lO
le
>
AKV-I Gp7
V:
A
A AR
_V eA?
_wp
_
O-bb
C A-Virus product Fgu 5-Tryptic peptides of gp7Os expressed in the AKR mouse. of Akv-1 gene locus (AKV-1). B-Virus product of Akv-2 gene locus (AKV-2). D-Free gp7O of the serum (serum gp7O). C-AKR xenotopic virus (AKR-X).
AKR-247 gpO
_e
AKR-13 gp70
a .
...
AI
D Fpe 6-Tryptic peptides of gp7O from four
env gene
recombinant (MCF) viruses of the AKR mouse.
