Standardized and simplified nomenclature for proteins common to all retroviruses.

We propose a revised standardized nomenclature for the proteins common to all retroviruses on the basis of biological function, enzymatic activity, and/or virion location data. (We do not discuss proteins specific for subfamilies or only some retroviruses.)     

Polymorphism of murine endogenous proviruses revealed by using virus class-specific oligonucleotide probes

Inbred mice contain three classes of endogenous nonecotropic murine leukemia virus-related sequences, namely xenotropic, polytropic, and modified polytropic proviruses. Oligonucleotide probes specific for the three different classes were prepared and used to examine the diversity of endogenous sequences present in eight different strains of mice: HRS/J, BALB/cJ, A/J, AKR/J, C57BL/6J, DBA/2J, C57L/J, and C3H/HeJ. A high degree of polymorphism was observed. Overall, the strains showed between 17% (A/J and HRS/J) and 65% (C57BL/6J and C57L/J) shared proviruses, and only four proviruses were present in all eight strains. The similarity among the strains is due in part to the few proviruses present in all of the strains but also represents the independent assortment of a limited set of proviruses. These oligonucleotides provide a basis for determining the stability, distribution, and mutagenic potential of nonecotropic proviruses within the mouse genome.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Characterization of endogenous and recombinant proviral elements of a highly tumorigenic AKR cell line

As an approach to evaluating the contribution of classes of endogenous viral sequences to leukemogenesis, a genomic library was prepared from the highly tumorigenic AKR SL12.3 cell line and screened for env-containing proviruses. An extensive battery of virus-derived probes and specific oligonucleotide probes were used to segregate 83 positive clones into related groups. The nonecotropic endogenous retroviruses were identified as members of the polytropic, modified polytropic, or xenotropic groups. At least three unique xenotropic proviruses were detected that differed from the published xenotropic sequence within a variable region of the 5' portion of env. Changes among the xenotropic proviruses included relative insertions and/or deletions that maintain an open reading frame and hence the potential to encode viable envelope gene products. Several recombinant viruses were also detected. Recombination was not random and primarily involved the formation of mink cell focus-inducing class I retroviruses via recombination between polytropic elements and ecotropic virus. One other recombinant was detected which contained ecotropic virus sequences in the 5' region encoding p15 of an otherwise xenotropic provirus. An interesting observation was the finding that certain clones contained more than one provirus within the average 20-kb cloned insert. This would not be expected if integration were totally random. The de novo recombinant proviruses identified here provide a series of potential candidates to be evaluated for their contribution to the tumorigencity of the SL12.3 cell line.     

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Molecular basis of host range variation in avian retroviruses.

Previous genetic analysis has localized the region of the Rous sarcoma virus (RSV) env gene responsible for host range specificity to that encoding the middle one-third of gp85. To better understand the host range determinants, the relevant regions of the genomes of infectious molecular clones of the transformation-defective Prague strain of RSV, subgroup B (Pr-RSV-B) and Rous-associated virus 0 (RAV-0) (subgroup E) were sequenced and compared with the sequence of Pr-RSV-C. This comparative analysis identified two variable regions of low amino acid sequence homology flanked by highly conserved amino acid sequences. The first variable region (hr1) begins at base 5654 in the Pr-RSV-C sequence and encodes 32 amino acids. The second variable region (hr2) begins at base 5846 and encodes 27 amino acids. To test the role of the variable regions in host range specificity, we determined the sequence of this region of the env gene of NTRE-4, a recombinant virus between Pr-RSV-B and RAV-0 which exhibits an extended host range. This analysis revealed that the recombinant subgroup-encoding region of NTRE-4 is composed of 200 bases of RAV-0 sequence, including hr2, flanked by sequences which are otherwise of Pr-RSV-B origin. This study indicates that hr1 and hr2 are the domains of gp85 responsible for host range determination in avian retroviruses.     

Determinants for receptor interaction and cell killing on the avian retrovirus glycoprotein gp85

The virion envelope glycoprotein gp85 confers a high degree of subgroup specificity for interaction with distinct cell receptors. Specific subgroups of gp85 have been associated with a cytopathic virus-cell interaction, most likely resulting from reduced resistance to superinfection, which allows the buildup of excessive amounts of viral DNA. Previous nucleotide sequence analysis of the gp85 coding region identified small regions of variable amino acid sequence within a conserved framework. To define the role of these variable regions we constructed a series of molecular clones carrying novel combinations of variable regions from viruses. Analysis of rescued virus shows that receptor binding is determined by the interaction of two major regions and one minor region in the middle of gp85. Cytopathogenicity is not associated with any specific variable region but rather with the ability to recognize the subgroup B receptor on chicken cells.     

Thymic epithelial genotype influences the production of recombinant leukemogenic retroviruses in mice

By using T1 oligonucleotide fingerprinting and mapping techniques, we analyzed the genomic structure of retroviruses produced by thymocytes and splenocytes of reciprocal bone marrow-and thymus-grafted chimeras. We found that the genetic factor(s) derived from NZB mice that suppresses the development of thymic leukemia in (AKR X NZB)F1 mice also prevents the formation of recombinant leukemogenic viruses and the expression of preleukemic changes in the (AKR X NZB)F1 thymocytes. The NZB mouse gene or genes appeared to exert this suppressive effect by acting on the thymic reticuloepithelial cells and not on the thymic lymphocytes of (AKR X NZB)F1 hybrids. Prospective studies with thymic epithelial grafts from young mice showed that the AKR thymic epithelium could mediate the formation and expression of leukemogenic recombinant viruses and preleukemic changes in thymocytes that lead to the development of thymic leukemia, whereas the (AKR X NZB)F1 thymic epithelium was deficient in this regard. Our results also confirmed a previous observation that during in vivo generation of recombinant leukemogenic viruses, the acquisition of polytropic virus-related sequences in the 3' portion of the p15E gene and the U3 region and in the 5' part of the gp70 gene can occur independently.