Discovery of a New Endogenous Type C Retrovirus (FcEV) in Cats: Evidence for RD-114 Being an FcEVGag-Pol/Baboon Endogenous Virus BaEVEnv Recombinant

Analysis of a cat genomic DNA library showed that cats harbor a previously unrecognized endogenous type C retrovirus, whose env gene has homology to the murine Fv-4 resistance gene. This unique retrovirus, designated FcEV (Felis catus endogenous retrovirus), has a type C pol gene, closely related to the primate Papio cynocephalus endogenous virus (PcEV) pol, not overlapping the env gene, unlike in other type C retroviruses, and is presumably present in a higher copy number than RD-114. Phylogenetic analysis of FcEV and RD-114 fragments amplified from cat species and comparison with baboon endogenous virus (BaEV) fragments from monkeys suggested that RD-114 does not represent the cat strain of BaEV but is actually a new recombinant between FcEV type C genes and the env gene of BaEV. Although BaEV did appear to have infected an ancestor of the domestic cat lineage, it was a de novo recombinant that made its way into the cat germ line.     

Structural Characteristics and Nucleotide Sequence Analysis of Genomic RNA from RD-114 Virus and Feline RNA Tumor Viruses

The results of molecular hybridization experiments have demonstrated that the RNA genome of RD-114 virus has extensive nucleotide sequence homology with the RNA genome of Crandell virus, an endogenous type C virus of cats, but only limited homology with the RNA genomes of feline sarcoma virus and feline leukemia virus. The genomic RNAs of RD-114 virus and Crandell virus also had identical sedimentation coefficients of 50S. A structural rearrangement of genomic RNA did not exist within released RD-114 virions, whereas a structural rearrangement of genomic RNA did occur within feline sarcoma virions and feline leukemia virions after release from virus-producing cells.     

Baboon Virus Isolate M-7 with Properties Similar to Feline Virus RD-114

A virus (M-7) isolated from baboon placental tissue demonstrates many similarities to endogenous feline virus RD-114. Immunodiffusion analysis shows a group-specific antigen (gs-1) line of identity between M-7 and RD-114. Anti-RD-114 DNA polymerase IgG inhibits M-7 polymerase by 57% compared to 97% for RD-114. M-7 virus has helper activity as demonstrated by rescue of murine sarcoma virus (MSV) from sarcoma-positive leukemia-negative human amnion cells. The host range of the rescued M-7 pseudotype of MSV, MSV (M-7), is similar to that of RD-114 virus. MSV (M-7) is also able to transform baboon cells and causes no detectable transformation of feline cells without addition of helper feline leukemia virus. Interference properties of M-7 and RD-114 virus are identical. Virus-specific neutralizing antisera, although partially cross-reacting, can distinguish MSV (M-7) from MSV (RD-114). These similarities and differences between RD-114 and M-7 viruses are best explained as type-specific differences between two viruses within the same strain.     

Monoclonal antibodies against baboon endogenous virus and against host cell antigens.

Monoclonal antibodies were produced by murine hybridomas after immunization with semipurified baboon endogenous virus. In a solid-phase radioimmunoassay, two antibodies (F12-9 and B9-18) reacted with viral antigen only. The antibodies A6-8 and C9-12 also reacted with virus-producing cells but not with control cells, whereas antibodies E4-6 and D12-2 bound to virus-free cells as well. The cytofluorometry technique confirmed these results and showed a competition between antibodies A6-8 and C9-12 for binding to virus-producing cells as well as a competition between antibodies D12-2 and E4-6 for binding to virus-free human cells. An immune precipitation assay with disrupted virions indicated that antibodies A6-8, B9-18, and C9-12 were directed against the gp70 glycoprotein, and that antibody F12-9 reacted with a viral antigen with a molecular weight of 18,000. The syncytia induced in RSa cells by baboon molecular weight of 18,000. The syncytia induced in RSa cells by baboon endogenous virus could be inhibited either when antibody A6-8 or C9-12 was combined to the virus or when the RSa cells were treated with the anticellular antibody D12-2 or E4-6. These two effects were not observed with Mason-Pfizer virus. Thus, of three antibodies with specificities for viral gp70, two (A6-8 and C9-12) were directed at viral sites responsible for syncytium formation. Another antiviral antibody (F12-9) reacted with a protein of unknown function with a molecular weight of 18,000. The two anticellular antibodies were directed at similar or neighboring epitopes, which may be situated within the receptor to the virus.     

Lack of Sequence Homology Between the 70S RNA of Various RNA Tumor Viruses and the DNA of Simian Virus 40 or Polyoma Virus

No significant hybridization was detected of DNA from simian virus 40 or polyoma virus, and of 70S RNA from avian myeloblastosis virus, murine leukemia virus (Rauscher), murine sarcoma virus (Kirsten), RD-114B, simian sarcoma virus-1, or Mason-Pfizer virus.     

Two active forms of RD-114 virus DNA polymerase in infected cells.

Two forms of DNA polymerase are present in RD-114-infected human, dog, and mink cells, but are not detectable in uninfected cells. The two enzymes are indistinguishable catalytically and immunologically, but differ with respect to molecular weight and elution position from (dT)12-18-cellulose and phosphocellulose. The large enzyme (equivalent 95,000 daltons) is found in the infected cells, but not the virions produced by these cells. The virions contain only the smaller enzyme (equivalent 70,000 daltons). The larger form may represent a mammalian viral equivalent to the beta subunit of avian RNA tumor virus DNA polymerase.     

Viral-specific antigen synthesis following de novo RD-114 virus infection of stationary cells

The cell cycle dependence of retrovirus replication was studied. Canine sarcoma (D-17) cells were infected de novo with the xenotropic feline retrovirus RD-114 under conditions previously reported to simultaneously inhibit virus replication and cell DNA synthesis and/or cell division. RD-114 viral antigen synthesis was observed under conditions previously reported to be inhibitory to avian and murine oncornavirus replication, including confluency and serum deprivation, X-irradiation, mitomycin C pretreatment, colchicine, and ethidium bromide treatments of cells. Several mechanisms that could account for viral antigen synthesis under the restrictive conditions used are discussed.     

Sodium-dependent neutral amino acid transporter type 1 is an auxiliary receptor for baboon endogenous retrovirus

The baboon endogenous retrovirus (BaEV) belongs to a large, widely dispersed interference group that includes the RD114 feline endogenous virus and primate type D retroviruses. Recently, we and another laboratory independently cloned a human receptor for these viruses and identified it as the human sodium-dependent neutral amino acid transporter type 2 (hASCT2). Interestingly, mouse and rat cells are efficiently infected by BaEV but only become susceptible to RD114 and type D retroviruses if the cells are pretreated with tunicamycin, an inhibitor of protein N-linked glycosylation. To investigate this host range difference, we cloned and analyzed NIH Swiss mouse ASCT2 (mASCT2). Surprisingly, mASCT2 did not mediate BaEV infection, which implied that mouse cells might have an alternative receptor for this virus. In addition, elimination of the two N-linked oligosaccharides from mASCT2 by mutagenesis, as substantiated by protein N-glycosidase F digestions and Western immunoblotting, did not enable it to function as a receptor for RD114 or type D retroviruses. Based on these results, we found that the related ASCT1 transporters of humans and mice are efficient receptors for BaEV but are relatively inactive for RD114 and type D retroviruses. Furthermore, elimination of the two N-linked oligosaccharides from extracellular loop 2 of mASCT1 by mutagenesis enabled it to function as an efficient receptor for RD114 and type D retroviruses. Thus, we infer that the tunicamycin-dependent infection of mouse cells by RD114 and type D retroviruses is caused by deglycosylation of mASCT1, which unmasks previously buried sites for viral interactions. In contrast, BaEV efficiently employs the glycosylated forms of mASCT1 that occur normally in untreated mouse cells.     

Localization of the receptor gene for type D simian retroviruses on human chromosome 19.

Simian retrovirus (SRV) serotypes 1 to 5 are exogenous type D viruses causing immune suppression in macaque monkeys. These viruses exhibit receptor interference with each other, with two endogenous type D viruses of the langur (PO-1-Lu) and squirrel monkey, and with two type C retroviruses, feline endogenous virus (RD114/CCC) and baboon endogenous virus (BaEV), indicating that each utilizes the same cell surface receptor (M. A. Sommerfelt and R. A. Weiss, Virology 176:58-69, 1990). Vesicular stomatitis virus pseudotype particles bearing envelope glycoproteins of RD114, BaEV, and the seven SRV strains were employed to detect receptors expressed in human-rodent somatic cell hybrids segregating human chromosomes. The only human chromosome common to all the susceptible hybrids was chromosome 19. By using hybrids retaining different fragments of chromosome 19, a provisional subchromosomal localization of the receptor gene was made to 19q13.1-13.2. Antibodies previously reported to be specific to a BaEV receptor (L. Thiry, J. Cogniaux-Leclerc, R. Olislager, S. Sprecher-Goldberger, and P. Burkens, J. Virol. 48:697-708, 1983) did not block BaEV, RD114, or SRV pseudotypes or syncytia. Antibodies to known surface markers determined by genes mapped to chromosome 19 did not block virus-receptor interaction. The identity of the receptor remains to be determined.     

Isolation of an RD-114-Like Oncornavirus from a Cat Cell Line

A clone of cells derived from a continuous line of cat cells (CCC) spontaneously produced an RNA C-type virus (CCC virus) which did not have the group-specific antigen of the standard strains of feline leukemia viruses but did have that of the RD-114 virus. Single-hit infection of a virus yielding CCC cell with only the feline leukemia virus pseudotype of murine sarcoma virus [MSV(FeLV)] resulted in the release of a pseudotype of MSV coated with the CCC virus envelope. Host range, transmission of virus, helper functions, interference properties, and specific neutralization showed that the CCC and the RD-114 isolates as well as their respective MSV pseudotypes are closely similar if not identical. Parental, virus-negative cells frozen before the existence of RD-114 were chemically induced to yield CCC-like virus de novo. Infection of susceptible human cells with the chemically induced virus resulted in interference with the CCC virus pseudotype of MSV but not with the FeLV pseudotype of MSV.     

A high-affinity monoclonal antibody (GIF-1) to human gamma-interferon: neutralization of interferon mediated inhibition of retrovirus production and 2'-5' (A) synthetase induction

An hybridoma clone secreting an IgG1 monoclonal antibody (GIF-1) specific for human gamma-interferon (HuIFN-gamma) has been generated using HAT medium supplemented with insulin (HIAT) at the initial stage of cell fusion. This antibody is capable of neutralizing the antiviral activity of HuIFN-gamma, the ability of HuIFN-gamma to inhibit retroviral replication in RD-114 cells, and the ability of HuIFN-gamma to induce the 2'-5' oligoadenylate (A) synthetase in RD-114 and HeLa cells. Eluate from an immunoaffinity column containing GIF-1 yielded two protein bands of molecular weight of 20 and 25 kd when subjected to SDS-PAGE.     

Differential association of transfer RNAs with the genomes of murine, feline and primate retroviruses

The tRNAs that are bound to the genomic RNAs of several murine, feline, and primate retroviruses have been identified. Transfer RNAs were divided into those loosely bound and those tightly bound by stepwise thermal dissociation of the 70 S RNA. They were then identified and semiquantitated by aminoacylation. Proline tRNA is the most tenaciously bound tRNA in several strains of murine leukemia virus, two strains of feline leukemia virus, and the primate viruses simian sarcoma, baboon endogenous, and gibbon ape lymphoma. In the feline xenotropic virus, RD-114, tRNAGly is enriched in the most tightly bound fraction. In Mason-Pfizer monkey virus, as in the murine mammary tumor virus, tRNALys is the tRNA most tenaciously bound to its genomic RNA. Besides the most tightly associated tRNA, one or more different tRNAs are found in relatively large amounts in association with the 70 S RNA. (For convenience, we refer to the largest RNA ccomplex (50-70 S) isolated from any of the retroviruses studies as '70 S' RNA.) These tRNAs can be distinguished from the most tightly bound tRNA by the fact that they can be dissociated at lower temperatures. However, they occur in the same relative abundance as the tightly bound tRNA.     

Isolation from cats of an endogenous type C virus with a novel envelope glycoprotein.

A search for variant endogenous cat viruses led to a novel isolate. Although the major envelope glycoprotein of this virus was similar in size to that of an RD-114-like virus that was coisolated, it was unrelated to RD-114 or feline leukemia virus by immunological and biological criteria. This degree of dissimilarity suggests a different evolutionary progenitor from that for the RD-114 and feline leukemia virus viral envelopes. The novel virus did, however, code for gag gene polypeptides which are closely related to RD-114 virus. Neither the novel isolate nor the RD-114-like coisolate induced foci in S+L- cat cells which restrict focus induction by RD-114 virus. This suggests that the two viruses share a common genomic target of restriction which resides outside of the env region.     

Protein blot analysis of virus receptors: identification and characterization of the Sendai virus receptor

Receptors for Sendai virions in human erythrocyte ghost membranes were identified by virus overlay of protein blots. Among the various erythrocyte polypeptides, only glycophorin was able to bind Sendai virions effectively. The detection of Sendai virions bound to glycophorin was accomplished either by employing anti-Sendai virus antibodies or by autoradiography, when 125I-labeled Sendai virions were used. The binding activity was associated with the viral hemagglutinin/neuraminidase (HN) glycoprotein, as inferred from the observation that the binding pattern of purified HN glycoprotein to human erythrocyte membranes was identical to that of intact Sendai virions. No binding was observed when blots, containing either human erythrocyte membranes or purified glycophorin, were probed with the viral fusion factor (F glycoprotein). Active virions competed effectively with the binding of 125I-labeled Sendai virions (or purified HN glycoprotein), whereas no competition was observed with inactivated Sendai virus. The results of the present work clearly show that protein blotting can be used to identify virus receptors in cell membrane preparations.     

Dynamic antibody specificities and virion concentrations in circulating immune complexes in acute to chronic HIV-1 infection

Understanding the interactions between human immunodeficiency virus type 1 (HIV-1) virions and antibodies (Ab) produced during acute HIV-1 infection (AHI) is critical for defining antibody antiviral capabilities. Antibodies that bind virions may prevent transmission by neutralization of virus or mechanically prevent HIV-1 migration through mucosal layers. In this study, we quantified circulating HIV-1 virion-immune complexes (ICs), present in approximately 90% of AHI subjects, and compared the levels and antibody specificity to those in chronic infection. Circulating HIV-1 virions coated with IgG (immune complexes) were in significantly lower levels relative to the viral load in acute infection than in chronic HIV-1 infection. The specificities of the antibodies in the immune complexes differed between acute and chronic infection (anti-gp41 Ab in acute infection and anti-gp120 in chronic infection), potentially suggesting different roles in immunopathogenesis for complexes arising at different stages of infection. We also determined the ability of circulating IgG from AHI to bind infectious versus noninfectious virions. Similar to a nonneutralizing anti-gp41 monoclonal antibody (MAb), purified plasma IgG from acute HIV-1 subjects bound both infectious and noninfectious virions. This was in contrast to the neutralizing antibody 2G12 MAb that bound predominantly infectious virions. Moreover, the initial antibody response captured acute HIV-1 virions without selection for different HIV-1 envelope sequences. In total, this study demonstrates that the composition of immune complexes are dynamic over the course of HIV-1 infection and are comprised initially of antibodies that nonselectively opsonize both infectious and noninfectious virions, likely contributing to the lack of efficacy of the antibody response during acute infection.     

RD 114 Virus-Specific Sequences in Feline Cellular RNA: Detection and Characterization

RNA extracted from cat cells contains sequences homologous to RD-114 viral RNA. The sequences are measured by molecular hybridization with a single-stranded DNA probe synthesized by the virion polymerase using the endogenous viral RNA as template. Viral-specific RNA has been detected in all cells of cat origin tested thus far, but not in cells of other animals, except for the virus-producing human rhabdomyosarcoma cell, RD-114. The extent of hybridization of the DNA probe to cellular RNA was equivalent to that obtained with viral 70S RNA indicating that an equal extent of viral specific sequences is present in all cat cells as well as in RD-114 cells. The amounts of this viral RNA reach approximately 100 copies per cell in cat cells, while virus-producing RD-114 cells contain about 1,000 copies per cell. The viral RNA is present in cat cells in two distinct sizes of about 35S and 18S, whereas in RD-114 cells virus-specific RNA is quite heterogeneous in size.     

Nucleotide composition of the RNA from RD-114 virions

The 70S and 4S RNA components of a C-type oncornavirus, RD-114, released from a human rhabdomyosarcoma cell line (RD) after transplantation in a kitten, were analyzed for nucleotide constituents. Minor nucleotides were detected only in the 4S RNA populations, and two of these nucleotides were identified as 5,6-dihydro-UMP and pseudo-UMP. The base composition of the RD-114 70S RNA differs from that of the 70S RNA from RD-FeLV (the virus released from the RD cell line after deliberate infection with a feline leukemia virus).