Analysis of the role of the bel and bet open reading frames of human foamy virus by using a new quantitative assay.

We have constructed a BHK-21-derived indicator cell line containing a single integrated copy of the beta-galactosidase (beta-Gal) gene under control of the human foamy virus (HFV) long terminal repeat promoter (from -533 to +20). These foamy virus-activated beta-Gal expression (FAB) cells can be used in a quantitative assay to measure the infectious titer of HFV. Our results show that the FAB assay is 50 times more sensitive than determination of the virus titer by the end-point dilution method. Using the FAB assay, we have found that HFV can productively replicate in several erythroblastoid cell lines as well as in the Jurkat T-cell line. We have also examined the roles of bel2, bet, and bel3 in viral replication by constructing proviral HFV clones in which the reading frame of Bel2, Bet, or Bel3 is disrupted by placement of translation stop codons. Analysis of these mutants reveals that while the bel3 gene is not required for viral replication in vitro, mutations in the bel2 or bet gene decrease cell-free viral transmission approximately 10-fold.     

An Evolutionarily Conserved Splice Generates a Secreted Env-Bet Fusion Protein during Human Foamy Virus Infection

Foamy viruses (spumaretroviruses) represent a retroviral genus which exhibits unusual features relating it to pararetroviruses. Previously, we reported the existence of a protein species harboring Env, Bel, and Bet epitopes in human foamy virus (HFV)-infected cells (M. L. Giron, F. Rozain, M. C. Debons-Guillemin, M. Canivet, J. Périès, and R. Emanoil-Ravier, J. Virol. 67:3596–3600, 1993). Here, we identify this protein as a 160-kDa Env-Bet fusion glycoprotein (gp160) translated from an mRNA species harboring a highly conserved splice site which deletes the membrane anchor domain of Env and fuses the env open reading frame with that of bel1/bet. While gp160 and Bet proteins were both secreted into the supernatant, only Bet was taken up by recipient cells. Since Bet plays a key role in the switch from lytic to chronic infection, secretion of Bet and gp160, together with cellular uptake of Bet, could be highly relevant for both immune response and development of HFV infection in vivo.     

Frequent disruption of the Nf1 gene by a novel murine AIDS virus-related provirus in BXH-2 murine myeloid lymphomas.

Evi-2, a common site of viral integration in BXH-2 myeloid lymphomas, is located within a large intron of the Nf1 tumor suppressor gene. Viral integration at Evi-2 appears to induce disease by disrupting normal Nf1 expression. During our attempts to characterize the nature of the proviruses located at Evi-2, we found that approximately half of the proviruses were defective nonecotropic proviruses (A. M. Buchberg, H. G. Bedigian, N. A. Jenkins, and N. G. Copeland, Mol. Cell. Biol. 10:4658-4666, 1990). This was surprising, since most proviruses characterized at other BXH-2 common integration sites are full-length ecotropic viruses. In the studies described here, we found that this defective provirus carries two large deletions, one in pol and one in env, and is structurally related to another murine retrovirus, the murine AIDS retrovirus. By using oligonucleotide probes specific for this defective provirus, designated MRV, we showed that MRV-related proviruses are carried as endogenous germ line proviruses in most inbred strains. In addition, we identified the endogenous MRV provirus that gives rise to the defective proviruses identified at Evi-2. We present a model that accounts for the positive selection of MRV proviruses at Evi-2, which may allow selective identification of common viral integration sites harboring tumor suppressor genes.     

Intra- and intercellular trafficking of the foamy virus auxiliary bet protein

The Bet protein of foamy viruses (FVs) is an auxiliary protein encoded by the 3' end of the viral genome. Although its function during the viral replication cycle is still unknown, Bet seems to play a key role in the establishment and/or maintenance of viral persistence, representing the predominant viral protein detected during chronic infection. To clarify the function of this viral protein, the subcellular distribution of Bet from the prototypic human foamy virus (HFV) was examined. We report here that this protein is distributed in both the cytoplasm and the nucleus of HFV-infected or Bet-transfected cells. The nuclear targeting results from the presence of a bipartite nuclear localization signal at the C-terminal region, sufficient to direct heterologous reporter proteins to the nucleus. Since HFV Bet spreads between cells, we show here that the secreted protein targets the nuclei of recipient cells. HFV Bet follows an unconventional route to exit the cell since its secretion is not affected by brefeldin A, a drug which disrupts the trafficking between the endoplasmic reticulum and the Golgi complex. Finally, these inter- and intracellular movements were also observed for the equine foamy virus Bet protein, strongly suggesting that these remarkable features are conserved among FVs.     

Retention or loss of v-mil sequences after propagation of MH2 virus in vivo or in vitro.

During propagation of the defective avian retrovirus MH2 in the presence of replication-competent helper virus, deletion of portions of the viral genome occurred frequently. After transformation of quail cells in vitro, v-mil sequences were lost, leading to populations of MH2 viruses which were highly deficient for mil gene expression but which could transform macrophage and fibroblast cells in vitro with high efficiency. In contrast, after induction of tumors in quail with mil-deficient MH2 viral stocks, a majority of the tumor DNAs contained mil+ proviruses, suggesting that there is selection for retention of the v-mil gene in vivo and that the mil protein may play a role in the oncogenicity of MH2 virus. We also isolated MH2-transformed cell lines which contained deleted proviruses arising from packaging and subsequent integration of the subgenomic v-myc-encoding mRNA. Some of these cell lines produced viruses which encoded abnormal v-myc proteins and had altered in vitro transforming properties. These altered phenotypes may be caused by mutations within the v-myc gene.     

Productive persistent infection of hematopoietic cells by human foamy virus.

Human foamy virus can establish persistent infections in human hematopoietic cell lines, such as H92.1.7 (erythroblastoid cells), Jurkat (CD4+ T cells), and U937 (myeloid-monocytic cells). The infection is characterized by constant production of infectious viruses (for > 2 1/2 years) with no cytopathic effects on the host cells. Electron microscopy of the infected cells showed a viral morphology similar to that observed for particles produced after acute infection. We have detected, in addition to the full-length form of bel1, a previously described deletion in the bel1 gene of the proviral DNA in these cells. RNA containing this 301-bp deletion, which mapped to the splice donor and acceptor sites of the intron of the bet gene, was also found in encapsidated virion RNA. However, the presence of this defective provirus harboring the deletion in bel1 does not prevent productive persistence in these chronically infected cells, since the virus titer does not decrease during cultivation.     

Foamy virus capsids require the cognate envelope protein for particle export

Unlike other subclasses of the Retroviridae the Spumavirinae, its prototype member being the so-called human foamy virus (HFV), require the expression of the envelope (Env) glycoprotein for viral particle egress. Both the murine leukemia virus (MuLV) Env and the vesicular stomatitis virus G protein, which efficiently pseudotype other retrovirus capsids, were not able to support export of HFV particles. Analysis of deletion and point mutants of the HFV Env protein revealed that the HFV Env cytoplasmic domain (CyD) is dispensable for HFV particle envelopment, release, and infectivity, whereas deletion of the membrane-spanning-domain (MSD) led to an accumulation of naked capsids in the cytoplasm. Neither alternative membrane association of HFV Env deletion mutants lacking the MSD and CyD via phosphoglycolipid anchor nor domain swapping mutants, with the MSD or CyD of MuLV Env and VSV-G exchanged against the corresponding HFV domains, could restore particle envelopment and the release defect of pseudotypes. However, replacement of the HFV MSD with that of MuLV led to budding of HFV capsids at the intracellular membranes. These virions were of apparently wild-type morphology but were not naturally released into the supernatant and they were noninfectious.     

Drosophila germline invasion by the endogenous retrovirus gypsy: involvement of the viral env gene

The endogenous retrovirus gypsy is expressed at high levels in mutant flamenco female flies. Gypsy viral particles extracted from such flies can infect naive flamenco individuals raised in the presence of these extracts mixed into their food. This results in the integration of new proviruses into the germline genome. These proviruses can then increase their copy number by (1) expression in the flamenco female somatic cells, (2) transfer into the oocyte and (3) integration into the genome of the progeny. Surprisingly, unlike the infection observed in the feeding experiments, this strategy of endogenous proviral multiplication does not seem to involve the expression of the viral env gene.     

Salt Tolerance of Glycinebetaine-Deficient and -Containing Maize Lines

Pairs of homozygous near-isogenic glycinebetaine-containing (Bet1/Bet1) and -deficient (bet1/bet1) F8 lines of Zea mays L. (maize) were tested for differences in salt (150 mM NaCl or 127.25 mM NaCl plus 22.5 mM CaCl2) tolerance. The Bet1/Bet1 lines exhibited less shoot growth inhibition (as measured by dry matter accumulation, leaf area expansion rate and/or, plant height extension rate) under salinized conditions in comparison to their nearisogenic bet1/bet1 sister lines. These growth differences were associated with maintenance of a significantly higher leaf relative water content, a higher rate of carbon assimilation, and a greater turgor in Bet1/Bet1 lines than in bet1/bet1 lines under salinized conditions. These results strongly suggest that a single gene conferring glycinebetaine accumulation (and/or a tightly linked locus) plays a key role in osmotic adjustment in maize.     

Epstein-Barr virus lytic infection contributes to lymphoproliferative disease in a SCID mouse model

Most Epstein-Barr virus (EBV)-positive tumor cells contain one of the latent forms of viral infection. The role of lytic viral gene expression in EBV-associated malignancies is unknown. Here we show that EBV mutants that cannot undergo lytic viral replication are defective in promoting EBV-mediated lymphoproliferative disease (LPD). Early-passage lymphoblastoid cell lines (LCLs) derived from EBV mutants with a deletion of either viral immediate-early gene grew similarly to wild-type (WT) virus LCLs in vitro but were deficient in producing LPD when inoculated into SCID mice. Restoration of lytic EBV gene expression enhanced growth in SCID mice. Acyclovir, which prevents lytic viral replication but not expression of early lytic viral genes, did not inhibit the growth of WT LCLs in SCID mice. Early-passage LCLs derived from the lytic-defective viruses had substantially decreased expression of the cytokine interleukin-6 (IL-6), and restoration of lytic gene expression reversed this defect. Expression of cellular IL-10 and viral IL-10 was also diminished in lytic-defective LCLs. These results suggest that lytic EBV gene expression contributes to EBV-associated lymphoproliferative disease, potentially through induction of paracrine B-cell growth factors.     

Genetic evidence for two distinct transactivation functions of the herpes simplex virus alpha protein ICP27.

Infected-cell protein 27 (ICP27) is a herpes simplex virus type 1 alpha, or immediate-early, protein involved in the regulation of viral gene expression. To better understand the function(s) of ICP27 in infected cells, we have isolated and characterized viral recombinants containing defined alterations in the ICP27 gene. The mutant virus d27-1 contains a 1.6-kilobase deletion which removes the ICP27 gene promoter and most of the coding sequences, while n59R, n263R, n406R, and n504R are mutants containing nonsense mutations which encode ICP27 molecules truncated at their carboxyl termini. All five mutants were defective for lytic replication in Vero cells. Analysis of the mutant phenotypes suggests that ICP27 has the following regulatory effects during the viral infection: (i) stimulation of expression of gamma-1 genes, (ii) induction of expression of gamma-2 genes, (iii) down regulation of expression of alpha and beta genes late in infection, and (iv) stimulation of viral DNA replication. Cells infected with the mutant n504R expressed wild-type levels of gamma-1 proteins but appeared to be unable to efficiently express gamma-2 mRNAs or proteins. This result suggests that ICP27 mediates two distinct transactivation functions, one which stimulates gamma-1 gene expression and a second one required for gamma-2 gene induction. Analysis of the mutant n406R suggested that a truncated ICP27 polypeptide can interfere with the expression of many viral beta genes. Our results demonstrate that ICP27 has a variety of positive and negative effects on the expression of viral genes during infection.     

A paradigm for virus-host coevolution: sequential counter-adaptations between endogenous and exogenous retroviruses

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of the host germline transmitted vertically from generation to generation. It is hypothesized that some ERVs are used by the host as restriction factors to block the infection of pathogenic retroviruses. Indeed, some ERVs efficiently interfere with the replication of related exogenous retroviruses. However, data suggesting that these mechanisms have influenced the coevolution of endogenous and/or exogenous retroviruses and their hosts have been more difficult to obtain. Sheep are an interesting model system to study retrovirus-host coevolution because of the coexistence in this animal species of two exogenous (i.e., horizontally transmitted) oncogenic retroviruses, Jaagsiekte sheep retrovirus and Enzootic nasal tumor virus, with highly related and biologically active endogenous retroviruses (enJSRVs). Here, we isolated and characterized the evolutionary history and molecular virology of 27 enJSRV proviruses. enJSRVs have been integrating in the host genome for the last 5-7 million y. Two enJSRV proviruses (enJS56A1 and enJSRV-20), which entered the host genome within the last 3 million y (before and during speciation within the genus Ovis), acquired in two temporally distinct events a defective Gag polyprotein resulting in a transdominant phenotype able to block late replication steps of related exogenous retroviruses. Both transdominant proviruses became fixed in the host genome before or around sheep domestication (approximately 9,000 y ago). Interestingly, a provirus escaping the transdominant enJSRVs has emerged very recently, most likely within the last 200 y. Thus, we determined sequentially distinct events during evolution that are indicative of an evolutionary antagonism between endogenous and exogenous retroviruses. This study strongly suggests that endogenization and selection of ERVs acting as restriction factors is a mechanism used by the host to fight retroviral infections.     

Role of the S4 gene in the establishment of persistent reovirus infection in L cells

We have studied the viral genes playing a role in persistence of reovirus in L cells. We established persistent infections by coinfecting L cells using wild-type reovirus type 2 and defective reovirus type 3. An analysis of the genomic double-stranded RNA pattern of the virus population selected during persistent infection revealed that the S4 gene was derived from the defective parent in three independently established lines, suggesting that the S4 gene plays an essential role in the establishment of persistent infection. The selection of recombinant viruses containing the S4 gene derived from the defective virus was specific for persistent infection, since it was not selected in lytic infections. Following the establishment of persistent infection, mutations in the S1 gene appeared in two of three cell lines. Thus mutations in the S4 gene play a critical role in the establishment of persistent infection, while mutations in the S1 gene play a role in the maintenance of the persistent infection.