A New Indicator Cell Line Established to Monitor Bovine Foamy Virus Infection

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro,we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR,from-7 to 1012).The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR.One clone,designated BFVL,was selected from ten neomycin-res...     

A new indicator cell line established to monitor bovine foamy virus infection

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro, we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR, from −7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone, designated BFVL, was selected from ten neomycin-resistant clones. BFVL showed a specific and inducible dose- and time-dependent luciferase activity in response to BFV infection. Although the changes in luciferase activity of BFVL peaked at 84 h post infection, it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL. Moreover, the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid, easy, sensitive, quantitative and specific for detection of BFV infection.     

Complete genome sequences of two novel European clade bovine foamy viruses from Germany and poland

Bovine foamy virus (BFV), or bovine spumaretrovirus, is an infectious agent of cattle with no obvious disease association but high prevalence in its host. Here, we report two complete BFV sequences, BFV-Riems, isolated in 1978 in East Germany, and BFV100, isolated in 2005 in Poland. Both new BFV isolates share the overall genetic makeup of other foamy viruses (FV). Although isolated almost 25 years apart and propagated in either bovine (BFV-Riems) or nonbovine (BFV100) cells, both viruses are highly related, forming the European BFV clade. Despite clear differences, BFV-Riems and BFV100 are still very similar to BFV isolates from China and the United States, comprising the non-European BFV clade. The genomic sequences presented here confirm the concept of high sequence conservation across most of the FV genome. Analyses of cell culture-derived genomes reveal that proviral DNA may specifically lack introns in the env-bel coding region. The spacing of the splice sites in this region suggests that BFV has developed a novel mode to express a secretory but nonfunctional Env protein.     

The promyelocytic leukemia protein does not mediate foamy virus latency in vitro

Spumaviruses, commonly called foamy viruses, are complex retroviruses that establish life-long persistent infections in the absence of accompanying pathology. Depending upon cell type, infection of cells in tissue culture cells can result in either lytic replication, persistence, or latency. The cellular factors that mediate foamy virus (FV) latency are poorly understood. In this study we show that the only known inhibitor of FV replication, the promyelocytic leukemia protein (PML), which binds the FV transactivator (Tas), does not play an important role in FV latency in vitro. We found no significant differences in PML levels in cells that supported lytic replication compared to those that were latently infected. Furthermore, endogenous PML levels did not change following exposure to phorbol myristate acetate (PMA), which induces FV replication. We demonstrated that FV replication proceeded in the presence of substantial levels of PML, both in fully permissive cells and during reactivation of latent FV. Endogenous PML did not efficiently colocalize with Tas, even after upregulation by alpha interferon (IFN-alpha) treatment. IFN-alpha did, however, partially suppress the reactivation of latent FV by PMA. Finally, depletion of endogenous PML by small interfering RNA did not promote activation of FV in cells that responded to PMA treatment. Taken together, these data indicate that endogenous PML does not play an important role in mediating FV latency.     

Interferon-inducible Myc/STAT-interacting protein Nmi associates with IFP 35 into a high molecular mass complex and inhibits proteasome-mediated degradation of IFP 35

Nmi is an interferon (IFN)-inducible protein homologous to IFN-inducible protein IFP 35. The homology consists of a novel Nmi/IFP 35 domain (NID) of 90-92 amino acids that is repeated in tandem in each protein and mediates Nmi-Nmi protein interactions and subcellular localization. In a yeast two-hybrid screen with a fragment of Nmi protein containing both NIDs, we identified an interaction between Nmi and IFP 35. Deletion derivatives of the proteins indicate that both NIDs are required for the interaction between Nmi and IFP 35. In mammalian cells, Nmi and IFP 35 co-immunoprecipitate and co-localize in large cytoplasmic speckles. Nmi and IFP 35 proteins associate into a high molecular mass complex of 300-400 kDa as determined by native gel electrophoresis and gel filtration. The association of Nmi and IFP 35 into a complex can be demonstrated in multiple cell lines and is not dependent on treatment with IFN. Short term and long term cultures of transfected HEK293 cells suggest that Nmi and IFP 35 proteins stabilize each other through complex formation. IFP 35 appears to be more labile because Nmi was stable in the absence of IFP 35, whereas IFP 35 was degraded in the absence of Nmi. A deletion analysis revealed that Nmi must interact with IFP 35 to prevent its degradation and that the amino terminus of Nmi is required, but not sufficient, for this function. Inhibition of the proteasome, but not other proteases, led to increased levels of IFP 35. Thus, we have shown that Nmi and IFP 35 associate into a protein complex, that IFP 35 is degraded in a proteasome-mediated process, and that a novel function of Nmi is to prevent IFP 35 degradation. The stabilization of IFP 35 by Nmi may serve to amplify the physiologic effects of IFNs.     

Similar Patterns of Infection with Bovine Foamy Virus in Experimentally Inoculated Calves and Sheep

Foamy viruses (FVs) are the least known retroviruses commonly found in primates, cats, horses, and cattle. Although FVs are considered apathogenic, simian and feline FVs have been shown to be associated with some transient health abnormalities in animal models. Currently, data regarding the course of infection with bovine FV (BFV) are not available. In this study, we conducted experimental infections of natural (cattle) and heterologous (sheep) hosts with the BFV₁₀₀ isolate and monitored infection patterns in both hosts during the early phase postinoculation as well as after long-term infection. Four calves and six sheep inoculated with BFV₁₀₀ showed no signs of pathology but developed persistent infection, as confirmed by virus rescue, consistent detection of BFV-specific antibodies, and presence of viral DNA. In both hosts, antibodies against BFV Gag and Bet appeared early after infection and persisted at high and stable levels while seroreactivity toward Env was consistently detectable only in BFV-infected sheep. Interestingly, the BFV proviral DNA load was highest in lung, spleen, and liver and moderate in leukocytes, while salivary glands contained either low or undetectable DNA loads in calves or sheep, respectively. Additionally, comparison of partial BFV sequences from inoculum and infected animals demonstrated very limited changes after long-term infection in the heterologous host, clearly less than those found in BFV field isolates. The persistence of BFV infection in both hosts suggests full replication competence of the BFV₁₀₀ isolate with no requirement of genetic adaptation for productive replication in the authentic and even in a heterologous host.     

Roles of a conserved proline in the internal fusion peptide of Ebola glycoprotein

The structural determinants underlying the functionality of viral internal fusion peptides (IFPs) are not well understood. We have compared EBOwt (GAAIGLAWIPYFGPAAE), representing the IFP of the Ebola fusion protein GP, and EBOwt (GAAIGLAWIPYFGRAAE) derived from a non-functional mutant with conserved Pro537 substituted by Arg. P537R substitution did not abrogate peptide-membrane association, but interfered with the ability to induce bilayer destabilization. Structural determinations suggest that Pro537 is required to preserve a membrane-perturbing local conformation in apolar environments.     

Bovine papillomavirus E1 protein is sumoylated by the host cell Ubc9 protein

Papillomavirus E1 protein is the replication initiator that recognizes and binds to the viral origin and initiates DNA strand separation through its ATP-dependent helicase activity. The E1 protein also functions in viral DNA replication by recruiting several cellular proteins to the origin, including host DNA polymerase alpha and replication protein A. To identify other cellular proteins that interact with bovine papillomavirus E1, an HeLa cDNA library was screened using a yeast two-hybrid assay. The host cell sumoylating enzyme, Ubc9, was found to interact specifically with E1 both in vitro and in vivo. Mapping studies localized critical E1 sequences for interaction to amino acids 315-459 and strongly implicated leucine 420 as critical for E1.Ubc9 complex formation. In addition to binding E1, Ubc9 catalyzed the covalent linkage of the ubiquitin-like protein, SUMO-1, to E1. An E1 mutant unable to bind Ubc9 showed normal intracellular stability, but was impaired for intranuclear distribution. Failure to accumulate in appropriate nuclear subdomains may account for the previously demonstrated replication defect of a human papillomavirus 16 E1 protein that was also unable to bind Ubc9 and suggests that sumoylation is a functionally important modification with regulatory implications for papillomavirus replication.     

Carboxy-terminal cleavage of the human foamy virus Gag precursor molecule is an essential step in the viral life cycle.

Foamy viruses (FVs) express the Gag protein as a precursor with a molecular mass of 74 kDa (pr74) from which a 70-kDa protein (p70) is cleaved by the viral protease. To gain a better understanding of FV Gag protein processing and function, we have generated and analyzed mutants in the C-terminal gag region of an infectious molecular clone. Our results show that p70 is an N-terminal cleavage product of pr74. However, we were unable to identify a p4 molecule. A virus mutant expressing p70 only was found to be replication competent, albeit at very low titers compared to those of wild-type virus. A strong tendency to synthesize and cleave a pr74 molecule was deduced from the occurrence of revertants upon transfection of this mutant. Substitution of the p6gag domain of human immunodeficiency virus type 1 for the p4 domain of FV resulted in a stable chimeric virus which replicated to titers 10 times lower than those of wild-type virus. FV Gag protein was found to be phosphorylated at serine residues. Mutagenesis of serines conserved in the p4 domain had no influence on viral replication in cell culture. The p70/p74 Gag cleavage was found to be required for viral infectivity, since mutagenesis of the putative cleavage site led to replication-incompetent virus. Interestingly, the cleavage site mutants were defective in the intracellular cDNA synthesis of virion DNA, which indicates that correct FV particle formation and the generation of virion DNA are functionally linked.