Chromatin tethering of incoming foamy virus by the structural Gag protein

Retroviruses hijack cellular machineries to productively infect their hosts. During the early stages of viral replication, proviral integration relies on specific interactions between components of the preintegration complex and host chromatin-bound proteins. Here, analyzing the fate of incoming primate foamy virus, we identify a short domain within the C-terminus of the structural Gag protein that efficiently binds host chromosomes, by interacting with H2A/H2B core histones. While viral particle production, virus entry and intracellular trafficking are not affected by mutation of this domain, chromosomal attachment of incoming subviral complexes is abolished, precluding proviral integration. We thus highlight a new function of the structural foamy Gag protein as the main tether between incoming subviral complexes and host chromatin prior to integration.     

Retrotransposition and cell-to-cell transfer of foamy viruses

A remarkable feature of the prototype foamy virus (PFV) replication pathway has been reported to consist of the ability to retrotranspose intracellularly with high efficiency (M. Heinkelein, T. Pietschmann, G. Jármy, M. Dressler, H. Imrich, J. Thurow, D. Lindemann, M. Bock, A. Moebes, J. Roy, O. Herchenr?der, and A. Rethwilm, EMBO J. 19:3436-3345, 2000). PFV intracellular retrotransposition (IRT) was reported to be enhanced by coexpression of fusion-defective envelope protein. To investigate the possibility of cell-to-cell transfer of PFV genomes, which could mimic IRT, we performed cocultivation experiments with cells transfected with an IRT-competent and marker gene-expressing PFV vector together with cells expressing a different marker and measured cells positive for both markers. The findings corroborated the initial report on IRT of Env-deficient PFV. Furthermore, they indicated that viral cores that have incorporated fusion-deficient Env can be transferred from cell to cell in a cell type-specific manor. One possible explanation consists of a minor alternative cleavage site in Env that can be used to expose the fusion peptide of the Env transmembrane protein, which appears to be required for virus uptake.     

Restriction of foamy viruses by primate Trim5alpha

Foamy viruses (FVs) are unconventional retroviruses with a replication strategy that is significantly different from orthoretroviruses and bears some homology to that of hepadnaviruses. Although some cellular proteins, such as APOBEC3, have been reported to block FVs, no restriction by Trim5alpha has been described to date. The sensitivity of three FV isolates of human-chimpanzee or prototypic (PFV), macaque (SFVmac), and feline (FFV) origin to a variety of primate Trim5alphas was therefore tested. PFV and SFVmac were restricted by Trim5alphas from most New World monkeys, but not from other primates, whereas FFV-based vectors were restricted by Trim5alphas from the great apes gorilla and orangutan. Trim5alphas from Old World monkeys did not restrict any FV isolate tested. Capuchin Trim5alpha was unique, as it restricted SFVmac and FFV but not PFV. Trim5alpha specificity for FVs was determined by the B30.2 domain, interestingly involving, in some instances, the same residues of the variable regions previously implicated as major determinants for human immunodeficiency virus type 1 restriction. FVs with chimeric Gags were made to map the viral determinants of sensitivity to restriction. The N-terminal half of the Gag molecule was found to contain the regions that control susceptibility. This region most likely corresponds to the capsid of conventional retroviruses. Due to their unique replication strategy, FVs should provide a valuable new system to examine the mechanism of retroviral restriction by Trim5alpha.     

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.     

Frequent and recent human acquisition of simian foamy viruses through apes' bites in central Africa

Human infection by simian foamy viruses (SFV) can be acquired by persons occupationally exposed to non-human primates (NHP) or in natural settings. This study aimed at getting better knowledge on SFV transmission dynamics, risk factors for such a zoonotic infection and, searching for intra-familial dissemination and the level of peripheral blood (pro)viral loads in infected individuals. We studied 1,321 people from the general adult population (mean age 49 yrs, 640 women and 681 men) and 198 individuals, mostly men, all of whom had encountered a NHP with a resulting bite or scratch. All of these, either Pygmies (436) or Bantus (1085) live in villages in South Cameroon. A specific SFV Western blot was used and two nested PCRs (polymerase, and LTR) were done on all the positive/borderline samples by serology. In the general population, 2/1,321 (0.2%) persons were found to be infected. In the second group, 37/198 (18.6%) persons were SFV positive. They were mostly infected by apes (37/39) FV (mainly gorilla). Infection by monkey FV was less frequent (2/39). The viral origin of the amplified sequences matched with the history reported by the hunters, most of which (83%) are aged 20 to 40 years and acquired the infection during the last twenty years. The (pro)viral load in 33 individuals infected by a gorilla FV was quite low (<1 to 145 copies per 10(5) cells) in the peripheral blood leucocytes. Of the 30 wives and 12 children from families of FV infected persons, only one woman was seropositive in WB without subsequent viral DNA amplification. We demonstrate a high level of recent transmission of SFVs to humans in natural settings specifically following severe gorilla bites during hunting activities. The virus was found to persist over several years, with low SFV loads in infected persons. Secondary transmission remains an open question.     

Differential pH-dependent cellular uptake pathways among foamy viruses elucidated using dual-colored fluorescent particles

ABSTRACT: BACKGROUND: It is thought that foamy viruses (FVs) enter host cells via endocytosis because all FV glycoproteins examined display pH-dependent fusion activities. Only the prototype FV (PFV) glycoprotein has also significant fusion activity at neutral pH, suggesting that its uptake mechanism may deviate from other FVs. To gain new insights into the uptake processes of FV in individual live host cells, we developed fluorescently labeled infectious FVs. RESULTS: N-terminal tagging of the FV envelope leader peptide domain with a fluorescent protein resulted in efficient incorporation of the fluorescently labeled glycoprotein into secreted virions without interfering with their infectivity. Double-tagged viruses consisting of an eGFP-tagged PFV capsid (Gag-eGFP) and mCherry-tagged Env (Ch-Env) from either PFV or macaque simian FV (SFVmac) were observed during early stages of the infection pathway. PFV Env, but not SFVmac Env, containing particles induced strong syncytia formation on target cells. Both virus types showed trafficking of double-tagged virions towards the cell center. Upon fusion and subsequent capsid release into the cytosol, accumulation of naked capsid proteins was observed within four hours in the perinuclear region, presumably representing the centrosomes. Interestingly, virions harboring fusion-defective glycoproteins still promoted virus attachment and uptake, but failed to show syncytia formation and perinuclear capsid accumulation. Non-fused or non-fusogenic viruses are rapidly cleared from the cells by putative lysosomal degradation. Monitoring the fraction of viruses containing both Env and capsid signals as a function of time demonstrated that PFV virions fused within the first few minutes, whereas fusion of SFVmac virions was less pronounced and observed over the entire 90 minutes measured. CONCLUSIONS: The characterized double-labeled FVs described here provide new mechanistic insights into FV early entry steps, demonstrating that productive viral fusion occurs early after target cell attachment and uptake. The analysis highlights apparent differences in the uptake pathways of individual FV species. Furthermore, the infectious double-labeled FVs promise to provide important tools for future detailed analyses on individual FV fusion events in real time using advanced imaging techniques.     

The phylogeography of orangutan foamy viruses supports the theory of ancient repopulation of Sumatra

Phylogenetic analysis of foamy virus sequences obtained from Bornean and Sumatran orangutans showed a distinct clustering pattern. One subcluster was represented by both Bornean and Sumatran orangutan simian foamy viruses (SFV). Combined analysis of host mitochondrial DNA and SFV phylogeny provided evidence for the hypothesis of the repopulation of Sumatra by orangutans from Borneo.     

Centrosomal association of histone macroH2A1.2 in embryonic stem cells and somatic cells

The histone 2A variant macroH2A1.2 is expressed in female and male mammals and is implicated in X-chromosome inactivation and autosomal gene silencing. In undifferentiated and early differentiating murine embryonic stem (ES) cells a cytosolic pool of macroH2A1.2 has recently been reported and found to be associated with the centrosome. Here, we show that the centrosomal association of macroH2A1.2 is a widespread phenomenon and is not restricted to undifferentiated and early differentiating ES cells. By indirect immunofluorescence we detect macroH2A1.2 protein in a juxtanuclear structure that duplicates once per cell cycle and colocalizes with centrosomal gamma-tubulin in both XX and XY ES cells prior to and throughout their differentiation. MacroH2A1.2 localization to the centrosome is also observed in female and male somatic cells, both in interphase and in mitosis. Biochemical analysis demonstrates that the association between macroH2A1.2 and the centrosome in somatic cells is stable, as macroH2A1.2 copurifies with centrosomes isolated from human lymphoblasts. Therefore, in addition to a nuclear pool of macroH2A1.2 a fraction of the histone is associated with the centrosome in various cell types and throughout ES cell differentiation.     

Potential sites of virus latency associated with indigenous pseudorabies viruses in feral swine

Free-ranging feral swine (Sus scrofa) are known to be present in at least 32 states of the USA and are continuously expanding their range. Infection with pseudorabies virus (PRV) occurs in feral swine and the primary route of transmission in free-living conditions seems to be venereal. Between 1995 and 1999, naturally infected feral swine and experimentally infected hybrid progeny of feral and domestic swine, were kept in isolation and evaluated for occurrence of latent PRV indigenous to feral swine in sacral and trigeminal ganglia and tonsil. Sacral ganglia were shown, by polymerase chain reaction (PCR) amplification of the thymidine kinase (TK) gene of PRV, to be the most frequent sites of latency of PRV. Nine (56%) of 16 sacral ganglia, seven (44%) of 16 trigeminal ganglia, and five (39%) of 13 tonsils from naturally infected feral swine were positive for PCR amplification of TK sequences of PRV. These tissues were negative for PRV when viral isolation was attempted in Vero cells. DNA sequencing of cloned TK fragments from the sacral ganglia of two feral swine, showed only one nucleotide difference between the two fragments and extensive sequence homology to fragment sequences from various domestic swine PRV strains from China, Northern Ireland, and the USA. The hybrid feral domestic swine, experimentally inoculated with an indigenous feral swine PRV isolate by either the genital or respiratory route, acquired the infection but showed no clinical signs of pseudorabies. Virus inoculated into either the genital or respiratory tract could, at times, be isolated from both these sites. The most common latency sites were the sacral ganglia, regardless of the route and dose of infection in these experimentally infected hybrids. Nine of 10 sacral ganglia, six of 10 trigeminal ganglia, and three of 10 tonsils were positive for PCR amplification of TK sequences. No virus was isolated from these tissues in Vero cells. The demonstration of the sacral ganglia as the most common sites of latency of pseudorabies viruses indigenous to feral swine, supports the hypothesis that these viruses are primarily transmitted venereally, and not by the respiratory route as is common in domestic swine, in which the trigeminal ganglia are the predominant sites of virus latency.     

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.