Involvement of a spliced and defective human foamy virus in the establishment of chronic infection.

Human foamy retrovirus (HFV) is found as two proviruses (HFV and delta HFV) which differ by a splice-induced deletion within the bel1 transactivator gene. The defective delta HFV (which lacks a functional Bel1 but harbors an intronless bet gene) is predominantly found in nonlytic infections in vitro as well as in vivo. Here, we show that infection of cell lines stably transduced by delta HFV DNA with the highly lytic HFV leads to chronic infections characterized by an absence of lysis, a balanced ratio of HFV to delta HFV, and a persistent Bet expression accompanied by a shutoff of structural genes. While this system only partially reflects the natural situation, in which target cells are infected by HFV and delta HFV simultaneously, it strongly suggests that delta HFV is a defective interfering retrovirus. Accordingly, previous or concomitant exposure to delta HFV viruses greatly enhances the formation of lysis-resistant clones in culture after HFV infection. The inability of delta HFV proviruses encoding a mutated bet gene to induce chronic infection suggests a role for Bet in this process. Through a specific, splice-induced, genomic deletion, resulting in a switch from Bel1 to Bet expression, the lytic properties of HFV are progressively lost. Such programmed inactivation of a key gene represents a new regulatory mechanism of gene expression in retroviruses.     

Human foamy virus polypeptides: identification of env and bel gene products.

Human foamy virus (HFV) proteins were identified in human cells cultured in vitro by immunoprecipitation and immunoblotting with specific antisera. Among several viral polypeptides, four glycoproteins of approximately 160, 130, 70, and 48 kDa were identified in HFV-infected cells. gp130 was shown to represent the intracellular env precursor, and gp70 and gp48 were shown to represent the external and transmembrane env proteins, respectively. The nature of gp160, which shares sequences with the env, bel1, and bel2 proteins, is not yet resolved. In addition, a p62 identified with bel1- and bel2-specific antisera likely corresponds to the bet gene product.     

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.     

Characterization of the R572T point mutant of a putative cleavage site in human foamy virus Env

A putative cleavage site of the human foamy virus (HFV) envelope glycoprotein (Env) was altered. Transient env expression revealed that the R572T mutant Env was normally expressed and modified by asparagine-linked oligosaccharide chains. However, this single-amino-acid substitution was sufficient to abolish all detectable cleavage of the gp130 precursor polyprotein. Cell surface biotinylation demonstrated that the uncleaved mutant gp130 was transported to the plasma membrane. The uncleaved mutant protein was incapable of syncytium formation. Glycoprotein-driven virion budding, a unique aspect of HFV assembly, occurred despite the absence of Env cleavage. We then substituted the R572T mutant env into a replication-competent HFV molecular clone. Transfection of the mutant viral DNA into BHK-21 cells followed by viral titration with the FAB (foamy virus-activated beta-galactosidase expression) assay revealed that proteolysis of the HFV Env was essential for viral infectivity. Wild-type HFV Env partially complemented the defective virus phenotype. Taken together, these experimental results established the location of the HFV Env proteolytic site; the effects of cleavage on Env transport, processing, and function; and the importance of Env proteolysis for virus maturation and infectivity.     

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.     

In vitro infection of primary and retrovirus-infected human leukocytes by human foamy virus.

The infectivity of human foamy virus (HFV) was examined in primary and cultured human leukocytes. Cell-free infectious viral stocks of HFV were prepared from the human kidney cell line 293 transfected with an infectious molecular clone of HFV. HFV productively infects a variety of human myeloid and lymphoid cell lines. In addition, primary cell cultures enriched for human CD4+, monocytes and brain-derived microglial cells, were readily infected by HFV. Interestingly, while infected primary CD4+ lymphocytes and microglial cells showed marked cytopathology characteristic of foamy virus, HFV-infected monocyte-derived macrophages failed to show any cytopathology. In addition, marked cytotoxicity due to HFV infection was seen in both human T-cell leukemia virus type 1- and human immunodeficiency virus type 1-infected T-cell lines and in human immunodeficiency virus type 1-infected monocytoid cell lines. Thus, HFV infection produces differential cytopathology in a wide host range of primary human leukocytes and hematopoietic cell lines.     

Multiple integrations of human foamy virus in persistently infected human erythroleukemia cells

Foamy viruses are complex retroviruses whose replication strategy resembles that of conventional retroviruses. However, foamy virus replication also resembles that of hepadnaviruses in many respects. Because hepadnaviruses replicate in an integrase-independent manner, we were interested in investigating the characteristics of human foamy virus (HFV) integration. We have shown that HFV requires a functional integrase protein for infectivity. Our analyses have revealed that in single-cell clones derived from HFV-infected erythroleukemia-derived cells (H92), there were up to 20 proviral copies per host cell genome as determined by Southern blot and fluorescent in situ hybridization analysis. Use of specific probes has also shown that a majority of the proviruses contain the complete tas gene, which encodes the viral transactivator, and are not derived from Deltatas cDNAs, which have been shown to arise rapidly in infected cells. To demonstrate that the multiple proviral sequences are due to integration instead of recombination, we have sequenced the junctions between the proviral sequences and the host genome and found that the proviruses have authentic long terminal repeat ends and that each integration is at a different chromosomal site. A virus lacking the Gag nuclear localization signal accumulates fewer proviruses, suggesting that nuclear translocation is important for high proviral load. Since persistently infected H92 clones are not resistant to superinfection, the relative importance of an intracellular versus extracellular mechanism in proviral acquisition has yet to be determined.     

Relationship of the env genes and the endonuclease domain of the pol genes of simian foamy virus type 1 and human foamy virus.

We have molecularly cloned and sequenced a portion of the simian foamy virus type 1 (SFV-1); open reading frames representing the endonuclease domain of the polymerase (pol) and the envelope (env) genes were identified by comparison with the human foamy virus (HFV). Unlike the HFV genomic organization, the SFV-1 pol gene overlaps the env gene; thus, the open reading frames reported for HFV between pol and env is not present in SFV-1. Comparisons of predicted amino acid sequences of HFV and SFV-1 reveal that the endonuclease domains of the pol genes are about 84% related. The region predicted to encode the SFV-1 extracellular env domain is 569 codons; SFV-1 and HFV have 64% amino acid similarity in this env domain. The predicted hydrophobic transmembrane env proteins of both HFV and SFV-1 show about 73% similarity. A total of 16 potential glycosylation sites are found in SFV-1 env, and 15 are found in HFV; 11 are shared. SFV-1 has 25 cysteine residues, and HFV has 23 residues; all 23 cysteine residues of HFV are conserved in SFV-1. This sequence analysis reveals that the human and simian foamy viruses are highly related.     

The carboxyl terminus of the human foamy virus Gag protein contains separable nucleic acid binding and nuclear transport domains.

The Gag protein of human foamy virus (HFV) lacks Cys-His boxes present in the nucleocapsid (NC) domains of other retroviruses; instead it contains three glycine-arginine-rich motifs (GR boxes). We have expressed the carboxyl end of HFV Gag containing the GR boxes (the NC domain equivalent) and analyzed its nucleic acid binding properties. Our results show that the NC domain of HFV Gag binds with high affinity to both RNA and DNA, in a sequence-independent manner, as determined by filter binding assays. Analysis of a mutant containing a heterologous sequence in place of GR box I indicates that this motif is required for nucleic acid binding and for viral replication. A mutant in GR box II still binds to RNA and DNA in vitro, but virus containing this mutation does not replicate and no nuclear staining of the Gag protein is found in transfected cells. Surprisingly, a revertant from this mutant that completely lacks GR box II and exhibits very little nuclear transport of Gag can readily replicate in tissue culture. This finding thus provides a direct evidence that although the sequences in GR box II can serve as a nuclear transport signal, they are not required for HFV replication and it is unlikely that nuclear localization of Gag protein plays any critical role during viral infection. Taken together, our results suggest that the Gag protein of HFV may be more analogous to the core protein of the hepatitis B virus family than to conventional retroviral Gag protein.     

Complementation analysis of the flavivirus Kunjin NS3 and NS5 proteins defines the minimal regions essential for formation of a replication complex and shows a requirement of NS3 in cis for virus assembly

We have previously reported successful trans-complementation of defective Kunjin virus genomic RNAs with a range of large lethal deletions in the nonstructural genes NS1, NS3, and NS5 (A. A. Khromykh et al., J. Virol. 74:3253-3263, 2000). In this study we have mapped further the minimal region in the NS5 gene essential for efficient trans-complementation of genome-length RNAs in repBHK cells to the first 316 of the 905 codons. To allow amplification and easy detection of complemented defective RNAs with deletions apparently affecting virus assembly, we have developed a dual replicon complementation system. In this system defective replicon RNAs with a deletion(s) in the nonstructural genes also encoded the puromycin resistance gene (PAC gene) and the reporter gene for beta-galactosidase (beta-Gal). Complementation of these defective replicon RNAs in repBHK cells resulted in expression of PAC and beta-Gal which allowed establishment of cell lines stably producing replicating defective RNAs by selection with puromycin and comparison of replication efficiencies of complemented defective RNAs by beta-Gal assay. Using this system we demonstrated that deletions in the C-terminal 434 codons of NS3 (codons 178 to 611) were complemented for RNA replication, while any deletions in the first 178 codons were not. None of the genome-length RNAs containing deletions in NS3 shown to be complementable for RNA replication produced secreted defective viruses during complementation in repBHK cells. In contrast, structural proteins produced from these complemented defective RNAs were able to package helper replicon RNA. The results define minimal regions in the NS3 and NS5 genes essential for the formation of complementable replication complex and show a requirement of NS3 in cis for virus assembly.     

Evaluation of consistency in quantification of gene copy number by real‐time reverse transcription quantitative polymerase chain reaction and virus titer by plaque‐forming assay for human respiratory syncytial virus

The plaque‐forming assay is the standard technique for determining viral titer, and a critical measurement for investigating viral replication. However, this assay is highly dependent on experimental technique and conditions. In the case of human respiratory syncytial virus (RSV) in particular, it can be difficult to objectively confirm the accuracy of plaque‐forming assay because the plaques made by RSV are often small and unclear. In recent studies, RT‐qPCR methods have emerged as a supportive procedure for assessment of viral titer, yielding highly sensitive and reproducible results. In this report, we compare the viral replication, as determined by plaque‐forming assay, and the copy numbers of RSV genes NS1, NS2, N, and F, as determined by RT‐qPCR. Two real‐time PCR systems, SYBR Green and TaqMan probe, gave highly similar results for measurement of copy numbers of RSV N genes of virus subgroups A. We determined the RSV gene copy numbers in the culture cell supernatant and cell lysate measured at various multiplicities of infection. We found that copy number of the RSV N gene in the culture supernatant and cell lysate was highly correlated with plaque‐forming units. In conclusion, RT‐qPCR measurement of RSV gene copy number was highly dependent on viral titer, and the detailed comparison between each gene copy number and virus titer should be useful and supportive in confirming RSV plaque‐forming assay and virus dynamics. The technique may also be used to estimate the amount of RSV present in clinical specimens.

     

Human foamy virus capsid formation requires an interaction domain in the N terminus of Gag

Retroviral Gag expression is sufficient for capsid assembly, which occurs through interaction between distinct Gag domains. Human foamy virus (HFV) capsids assemble within the cytoplasm, although their budding, which mainly occurs in the endoplasmic reticulum, requires the presence of homologous Env. Yet little is known about the molecular basis of HFV Gag precursor assembly. Using fusions between HFV Gag and a nuclear reporter protein, we have identified a strong interaction domain in the N terminus of HFV Gag which is predicted to contain a conserved coiled-coil motif. Deletion within this region in an HFV provirus abolishes viral production through inhibition of capsid assembly.