Prevalence of human foamy virus-related sequences in the Korean population

The possible association of human foamy virus (HFV) with human thyroid disorders such as Graves' disease (GD) has been a topic of controversy due to the inconsistent results reported by several groups of investigators. Here we report the investigation of the presence of HFV-related sequences in the Korean population. DNA was obtained from peripheral blood lymphocytes from 24 GD patients and 23 healthy blood donors and subjected to PCR amplification using three sets of nested primers derived fromgag, env, and LTR regions of the HFV genome. Contrary to previously reported studies, our analysis identified HFV-related sequences in the genomes of both healthy individuals and the GD patients. However, the nature of the HFV genome present in each group appeared to be different. We detected all 3 regions of HFV-related sequences in 29% of the HFV-positive GD patients, while no samples in the control group amplified all three regions. This suggests that the LTR may be used as a tool for screening for HFV in GD patients. Our data favor the hypothesis of a relationship between GD and the presence of HFV-related sequences, though in a complex way.     

Comparison of enzymatic activities of the HIV-1 and HFV integrases to their U5 LTR substrates.

The human immunodeficiency virus type 1 (HIV-1) and human foamy virus (HFV) integrase proteins were overexpressed in Escherichia coli, and purified to a near homogeneity by one- or two-step purification scheme. The endonucleolytic, integration, and disintegration activities for the HIV-1 and HFV integrases were characterized in vitro. The endonucleolytic activities for the HIV-1 and HFV integrases were found only on their own substrates, respectively, indicating that the cognate U5 LTR sequences in the substrates is critical for specific cleavage. However, the integration and disintegration activities showed less specificity on the substrate usage. Our results suggest that the disintegration activity have more preference for substrates based on Y-shaped structure rather than on viral donor DNA sequence.     

Analysis of the rat JE gene promoter identifies an AP-1 binding site essential for basal expression but not for TPA induction.

We have cloned the immediate-early serum-reponsive JE gene from the rat in order to study the regulation of this gene. We show that sequences of the JE promoter region confer serum-inducibility to a reporter gene. Analysis of the promoter in transient assays reveals that: i) the -141/-88 region is required for the response to the phorbol ester TPA, ii) the -70/-38 region is essential for basal activity. This latter region harbors the sequence TGACTCC, which resembles the consensus site for AP-1 binding, TGACTCA. DNA-protein binding assays indicate that the JE AP-1 site and the consensus AP-1 site have an overlapping but not identical binding spectrum for AP-1 proteins. Our data suggest that the inability of some AP-1 sites to respond to TPA is caused by subtle differences in affinity for AP-1 proteins.     

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.     

Dispensable role of the NF-kappa B sites in the UV-induction of the HIV-1 LTR in transgenic mice.

We have previously reported the epidermis-specific expression of the HIV-1 LTR in transgenic mice and its induction by UV-B rays. To dissect the underlying mechanism of the UV induction of the LTR in mice, we developed two approaches. We first demonstrated by gel mobility shift analysis, using mice epidermal extracts, that the NF-kappa B sites of the HIV-1 LTR were one of the targets of the UV induction. The Sp-1 sites and the potential AP-1 sites of the LTR were not involved in this phenomenon. The transient transfection assays of modified LTR in HeLa cells also demonstrated the involvement of the NF-kappa B sites in the UV induction and were consistent with previously published data. Secondly, to study the regulation acting on an integrated gene, we generated transgenic mice carrying the lacZ gene under the control of the partially deleted LTR. All the transgenic lines and unexpectedly those carrying the LTR deleted for the kappa B sites displayed a UV-inducible epidermal expression. This suggests that, in mice, the UV induction might be mediated through other sites than the kappa B sites and may also depend on changes of the chromatin state.     

ERK MAP kinase links cytokine signals to activation of latent HIV-1 infection by stimulating a cooperative interaction of AP-1 and NF-kappaB.

Human immunodeficiency virus type 1 (HIV-1) can establish latent infection following provirus integration into the host genome. NF-kappaB plays a critical role in activation of HIV-1 gene expression by cytokines and other stimuli, but the signal transduction pathways that regulate the switch from latent to productive infection have not been defined. Here, we show that ERK1/ERK2 mitogen-activated protein kinase (MAPK) plays a central role in linking signals at the cell surface to activation of HIV-1 gene expression in latently infected cells. MAPK was activated by cytokines and phorbol 12-myristate 13-acetate in latently infected U1 cells. The induction of HIV-1 expression by these stimuli was inhibited by PD98059 and U0126, which are specific inhibitors of MAPK activation. Studies using constitutively active MEK or Raf kinase mutants demonstrated that MAPK activates the HIV-1 long terminal repeat (LTR) through the NF-kappaB sites. Most HIV-1 inducers activated NF-kappaB via a MAPK-independent pathway, indicating that activation of NF-kappaB is not sufficient to explain the activation of HIV-1 gene expression by MAPK. In contrast, all of the stimuli activated AP-1 via a MAPK-dependent pathway. NF-kappaB and AP-1 components c-Fos and c-Jun were shown to physically associate by yeast two-hybrid assays and electrophoretic mobility shift assays. Coexpression of NF-kappaB and c-Fos or c-Jun synergistically transactivated the HIV-1 LTR through the NF-kappaB sites. These studies suggest that MAPK acts by stimulating AP-1 and a subsequent physical and functional interaction of AP-1 with NF-kappaB, resulting in a complex that synergistically transactivates the HIV-1 LTR. These results define a mechanism for signal-dependent activation of HIV-1 replication in latently infected cells and suggest potential therapeutic strategies for unmasking latent reservoirs of HIV-1.