C-C Chemokine receptor-like 2 (CCRL2) acts as coreceptor for human immunodeficiency virus-2.

INTRODUCTION: Most of the typical chemokine receptors (CKRs) have been identified as coreceptors for a variety of human and simian immunodeficiency viruses (HIVs and SIVs). This study evaluated CCRL2 to examine if it was an HIV/SIV coreceptor. METHODS: The Human glioma cell line, NP-2, is normally resistant to infection by HIV and SIV. The cell was transduced with amplified cluster of differentiation 4 (CD4) as a receptor and CCR5, CXCR4 and CCRL2 as coreceptor candidates to produce NP-2/CD4/coreceptor cells (). The cells were infected with multiplicity of infection (MOI) 1.0. Infected cells were detected by indirect immunofluorescence assay (IFA). Multinucleated giant cells (MGC) in syncytia were quantified by Giemsa staining. Proviral DNA was detected by polymerase chain reaction (PCR), and reverse transcriptase (RT) activity was measured. RESULTS: IFA detected viral antigens of the primary isolates, HIV-1HAN2 and HIV-2MIR in infected NP-2/CD4/CCRL2 cells, indicated CCRL2 as a functional coreceptor. IFA results were confirmed by the detection of proviral DNA and measurement of RT-activity in the spent cell supernatants. Additionally, MGC was detected in HIV-2MIR-infected NP-2/CD4/CCCRL2 cells. HIV-2MIR were found more potent users of CCRL2 than HIV-1HAN2. Moreover, GWAS studies, gene ontology and cell signaling pathways of the HIV-associated genes show interaction of CCRL2 with HIV/SIV envelope protein. CONCLUSIONS: In vitro experiments showed CCRL2 to function as a newly identified coreceptor for primary HIV-2 isolates conveniently. The findings contribute additional insights into HIV/SIV transmission and pathogenesis. However, its in vivo relevance still needs to be evaluated. Confirming in vivo relevance, ligands of CCRL2 can be investigated as potential targets for HIV entry-inhibitor drugs.

CKR-L3, a deletion version CCR6-isoform shows coreceptor-activity for limited human and simian immunodeficiency viruses.

BACKGROUND: The chemokine receptors (CKRs), mainly CCR5 and CXCR4 function as major coreceptors in infections caused by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Approximately 20 G protein-coupled receptors (GPCRs) have been identified as minor coreceptors, alike CCR6 that we reported recently. Since CKR-L3 is indentified as a natural isoform of CCR6, we attempted in this study to explore the coreceptor function of CKR-L3. METHODS: NP-2 cells transduced with CD4-receptor (NP-2/CD4) normally remain resistant to HIV or SIV infection. However, the introduction of functional coreceptors can make these cells susceptible to these viruses. NP-2/CD4/CKR-L3 cells were produced to examine the coreceptor activity of CKR-L3. Likely, CCR6-isoform and the major coreceptors, CCR5 and CXCR4 were also examined in parallel. Presence of viral antigen in infected NP-2/CD4/coreceptor cells was detected by indirect immunofluorescence assay (IFA). The results were validated by detection of syncytia, proviral DNA and by measuring reverse transcriptase (RT) activities. RESULTS: HIV-2MIR and SIVsmE660 were found to infect NP-2/CD4/CKR-L3 cells, indicative of the coreceptor function of CKR-L3. Viral antigens appeared faster in NP-2/CD4/CKR-L3 cells than in NP-2/CD4/CCR6, indicating that CKR-L3 is a more efficient coreceptor. Moreover, syncytia formation was more rapid and RT release evidenced earlier and at higher levels with CKR-L3 than with CCR6. Sequence analysis in the C2-V3 envelope region of HIV-2MIR replicated through CKR-L3 and CCR6 coreceptor showed two and three amino acid substitutions respectively, in the C2 region compared to the CCR5-variant. The SIVsmE660-CKRL3 variant showed three amino acid substitutions in the V1 region, one change in the V2 and two changes in the C2 region. The SIVsmE660-CCR6 variant produced two changes in the V1 region, and three in the C2 region. CONCLUSIONS: Isoform CKR-L3 exhibited coreceptor activity for limited primary HIV and SIV isolates with better efficiency than the parent CCR6-isoform. Amino acid substitutions in the envelope region of these viruses may confer selective pressure towards CKR-L3-use. CKR-L3 with other minor coreceptors may contribute to HIV and SIV pathogenesis including dissemination, trafficking and latency especially when major coreceptors become compromised. However, further works will be required to determine its clinical significance in HIV and SIV infection.

Entry of human T-cell leukemia virus type 1 is augmented by heparin sulfate proteoglycans bearing short heparin-like structures.

Three molecules have been identified as the main cellular factors required for binding and entry of human T-cell leukemia virus type 1 (HTLV-1): glucose transporter 1 (GLUT1), heparan sulfate (HS), and neuropilin 1 (NRP-1). However, the precise mechanism of HTLV-1 cell tropism has yet to be elucidated. Here, we examined the susceptibilities of various human cell lines to HTLV-1 by using vesicular stomatitis virus pseudotypes bearing HTLV-1 envelope proteins. We found that the cellular susceptibility to HTLV-1 infection did not correlate with the expression of GLUT1, HS, or NRP-1 alone. To investigate whether other cellular factors were responsible for HTLV-1 susceptibility, we conducted expression cloning. We identified two HS proteoglycan core proteins, syndecan 1 and syndecan 2, as molecules responsible for susceptibility to HTLV-1. We found that treatment of syndecan 1-transduced cells (expressing increased HS) with heparinase, a heparin-degradative enzyme, reduced HTLV-1 susceptibility without affecting the expression levels of HS chains. To further elucidate these results, we characterized the expression of HS chains in terms of the mass, number, and length of HS in several syndecan 1-transduced cell clones as well as human cell lines. We found a significant correlation between HTLV-1 susceptibility and the number of HS chains with short chain lengths. Our findings suggest that a combination of the number and the length of HS chains containing heparin-like regions is a critical factor which affects the cell tropism of HTLV-1.

Transient heating of expressed breast milk up to 65°C inactivates HIV-1 in milk: a simple, rapid, and cost-effective method to prevent postnatal transmission.

The postnatal transmission of human immunodeficiency virus (HIV) from mothers to children occurs through breastfeeding. Although heat treatment of expressed breast milk is a promising approach to make breastfeeding safer, it is still not popular, mainly because the recommended procedures are difficult to follow, or time-consuming, or because mothers do not know which temperature is sufficient to inactivate HIV without destroying the nutritional elements of milk. To overcome these drawbacks, a simple and rapid method of heat treatment that a mother could perform with regular household materials applying her day-to-day art of cooking was examined. This structured experiment has demonstrated that both cell-free and cell-associated HIV type 1 (HIV-1) in expressed breast milk could be inactivated once the temperature of milk reached 65°C. Furthermore, a heating method as simple as heating the milk in a pan over a stove to 65°C inhibited HIV-1 transmission retaining milk's nutritional key elements, for example, total protein, IgG, IgA, and vitamin B(12) . This study has highlighted a simple, handy, and cost-effective method of heat treatment of expressed breast milk that mothers infected with HIV could apply easily and with more confidence.

X4-tropic human immunodeficiency virus IIIB utilizes CXCR4 as coreceptor, as distinct from R5X4-tropic viruses.

Human immunodeficiency viruses initiate infections via CCR5 coreceptors and then change their tropism to C-X-C chemokine receptor type 4 (CXCR4), this change being associated with rapid disease progression. HIV-1IIIB, a widely described pure X4-tropic strain, is distinct from R5X4-tropic viruses. In this study, the requirement for amino terminal regions (NTRs) of CXCR4 for entry of HIV-1IIIB virus into host cells was examined and compared to that of R5X4-tropic viruses. CXCR4 and its deletion mutant (CXCR4ΔNTR23; first 23 amino acids removed from NTR) were amplified to examine their coreceptor activities. NP-2/CD4/CXCR4 and NP-2/CD4/CXCR4ΔNTR23 cell lines were prepared accordingly. Indirect immune fluorescence assay (IFA), PCR, and reverse transcriptase (RT) activity were used to compare the process of infection of host cells by HIV-1IIIB virus, one R5-tropic and five other R5X4-tropic viruses. All the R5X4-tropic HIVs were found to utilize both CCR5 and CXCR4 but unable to use CXCR4ΔNTR23 as coreceptors. In contrast, X4-tropic HIV-1IIIB was found to preferentially infect through CXCR4ΔNTR23. Viral antigens in infected NP-2/CD4/CXCR4ΔNTR23 cells were detected by IFA and confirmed by detection of proviral DNA and by performing RT assays on the spent cell-supernatants. In dual tropic viruses, deletion of 23 amino acids from NTR abrogates the coreceptor activity of CXCR4. This observation demonstrates that NTR of CXCR4 have an obligatory coreceptor role for dual tropic viruses. However, HIV-1IIIB may have different requirements for NTR than R5X4 viruses or may infect host cells independent of NTR of CXCR4.

Tax1-expressing feline 8C cells are useful to monitor the life cycle of human T-cell leukemia virus type I.

Extremely low infectivity has hampered direct (cell-free) infection studies of human T-cell leukemia virus type I (HTLV-I). In order to break through this barrier, we examined the susceptibility of many kinds of cells to HTLV-I and found a feline kidney cell line, 8C, that is highly susceptible to HTLV-I and produced remarkable amounts of infectious progeny viruses. Tax1 protein encoded by HTLV-I is known as a transcription activator for viral and cellular genes. We found that the 8C cells expressing the Tax1 protein (8C/TaxWT cells) can produce more progeny viruses than 8C cells when the cells were exposed to cell-free HTLV-I. A large number of syncytia were also induced in these cells. Here, we propose 8C/TaxWT cells as a useful tool to study the cell-free HTLV-I infection.

Human T-cell leukemia viruses are highly unstable over a wide range of temperatures.

The biological properties of human T-cell leukemia virus type I (HTLV-I) and HTLV type II (HTLV-II) are not well elucidated as cell-free viruses. We established new assay systems to detect the infectivity of cell-free HTLVs and examined the stability of cell-free HTLVs at different temperatures. HTLVs lost infectivity more rapidly than did bovine leukemia virus (BLV), which is genetically related to HTLVs. The half-lives of three HTLV-I strains (two cosmopolitan strains and one Melanesian strain) at 37 °C were approximately 0.6 h, whereas the half-life of a BLV strain was 8.5 h. HTLV-I rapidly lost infectivity unexpectedly at 0 and 4 °C. We examined the stability of vesicular stomatitis virus pseudotypes with HTLV-I, HTLV-II or BLV Env proteins, and the Env proteins of HTLVs were found to be more unstable at 4 and 25 °C than the Env proteins of the BLV. Over the course of the viral life cycle, heat treatment inhibited HTLV-I infection at the phase of attachment to the host cells, and inhibition was more marked upon entry into the cells. The HTLV-I Env surface (SU) protein (gp46) was easily released from virions during incubation at 37 °C. However, this release was inhibited by pre-treatment of the virions with N-ethylmaleimide, suggesting that the inter-subunit bond between gp46 SU and gp21 transmembrane (TM) proteins is rearranged by disulfide bond isomerization. HTLVs are highly unstable over a wide range of temperatures because the disulfide bonds between the SU and TM proteins are labile.

Ionising irradiation alters the dynamics of human long interspersed nuclear elements 1 (LINE1) retrotransposon.

It is important to identify the mechanism by which ionising irradiation induces various genomic alterations in the progeny of surviving cells. Ionising irradiation activates mobile elements like retrotransposons, although the mechanism of its phenomena consisting of transcriptions and insertions of the products into new sites of the genome remains unclear. In this study, we analysed the effects of sparsely ionising X-rays and densely ionising carbon-ion beams on the activities of a family of active retrotransposons, long interspersed nuclear elements 1 (L1). We used the L1/reporter knock-in human glioma cell line, NP-2/L1RP-enhanced GFP (EGFP), that harbours full-length L1 tagged with EGFP retrotransposition detection cassette (L1RP-EGFP) in the chromosomal DNA. X-rays and carbon-ion beams similarly increased frequencies the transcription from L1RP-EGFP and its retrotransposition. Short-sized de novo L1RP-EGFP insertions with 5'-truncation were induced by X-rays, while full-length or long-sized insertions (>5 kb, containing ORF1 and ORF2) were found only in cell clones irradiated by the carbon-ion beams. These data suggest that X-rays and carbon-ion beams induce different length of de novo L1 insertions, respectively. Our findings thus highlight the necessity to investigate the mechanisms of mutations caused by transposable elements by ionising irradiation.

CADM1 interacts with Tiam1 and promotes invasive phenotype of human T-cell leukemia virus type I-transformed cells and adult T-cell leukemia cells.

CADM1 encodes a multifunctional immunoglobulin-like cell adhesion molecule whose cytoplasmic domain contains a type II PSD95/Dlg/ZO-1 (PDZ)-binding motif (BM) for associating with other intracellular proteins. Although CADM1 lacks expression in T lymphocytes of healthy individuals, it is overexpressed in adult T-cell leukemia-lymphoma (ATL) cells. It has been suggested that the expression of CADM1 protein promotes infiltration of leukemic cells into various organs and tissues, which is one of the frequent clinical manifestations of ATL. Amino acid sequence alignment revealed that Tiam1 (T-lymphoma invasion and metastasis 1), a Rac-specific guanine nucleotide exchange factor, has a type II PDZ domain similar to those of membrane-associated guanylate kinase homologs (MAGUKs) that are known to bind to the PDZ-BM of CADM1. In this study, we demonstrated that the cytoplasmic domain of CADM1 directly interacted with the PDZ domain of Tiam1 and induced formation of lamellipodia through Rac activation in HTLV-I-transformed cell lines as well as ATL cell lines. Our results indicate that Tiam1 integrates signals from CADM1 to regulate the actin cytoskeleton through Rac activation, which may lead to tissue infiltration of leukemic cells in ATL patients.

c-Maf suppresses human T-cell leukemia virus type 1 Tax by competing for CREB-binding protein.

Latent infection of human T-cell leukemia virus type 1 (HTLV-1) is considered to be preferentially associated with CCR4(+) CD4(+) T cells. Here we report that c-Maf, one of the critical transcription factors for Th2 differentiation, suppresses the transcriptional activity of HTLV-1 Tax by competing for CREB-binding protein. Notably, c-maf expression is selectively induced in a fraction of CCR4(+) CD4(+) T cells upon activation. Furthermore, c-Maf significantly decreases Tax-induced HTLV-1 envelope gp46 gene expression from an infectious HTLV-1 molecular clone and tax expression in a cell-free HTLV-1 infection system. Collectively, c-Maf may play a role in latent infection of HTLV-1 in CCR4(+) CD4(+) T cells by negatively regulating Tax activity.

Effects of respiratory syncytial virus infection and major basic protein derived from eosinophils in pulmonary alveolar epithelial cells (A549).

RSV (respiratory syncytial virus)-induced pneumonia and bronchiolitis may be associated with hyperresponsive conditions, including asthma. Eosinophilic proteins such as MBP (major basic protein) may also be associated with the pathophysiology of asthma. To elucidate the roles of RSV infection and MBP in the pathogenesis of pneumonia with hyperresponsiveness, we investigated the effects of RSV infection and MBP on A549 (alveolar epithelial) cells. CPE (cytopathic effects) in A549 cells were observed by microscopy. Apoptosis and cell death was evaluated by flow cytometric analysis and modified MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. We also measured 15 types of cytokines and chemokines in A549 cell supernatants. Although RSV alone did not affect the CPE of A549, high concentrations of MBP resulted in cell death within 24 h. Combinations of RSV and MBP synergistically induced cell death. In A549 cells infected with RSV alone, the release of GM-CSF (granulocyte-macrophage colony-stimulating factor) was significantly enhanced compared with control cells (no infection). In the cells treated with MBP alone, the production of IL (interleukin)-2, 4, 5, 7, 10, 12, 13, 17, IFN (interferon)-γ, GM-CSF, G-CSF (granulocyte colony-stimulating factor) and MIP (macrophage inflammatory protein)-1ß was significantly increased compared with control cells. Notably, the levels of GM-CSF and IL-17 in RSV/MBP-treated cells were significantly higher than those treated with MBP alone. These results suggest that MBP synergistically enhanced the release of various cytokines/chemokines and the cell death of RSV-infected A549 cells, indicating that MBP may be closely associated with the pathophysiology of allergic reactions in bronchiolitis/pneumonia due to RSV.

A formylpeptide receptor, FPRL1, acts as an efficient coreceptor for primary isolates of human immunodeficiency virus.

BACKGROUND: More than 10 members of seven-transmembrane G protein-coupled receptors (GPCRs) have been shown to work as coreceptors for human immunodeficiency virus type 1 (HIV-1), HIV type 2 (HIV-2), and simian immunodeficiency viruses (SIVs). As a common feature of HIV/SIV coreceptors, tyrosine residues are present with asparagines, aspartic acids or glutamic acids in the amino-terminal extracellular regions (NTRs). We noticed that a receptor for N-formylpeptides, FPRL1, also contains two tyrosine residues accompanied by glutamic acids in its NTR. It was reported that monocytes expressing CCR5 and FPRL1 in addition to CD4 are activated by treatment with ligands or agonists of FPRL1. Activated monocytes down-modulate CCR5 and become resistant to infection by HIV-1 strains. Thus, FPRL1 plays important roles in protection of monocyptes against HIV-1 infection. However, its own coreceptor activity has not been elucidated yet. In this study, we examined coreceptor activities of FPRL1 for HIV/SIV strains including primary HIV-1 isolates. RESULTS: A CD4-transduced human glioma cell line, NP-2/CD4, is strictly resistant to HIV/SIV infection. We have reported that when NP-2/CD4 cells are transduced with a GPCR having coreceptor activity, the cells become susceptible to HIV/SIV strains. When NP-2/CD4 cells were transduced with FPRL1, the resultant NP-2/CD4/FPRL1 cells became markedly susceptible to some laboratory-adapted HIV/SIV strains. We found that FPRL1 is also efficiently used as a coreceptor by primary HIV-1 isolates as well as CCR5 or CXCR4. Amino acid sequences linked to the FPRL1 use could not be detected in the V3 loop of the HIV-1 Env protein. Coreceptor activities of FPRL1 were partially blocked by the forymyl-Met-Leu-Phe (fMLF) peptide. CONCLUSION: We conclude that FPRL1 is a novel and efficient coreceptor for HIV/SIV strains. FPRL1 works as a bifunctional factor in HIV-1 infection. Namely, the role of FPRL1 in HIV-1 infection is protective and/or promotive in different conditions. FPRL1 has been reported to be abundantly expressed in the lung, spleen, testis, and neutrophils. We detected mRNA expression of FPRL1 in 293T (embryonal kidney cell line), C8166 (T cell line), HOS (osteosarcoma cell line), Molt4#8 (T cell line), U251MG (astrocytoma cell line), U87/CD4 (CD4-transduced glioma cell line), and peripheral blood lymphocytes. Roles of FPRL1 in HIV-1 infection in vivo should be further investigated.

CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates.

BACKGROUND: CD4-independence has been taken as a sign of a more open envelope structure that is more accessible to neutralizing antibodies and may confer altered cell tropism. In the present study, we analyzed SIVsm isolates for CD4-independent use of CCR5, mode of CCR5-use and macrophage tropism. The isolates have been collected sequentially from 13 experimentally infected cynomolgus macaques and have previously been shown to use CCR5 together with CD4. Furthermore, viruses obtained early after infection were neutralization sensitive, while neutralization resistance appeared already three months after infection in monkeys with progressive immunodeficiency. RESULTS: Depending whether isolated early or late in infection, two phenotypes of CD4-independent use of CCR5 could be observed. The inoculum virus (SIVsm isolate SMM-3) and reisolates obtained early in infection often showed a pronounced CD4-independence since virus production and/or syncytia induction could be detected directly in NP-2 cells expressing CCR5 but not CD4 (CD4-independent-HIGH). Conversely, late isolates were often more CD4-dependent in that productive infection in NP-2/CCR5 cells was in most cases weak and was revealed only after cocultivation of infected NP-2/CCR5 cells with peripheral blood mononuclear cells (CD4-independent-LOW). Considering neutralization sensitivity of these isolates, newly infected macaques often harbored virus populations with a CD4-independent-HIGH and neutralization sensitive phenotype that changed to a CD4-independent-LOW and neutralization resistant virus population in the course of infection. Phenotype changes occurred faster in progressor than long-term non-progressor macaques. The phenotypes were not reflected by macrophage tropism, since all isolates replicated efficiently in macrophages. Infection of cells expressing CCR5/CXCR4 chimeric receptors revealed that SIVsm used the CCR5 receptor in a different mode than HIV-1. CONCLUSION: Our results show that SIVsm isolates use CCR5 independently of CD4. While the degree of CD4 independence and neutralization sensitivity vary over time, the ability to productively infect monocyte-derived macrophages remains at a steady high level throughout infection. The mode of CCR5 use differs between SIVsm and HIV-1, SIVsm appears to be more flexible than HIV-1 in its receptor requirement. We suggest that the mode of CCR5 coreceptor use and CD4-independence are interrelated properties.

Construction and in vitro characterization of a chimeric simian and human immunodeficiency virus with the RANTES gene.

Chimeric simian-human immunodeficiency virus (SHIV) containing the env gene of HIV-1 infects macaque monkeys and provides basic information that is useful for the development of HIV-1 vaccines. Regulated-on-activation-normal-T-cell-expressed-and-secreted (RANTES), a CC-chemokine, enhances antigen-specific T helper type-1 responses against HIV-1. With the final goal of testing the adjuvant effects of RANTES in SHIV-macaque models, we constructed a SHIV having the RANTES gene (SHIV-RANTES) and characterized its properties in vitro. SHIV-RANTES replicated both in human and monkey T cell lines. Along with SHIV-RANTES replication, RANTES was detected in the supernatant of human and monkey cell cultures, at maximal levels of 98.5 and 4.1 ng/ml, respectively. A flow cytometric analysis showed that the expressed RANTES down-modulated CC-chemokine receptor 5 (CCR5) on PM1 cells, which was restored by adding anti-RANTES antibody. UV-irradiated culture supernatants from the SHIV-RANTES-infected cells suppressed replication of CCR5-tropic HIV-1 BaL in PM-1 cells. Differentiating real-time RT-PCR showed that pre-infection of SHIV-RANTES in C8166 cells expressing CCR5 suppressed the replication of HIV-1 BaL. Biological activity of the expressed RANTES and the inserted RANTES gene in SHIV-RANTES remained stable after 10 passages. These results suggest that SHIV-RANTES is worth testing in macaque models.

Suppression of HIV-1 Infectivity by Human Glioma Cells.

HIV-1 infection to the central nervous system (CNS) is very common in AIDS patients. The predominant cell types infected in the brain are monocytes and macrophages, which are surrounded by several HIV-1-resistant cell types, such as astrocytes, oligodendrocytes, neurons, and microvascular cells. The effect of these HIV-1-resistant cells on HIV-1 infection is largely unknown. In this study, we examined the stability of HIV-1 cultured with several human glioblastoma cell lines, for example, NP-2, U87MG, T98G, and A172, to determine whether these HIV-1-resistant brain cells could enhance or suppress HIV-1 infection and thus modulate HIV-1 infection in the CNS. The HIV-1 titer was determined using the MAGIC-5A indicator cell line as well as naturally occurring CD4(+) T cells. We found that the stability of HIV-1 incubated with NP-2 or U87MG cells at 37°C was significantly shorter (half-life, 2.5-4 h) compared to that of HIV-1 incubated with T98G or A172 cells or in culture medium without cells (half-life, 8-18 h). The spent culture media (SCM) of NP-2 and U87MG cells had the ability to suppress both R5- and X4-HIV-1 infection by inhibiting HIV-1 attachment to target cells. This inhibitory effect was eliminated by the treatment of the SCM with chondroitinase ABC but not heparinase, suggesting that the inhibitory factor(s) secreted by NP-2 and U87MG cells was chiefly mediated by chondroitin sulfate (CS) or CS-like moiety. Thus, this study reveals that some but not all glioma cells secrete inhibitory molecules to HIV-1 infection that may contribute in lowering HIV-1 infection in the CNS in vivo.

Efficient formation of vesicular stomatitis virus pseudotypes bearing the native forms of hepatitis C virus envelope proteins detected after sonication.

Hepatitis C virus (HCV) causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma in addition to acute hepatitis. The HCV genome encodes two envelope glycoproteins, E1 and E2. To investigate the role of E1 and E2 in HCV infection, we used a recombinant vesicular stomatitis virus (VSV), VSVdeltaG*, harboring the green fluorescent protein gene instead of the VSV G envelope protein gene. It was complemented with the native form of E1 and E2, or E1 or E2 alone, to make HCV pseudotypes VSVdeltaG*(HCV), VSVdeltaG*(E1), and VSVdeltaG*(E2). Neither E1 nor E2 expression was detected on the cell surface, as reported. Unlike previous reports, infectious activities of VSVdeltaG*(HCV), VSVdeltaG*(E1) and VSVdeltaG*(E2) pseudotypes were detected under conditions where VSV was completely neutralized by anti-VSV. We could enhance the infectious titers 100-fold by sonication upon virus harvest. Bovine lactoferrin efficiently inhibited infection by VSVdeltaG*(HCV) as well as VSVdeltaG*(E2), as the interaction between E2 and lactoferrin has been thought to contribute to the inhibition of HCV infectivity. VSVdeltaG*(HCV) infected many adherent cell lines, including hepatic cell lines, but not most hematopoietic cell lines. Treatment of cells with trypsin, tunicamycin, or sulfated polysaccharides before infection reduced the infectivity of VSVdeltaG*(HCV) by about 90%, suggesting that a cell surface protein(s) with sugar chains plays an important role in HCV infection. The VSV pseudotypes developed here would be useful for analyzing the early stages of HCV infection.

Identification of HIV type 2 subtype B transmission in East Asia.

We isolated an HIV-2 strain (01JP-IMCJ/KR020) from a Korean patient who has lived in Japan for the past 6 years. He was infected with HIV-2 through heterosexual contacts in either Korea or Japan. The phylogenetic analyses based on the near full-length nucleotide sequence revealed that 01JP-IMCJ/KR020 belongs to HIV-2 subtype B cluster with high bootstrap support. This isolate harbors a 13-nucleotide insertion upstream of the NF-KB site, which is one of the sequence signatures specific to HIV-2 subtype B. This represents the first report of HIV-2 subtype B transmission in East Asia; three cases of HIV-2 subtype A infection have been reported in Korea in 1991-1998. This suggests that at least sporadic transmission of HIV-2, including both subtypes A and B, occurs in East Asia. It is necessary to keep monitoring HIV-2 to see whether there is an increasing trend toward HIV-2 infection in this particular area in Asia.

Isolation and characterization of an infectious HIV type 1 molecular clone from a patient with primary infection.

We isolated a subtype B infectious DNA clone of human immunodeficiency virus type 1 (HIV-1) from a seronegative patient with acute infection and determined the entire nucleotide sequence. All the reading frames encoding the structural proteins (Gag, Pol, and Env) and nonstructural proteins (Tat, Rev, Vpr, Vif, and Nef) were found. Although moat functional domains in these proteins were conserved, we identified a duplication of the T cell factor lei (TCF-1alpha) element in the long terminal repeat and many variations in the N-linked glycosylation sites in the V4-V5 region but not in the V1-V3 loop of Env, compared with prototype subtype B clones. Furthermore, phylogenetic analysis of the entire nucleotide sequence indicated that this HIV-1 was distinct from the prototype subtype B clones, suggesting that transmitted viruses can be variants. This HIV-1 DNA done will be a useful prototype for investigating the mechanism of HIV-1 transmission.

[Coreceptors of HIV infection and the development of HIV entry inhibitors: overview and targets].

In 1996 CXCR4 was identified as a coreceptor for HIV-1. This finding has lead to further identification of more than ten G-protein-coupled receptors (GPCRs) as coreceptors for HIV/SIV. Cell tropisms and coreceptor uses of HIV during the course of HIV infection are summarized. Promiscuous properties of correlations between chemokines and their chemokine receptor uses and also between variable amino acid sequences in the V3 region of HIV gp120 Env and HIV coreceptor uses are discussed. This promiscuous property of HIV-1 is claimed to be a possible cause of a difficulty in developing highly effective entry inhibitors and in addition to allow rapid appearance of immune escape HIV mutants. Representative agents that inhibit HIV entry with a special reference to inhibitors of coreceptor use and gp41 function are summarized. gp41 is discussed as a promising target for the development of effective entry inhibitors.

Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells.

The synthesis and subsequent genomic integration of DNA that is complementary to the genomes of non-retroviral RNA viruses are rarely observed. However, upon infection of various human cell lines and primary fibroblasts with the vesicular stomatitis virus (VSV), we detected DNA complementary to the VSV RNA. The VSV DNA was detected in the cytoplasm as single-stranded DNA fully complementary to the viral mRNA from the poly(A) region to the 7-methyl guanosine cap. The formation of this DNA was cell-dependent. Experimentally, we found that the transduction of cells that do not produce VSV DNA with the long interspersed nuclear element 1 and their infection with VSV could lead to the formation of VSV DNA. Viral DNA complementary to other RNA viruses was also detected in the respective infected human cells. Thus, the genetic information of the non-retroviral RNA virus genome can flow into the DNA of mammalian cells expressing LINE-1-like elements.