Gene expression during osteoclast-like cell formation induced by antifusion regulatory protein-1/CD98/4F2 monoclonal antibodies (MAbs): c-src is selectively induced by anti-FRP-1 MAb.

Human blood monocytes can differentiate into osteoclast-like cells when they are cultured in the presence of anti-FRP-1. Messenger (mRNA) expression of markers related to osteoclasts was analyzed during differentiation of osteoclasts from monocytes. As markers related to osteoclasts, we selected cathepsin-K, carbonic anhydrase (CA) II, vacuolar H(+)-ATPase (v-ATPase), vitronectin receptor (VNR), tartrate-resistant acid phosphatase (TRAP), osteopontin (OPN), galectin-3, c-src, c-fos, and c-fms. The mRNAs other than c-src mRNA were expressed in freshly isolated monocytes or monocytes incubated with control antibody or anti-FRP-1 monoclonal antibody (MAb) for 14 days. Of these mRNAs, cathepsin-K, CA II, v-ATPase, VNR, TRAP, OPN, and c-fms mRNAs were expressed at higher levels in the osteoclast-like cells than those in monocytes cultured with control antibody. On the other hand, galectin-3 mRNA was expressed at lower levels in the osteoclast-like cells, and there was no significant difference in c-fos mRNA expression between the monocytes cultured with control antibody and anti-FRP-1 MAb. c-src mRNA could not be detected in monocytes freshly isolated or incubated with control antibody. Surprisingly, expression of c-src mRNA was induced in monocytes by anti-FRP-1 MAb and was detectable as early as 3 h after anti-FRP-1 MAb treatment, indicating that c-src is selectively induced by anti-FRP-1 MAb treatment. Furthermore, the osteoclast-like cells expressed calcitonin receptor. Receptor activator of NF-kappaB (RANK) mRNA was detectable in freshly isolated monocytes or monocytes cultured with control antibody or anti-FRP-1 MAbs. Maximal expression of RANK was observed in osteoclast-like cells. On the other hand, no receptor activator of NF-KB ligand (RANKL) mRNA was detectable in any of the samples, suggesting that anti-FRP-1 mAb can induce osteoclast-like cells from blood monocytes without RANKL.

Isolation and characterization of a full-length molecular DNA clone of Ghanaian HIV type 1 intersubtype A/G recombinant CRF02_AG, which is replication competent in a restricted host range.

We have isolated a replication-competent, full-length molecular clone of HIV-1 CRF02_AG, designated p97GH-AG1, by reconstituting two separately amplified genomic regions of an HIV-1 provirus of a 1997 Ghanaian isolate. The phylogenetic and recombination breakpoint analyses revealed that 97GH-AG1 had an A/G recombinant structure similar to that of prototype Nigerian isolate IbNG. The 17-nucleotide insertion downstream of the primer-binding site appeared to be a common sequence signature specific to most CRF02_AG strains, including 97GH-AG1. 97GH-AG1 showed an R5 phenotype and exerted productive infection in both HOS and NP2 cell infectivity assays, whereas it failed to show a detectable level of progeny production in peripheral blood mononuclear cells (PBMCs). The data may suggest the presence of unknown determinant(s) that dictate efficient replication in PBMCs, but that are not required for replication in immortalized cell lines.

Identification and molecular characterization of human T lymphotropic virus type II infections in intravenous drug abusers in the former South Vietnam.

Previous serological studies have demonstrated that some 60% of intravenous drug abusers (IVDAs) in urban areas of the former South Vietnam are infected with HTLV-II. In the present report we have attempted to characterize the viruses using restriction fragment length polymorphism (RFLP) and nucleotide sequence analysis of the provirus long terminal repeat (LTR) region. RFLP analysis of nine samples demonstrated that all were infected with the HTLV-IIb subtype. The HTLV-IIa subtype was not detected. Phylogenetic analysis of the nucleotide sequences demonstrated that the viruses clustered closely with HTLV-IIb isolates present in IVDAs from the New York City area. The present molecular analysis together with the previously reported absence of HTLV-II infection in North Vietnam supports the view that HTLV-II may have been introduced from the United States to this part of Asia by military personnel during the Vietnam conflict.

Genetic and serologic characterization of HIV type 1 prevailing in Myanmar (Burma).

To study the molecular epidemiology of HIV-1 spread in Myanmar and the interplay with the epidemic in surrounding Southeast Asian countries, we determined the HIV-1 subtypes prevailing in Myanmar. Thirty HIV-positive blood specimens were sampled in the capital city, Yangon, and an additional 459 sera were collected nationwide in 1995. Genetic subtyping based on the env C2/V3 sequence and serologic data, using a V3 peptide enzyme immunoassay (PEIA), revealed three patterns of HIV spread in different geographic regions in Myanmar: (1) in the capital city, Yangon, HIV-1 subtype B' ("Thai-B" cluster within subtype B) predominated both in IDUs and heterosexuals; (2) in the cities near the border with Thailand, including Tachelaik and Kawthaung, where heterosexual transmission is a major pathway of HIV-1 spread, HIV-1 subtype E was predominantly distributed among the commercial sex workers and heterosexuals; (3) in central and northeast Myanmar, both HIV-1 subtypes B' and E occurred in a mixed distribution, without showing any significant segregation by risk group. In addition, the PEIA data implied the occurrence of other subtype(s) in these areas. The interperson nucleotide sequence variations in env C2/V3 regions of B' and E, prevailing in Yangon, were 6.7 +/- 2.1 and 7.1 +/- 0.7%, respectively. They were similar to those levels observed in Thailand. These findings are consistent with the view that HIV spread in Myanmar might have taken place at about the same time as that in Thailand, and that multiple entries and exchanges of HIV-1 with neighboring countries are important factors contributing to the current distribution of subtypes in Myanmar.

Emergence of new forms of human immunodeficiency virus type 1 intersubtype recombinants in central Myanmar.

We have previously shown that HIV-1 env subtypes B' (a Thai-B cluster within subtype B) and E (CRF01_AE) are distributed in Yangon, the capital city of Myanmar. However, HIV strains from the rest of country have not yet been genetically characterized. In the present study, we determined env (C2/V3) and gag (p17) subtypes of 25 specimens from central Myanmar (Mandalay). Phylogenetic analyses identified 5 subtype C (20%), in addition to 10 CRF01_AE (40%) and 4 subtype B' (16%). Interestingly, the remaining six specimens (24%) showed discordance between gag and env subtypes; three gag subtype B'/env subtype C, one gag subtype B'/env subtype E, one gag subtype C/env subtype B', and one gag subtype C/env subtype E. These discordant specimens were found frequently among injecting drug users (4 of 12, 33%) and female commercial sex workers (2 of 8, 25%) engaging in high-risk behaviors. The recombinant nature of these HIV-1 strains was verified in three specimens, indicating the presence of new forms of HIV-1 intersubtype C/B' and C/B'/E recombinants with different recombination breakpoints. The data suggest that multiple subtypes of B', C, and CRF01_AE are cocirculating in central Myanmar, leading to the evolution of new forms of intersubtype recombinants among the risk populations exhibiting one of the highest HIV infection rates in the region.

Closely related HIV-1 CRF01_AE variant among injecting drug users in northern Vietnam: evidence of HIV spread across the Vietnam-China border.

To investigate the nature of recent HIV outbreaks among injecting drug users (IDUs) near the Vietnam-China border, we genetically analyzed 24 HIV-positive blood specimens from 2 northern provinces of Vietnam (Lang Son and quang Ninh) adjacent to the China border, where HIV outbreaks among IDUs were first detected in late 1996. Genetic subtyping based on gag (p17) and env (C2/V3) sequences revealed that CRF01_AE is a principal strain circulating throughout Vietnam, including the provinces near the China border. The majority of CRF01_AE sequences among IDUs in Quang Ninh and Lang Son showed significant clustering with those found in nearby Pingxiang City of China's Guangxi Province, sharing a unique valine substitution 12 amino acids downstream of the V3 loop. This particular subtype E variant, uniquely found among IDUs in northern Vietnam and southeastern China, is designated E(v). The genetic diversity of CRF01_AE distributed in Quang Ninh (1.5 +/- 0.6%) and Pingxiang City (1.9 +/- 1.2%) was remarkably low, indicating the emerging nature of HIV spread in these areas. It is also noted that the genetic diversity of CRF01_AE among IDUs was consistently lower than that in persons infected sexually, suggesting that fewer closely related CRF01_AE variants were introduced into IDUs and, conversely, that multiple strains of CRF01_AE had been introduced via the sexual route. The data in the present study provide additional evidence that HIV outbreaks among IDUs in northern Vietnam were caused by the recent introduction of a highly homogeneous CRF01_AE variant (E(v)) closely related to that prevailing in nearby southern China.

Genetic similarity of HIV type 1 subtype E in a recent outbreak among injecting drug users in northern Vietnam to strains in Guangxi Province of southern China.

To investigate the molecular epidemiology of a recent HIV-1 outbreak in northern Vietnam and its relation to the epidemic in surrounding areas, we analyzed 17 HIV-positive blood specimens from 3 heterosexuals, 2 sexually transmitted disease patients, and 12 injecting drug users (IDUs), collected in 4 provinces near Hanoi in 1998. These were compared with the specimens from Ho Chi Minh City (n = 10) and An Giang Province (n = 10) in southern Vietnam and with published sequences from neighboring countries. Genetic subtyping based on the env C2/V3 sequences revealed that HIV-1 subtype E predominated throughout Vietnam in all risk populations; the exception was one typical United States-European-type HIV-1 subtype B detected in a patient in Ho Chi Minh City, the first case of HIV infection identified in Vietnam in 1990. The HIV-1 subtype E sequences identified in 9 of the 12 IDUs from northern provinces were closely related phylogenetically to those in IDUs in nearby Guangxi Province of China, and also shared a common amino acid signature downstream of the env V3 loop region. The low interperson nucleotide diversity among IDUs in northern Vietnam supports the view that HIV-1 subtype E was introduced recently among IDUs in northern Vietnam. These data indicate a linkage between HIV-1 circulating among IDUs in northern Vietnam and southern China, and suggest recent transborder introductions as the likely source of HIV-1 subtype E in northern Vietnam.

Sendai virus-based production of HIV type 1 subtype B and subtype E envelope glycoprotein 120 antigens and their use for highly sensitive detection of subtype-specific serum antibodies.

We previously described a Sendai virus (SeV)-based expression system for the recombinant gp120 of HIV-1 subtype B (rgp120-B), which has permitted the production of antigenetically and functionally authentic gp120 at a concentration as high as 6 microg/ml of culture supernatant (Yu D et al.: Genes Cells 1997;2:457-466). Here the same procedure was successfully applied to the production of HIV-1 subtype E gp120 (rgp120-E). The remarkable production of the proteins by the SeV expression system enabled us to use crude culture supernatants for serological and functional studies of gp120s. The immunological authenticity of rgp120-E was verified by patient sera and anti-V3 loop monoclonal antibodies specific for HIV-1 subtypes B and E. CD4-binding properties were corroborated by FACS analyses. The rgp120s were then used in an enzyme immunoassay (rgp120-EIA) to detect antibodies in the sera of HIV-1-infected individuals, and the performance was assessed in comparison with a conventional V3 loop peptide EIA (V3-EIA). The initial evaluation of a serum panel (n = 164) consisting of 76 subtype E and 88 subtype B sera revealed that the rgp120-EIA was nearly 1000-fold more sensitive than the V3-EIA and was able to detect subtype-specific antibody with 100% sensitivity and with a complete correlation with the genotypes, whereas the V3-EIA failed to detect 9 and 24% of the same subtype E and B sera, respectively. Furthermore, a study employing a panel of 28 international sera with known genotypes (HIV-1 subtypes A through F) confirmed the remarkable specificity of this method. An EIA reactivity higher than 1.0 was an unambiguous predictor of HIV-1 subtype E and B infections. The data imply the presence of strong subtype-specific epitopes for antibody bindings to these rgp120s.

Characterization of Bangor virus proteins by using monoclonal antibodies.

A new virus was isolated from a finch in quarantine in Northern Ireland in 1973. The virus had the morphological characteristics of a paramyxovirus, and was named Bangor virus (BaV). In order to identify the structural proteins of BaV and to investigate the biological characterization of the virus, 28 monoclonal antibodies (mAbs) directed against BaV were prepared. Eight of these mAbs reacted with the nucleocapsid protein (NP), 10 with hemagglutinin-neuraminidase (HN) protein, and 10 with fusion (F) protein. With the aid of these mAbs, the structural proteins of BaV were determined, namely, p52, gp74, gp63, and gp51 were identified as the NP, HN, F0, and F1 proteins, respectively. The biological activities of the mAbs directed against the envelope glycoproteins of BaV were examined. Intriguingly, it was found in the neutralization assay that four mAbs directed against the HN protein of BaV can enhance the fusion of HeLa cells infected with BaV, showing the presence of a potential third function of the HN protein that affects the fusion activity of the F protein. Furthermore, all of the anti-F protein mAbs showed neutralizing activity.

Isolation of monoclonal antibodies directed against the V protein of human parainfluenza virus type 2 and localization of the V protein in virus-infected cells.

Two monoclonal antibodies (mAbs) specific for the human parainfluenza virus type 2 (hPIV-2) V protein were obtained by immunizing mice with the V protein recombinantly expressed in Escherichia coli. Both mAbs were found to react with the V protein in ELISA and in Western blot analysis. Using these mAbs and previously obtained mAbs specific for hPIV-2 nucleoprotein (NP) or hPIV-2 phospho-(P) protein, we examined the intracellular distributions of the V, P and NP proteins in hPIV-2-infected cells by indirect immunofluorescence analyses. The P and NP proteins were organized in numerous granules in the cytoplasm of hPIV-2 infected cells. In contrast, the V protein showed diffuse nuclear and cytoplasm distributions.

Role of a single amino acid at the amino terminus of the simian virus 5 F2 subunit in syncytium formation.

The fusion (F) protein of simian virus 5 (strain W3A) induced extensive cell fusion in BHK cells when expressed alone, while that of strain WR did not. Mutational analysis demonstrated that the fusing activity can be transferred to the WR F protein by a proline residue at position 22 of subunit W3A F2.

DNA vaccine-encapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration.

Delivery of foreign genes to the digestive tract mucosa by oral administration of nonreplicating gene transfer vectors would be a very useful method for vaccination and gene therapy. However, there have been few reports on suitable vectors. In the present study, we found that plasmid DNA can be packaged in vitro into a virus-like particle (VLP) composed of open reading frame 2 of hepatitis E virus, which is an orally transmissible virus, and that these VLPs can deliver this foreign DNA to the intestinal mucosa in vivo. The delivery of plasmid DNA to the mucosa of the small intestine was confirmed by the results of immunohistochemical analyses using an expression plasmid encoding human immunodeficiency virus env (HIV env) gp120. After oral administration of VLPs loaded with HIV env cDNA, significant levels of specific IgG and IgA to HIV env in fecal extracts and sera were found. Moreover, mice used in this study exhibited cytotoxic T-lymphocyte responses specific to HIV env in the spleen, Payer's patches and mesenteric lymph nodes. These findings suggest that VLPs derived from orally transmissible viruses can be used as vectors for delivery of genes to mucosal tissue by oral administration for the purpose of DNA vaccination and gene therapy.

Cutting edge: primary structure of the light chain of fusion regulatory protein-1/CD98/4F2 predicts a protein with multiple transmembrane domains that is almost identical to the amino acid transporter E16.

The CD98 light chain (CD98LC) was copurified from HeLa S3 cells by an affinity chromatography using a mAb specific for the fusion regulatory protein-1 (FRP-1) which is identical to the CD98 heavy chain. On the basis of the N-terminal sequence (63 amino acids) of purified CD98LC polypeptide, we have cloned a PCR fragment (155 bp) from a HeLa S3 cDNA library and finally obtained a full cDNA clone encoding the CD98LC. Fluorescence in situ hybridization analysis using the cDNA assigned the CD98LC gene to the long arm of human chromosome 16 (16q24). The predicted amino acid sequence suggested that CD98LC is a protein with multiple transmembrane domains and is almost identical to the amino acid transporter E16. Resting monocytes and lymphocytes expressed CD98LC as analyzed by a newly isolated anti-CD98LC mAb, which showed cross-reactivity with insect Sf9 cells as well as with various mammalian cell lines.

Sequence determination of the P gene of simian virus 41: presence of irregular deletions near the RNA-editing sites of paramyxoviruses.

The complete nucleotide sequence of the P gene of simian virus 41 (SV41) was determined. The gene was found to be 1406 nucleotides long and to contain a relatively small open reading frame encoding a cysteine-rich V protein with a calculated M(r) of 24076. We have demonstrated that RNA-editing events occur in SV41 P gene transcripts and that the ratio of edited mRNAs to faithfully copied mRNA (P-mRNA:V-mRNA) is about 1:5 at either 24 or 40 h post-infection. The mRNA with two G insertions was capable of encoding a P protein of 395 amino acids with a predicted M(r) of 41,992. A kinetic study of P and V proteins by Western blot analysis showed that in virus-infected cells the amounts of both proteins were almost equal although the V-mRNA was considerably more abundant than the P-mRNA. Alignment of the SV41 P and V proteins with those of nine other paramyxoviruses demonstrated that irregular gaps were present around the RNA-editing sites.

A cell fusion-inhibiting monoclonal antibody binds to the presumed stalk domain of the human parainfluenza type 2 virus hemagglutinin-neuraminidase protein.

Previously, we obtained a neutralizing monoclonal antibody directed against the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 2 virus (PIV2), which was able to prevent cell fusion without affecting the hemagglutinating and neuraminidase activities. In this study, four escape mutants of PIV2 have been obtained under pressure of the monoclonal antibody. Intriguingly, the HN protein of each mutant proved to have two amino acid substitutions, one of which is at 83Asn or 91Lys, and another one is at 150Leu, 160Ala, or 186Met. One mutant designated F13, which has substitutions at 83Asn and 186Met in the HN protein, could not cause cell fusion in HeLa cells despite its multiple replication, while the other mutants formed typical syncytial cells. The deduced amino acid sequence of F13 fusion (F) protein proved to be identical to that of wild-type F protein, and furthermore, protein expression analyses have revealed that the low-fusion phenotype of F13 was due to its mutated HN protein, whose antigenicity to the monoclonal antibody was abolished by the single mutation at 83Asn. These observations have suggested that the principal epitope for the monoclonal antibody resides in the presumed stalk domain of the HN protein, which may play an important role in promoting cell fusion.

Identification of the sequences responsible for nuclear targeting of the V protein of human parainfluenza virus type 2.

In human parainfluenza virus type 2 (hPIV-2)-infected cells, anti-phosphoprotein (P)-specific monoclonal antibody (MAb) densely stained the perinuclear regions of infected cells throughout infection, indicating that the P protein was localized exclusively in the cell cytoplasm. By contrast, antigens recognized by MAbs directed against the P-V-common domain of hPIV-2 were located predominantly in the cytoplasm, but in some hPIV-2-infected cells they were also found in the nuclei, suggesting that a fraction of hPIV-2 V protein is localized there. hPIV-2 V protein expressed from a cDNA clone was localized in the nuclei of transfected cells. By using indirect immunofluorescence analyses, we examined the intracellular localization of various sequentially deleted V proteins, to determine the nuclear localization signals (NLS) of the V protein. Two noncontiguous regions in the V protein were required for nuclear localization and retention, since deletion of these regions [region I (aa 1-46) and region II (aa 175-196)] resulted in cytoplasmic localization. Both regions resulted in nuclear localization independently. A nucleoplasmin-like NLS was identified in region II but no consensus targeting sequence could be found in region I. When NP protein was co-expressed with V protein or the N-terminal fragment (aa 1-46) of V protein, a fraction of the NP protein was translocated into cell nuclei.

Nucleotide sequence analysis of the simian virus 41 gene encoding the large (L) protein and construction of a phylogenetic tree for the L proteins of paramyxoviruses.

The complete nucleotide sequence of the simian virus 41 (SV41) large (L) protein gene was determined. The L gene spanned 6883 nucleotides including a putative trailer RNA, and the L mRNA contained a single large open reading frame encoding a polypeptide of 2269 amino acids. Dot-matrix comparisons under stringent conditions identified domains highly conserved among paramyxoviruses. Domain 3 is the most highly conserved, and has been hypothesized to be the RNA polymerase active site. A phylogenetic tree was constructed from the sequences of the L proteins of seven paramyxoviruses. SV41 was most closely related to human parainfluenza virus type 2 (HPIV-2), and SV41, HPIV-2 and SV5 form a subgroup. The intergenic sequences at the nucleocapsid protein-phosphoprotein and haemagglutinin-neuraminidase-L protein gene junctions, and the 5' trailer sequence of SV41 were also determined, and it was shown that the first 13 nucleotides of the 5' trailer sequence are complementary to those of the 3' leader sequence. The intergenic, and gene-start and -end sequences of SV41, HPIV-2 and SV5 are shown.

Sequence analysis of the large (L) protein of simian virus 5.

The complete nucleotide sequence of the large (L) protein gene of simian virus 5 (SV5) was determined from cDNA of the genomic RNA and mRNA, and found to be 6804 bases in length, exclusive of a poly(A) tract. The sequence contained an open reading frame of 6765 nucleotides encoding 2255 amino acids. Results of dot matrix comparisons of the L protein of SV5 with those of human parainfluenza type 3 virus and Sendai virus indicated that there are five conserved domains, and that each domain contains characteristic sequence(s). The L protein of SV5 was detected in purified virions using antiserum directed against an oligopeptide corresponding to the N-terminal region.