Adoptive immunotherapy with functional T cells is potentially an effective therapeutic strategy for combating many types of cancer and viral infection. However, exhaustion of antigen-specific T cells represents a major challenge to this type of approach. In an effort to overcome this problem, we reprogrammed clonally expanded antigen-specific CD8(+) T cells from an HIV-1-infected patient to pluripotency. The T cell-derived induced pluripotent stem cells were then redifferentiated into CD8(+) T cells that had a high proliferative capacity and elongated telomeres. These "rejuvenated" cells possessed antigen-specific killing activity and exhibited T cell receptor gene-rearrangement patterns identical to those of the original T cell clone from the patient. We also found that this method can be effective for generating specific T cells for other pathology-associated antigens. Thus, this type of approach may have broad applications in the field of adoptive immunotherapy.

The recently reported Middle East respiratory syndrome coronavirus (MERS-CoV) is phylogenetically closely related to the bat coronaviruses (BatCoVs) HKU4 and HKU5. However, the evolutionary pathway of MERS-CoV is still unclear. A receptor binding domain (RBD) in the MERS-CoV envelope-embedded spike protein specifically engages human CD26 (hCD26) to initiate viral entry. The high sequence identity in the viral spike protein prompted us to investigate if HKU4 and HKU5 can recognize hCD26 for cell entry. We found that HKU4-RBD, but not HKU5-RBD, binds to hCD26, and pseudotyped viruses embedding HKU4 spike can infect cells via hCD26 recognition. The structure of the HKU4-RBD/hCD26 complex revealed a hCD26-binding mode similar overall to that observed for MERS-RBD. HKU4-RBD, however, is less adapted to hCD26 than MERS-RBD, explaining its lower affinity for receptor binding. Our findings support a bat origin for MERS-CoV and indicate the need for surveillance of HKU4-related viruses in bats.

RANTES (regulated on activation normal T cell expressed and secreted) is one of the natural ligands for the chemokine receptor CCR5 and potently suppresses in vitro replication of the R5 strains of HIV-1, which use CCR5 as a coreceptor. Previous studies showed that peripheral blood mononuclear cells or CD4 ؉ lymphocytes obtained from different individuals had wide variations in their ability to secrete RANTES. These findings prompted us to analyze the upstream noncoding region of the RANTES gene, which contains cis-acting elements involved in RANTES promoter activity, in 272 HIV-1-infected and 193 non-HIV-1-infected individuals in Japan. Our results showed that there were two polymorphic positions, one of which was associated with reduced CD4 ؉ lymphocyte depletion rates during untreated periods in HIV-1-infected individuals. This mutation, RANTES؊28G, occurred at an allele frequency of Ϸ17% in the non-HIV-1-infected Japanese population and exerted no inf luence on the incidence of HIV-1 infection. Functional analyses of RANTES promoter activity indicated that the RANTES؊28G mutation increases transcription of the RAN-TES gene. Taken together, these data suggest that the RANTES؊28G mutation increases RANTES expression in HIV-1-infected individuals and thus delays the progression of the HIV-1 disease.The chemokine receptor CCR5 is an essential coreceptor for the cellular entry of R5 strains (macrophage tropic͞non-syncytium-inducing strains) of HIV-1 (1-6), which predominate in the early stages of infection (7). During the course of infection, variants called X4 strains (T cell-line tropic͞ syncytium-inducing strains) emerge (1, 8-11), which use CXCR4 as a coreceptor (12). In vitro replication of R5 strains can be blocked by the ligands for CCR5, macrophage inflammatory peptide-1␣ and -1␤, and RANTES (regulated on activation normal T cell expressed and secreted; refs. 13 and 14), whereas that of X4 strains can be blocked by the CXCR4 ligands stromal cell derived factor-1␣ and -1␤ (15, 16).Mutations in HIV-1 coreceptors and their natural ligand genes have been shown to modify HIV-1 transmission and disease progression. Individuals homozygous for a 32-nt deletion in the CCR5 coding region were resistant to HIV-1 infection (17, 18), whereas heterozygosity delays disease progression (19,20). A single V-to-I substitution in the first transmembrane segment of CCR2, a minor coreceptor for dual tropic R5X4 strains (3, 5), has a significant impact on disease progression but not on HIV-1 transmission in cohorts of seroconverters (21,22). Finally, homozygosity of a single G-to-A mutation in the 3Ј noncoding region of the stromal cell derived factor-1␤ gene also showed a disease-retarding effect (23), although later studies could not confirm this effect (24,25).Among three natural CCR5 ligands, RANTES showed the highest potency to suppress in vitro replication of R5 strains of HIV-1 (13). Phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) or CD4 ϩ enriched lymphocytes obtained from different...

Many types of human papillomavirus (HPV) are associated with genital lesions. In order to develop simple and sensitive diagnostic procedures for HPV infection, we took advantage of the polymerase chain reaction (PCR). We compared the published nucleotide sequences of the LI region from six genital HPV types and designed a pair of consensus primers for LI region. The PCR with the consensus primers for LI region (Ll‐PCR) could amplify at least nine genital HPV types, 6,11, 16, 18, 31, 33, 42, 52 and 58, and the amplified HPV DNA could be typed by subsequent restriction mapping. Ll‐PCR was compared to Southern blot analysis and also to the consensus primer‐mediated PCR for E6 region (E6‐PCR) described before. Although both our PCR systems are nonradioactive, the sensitivity in detecting HPV DNA was even better than that obtained by using Southern blot analysis. By means of the PCR systems we detected HPV DNA in 100% of cervical condylomas (10/10), 92% of cervical intraepithelial neoplasias (33/36) and 96% of invasive cervical carcinomas (53/55), while we detected HPV DNA in 12% of normal cervices (12/102).

A human T-cell line, MOLT-4, either uninfected or infected with murine retroviruses, was tested for its susceptibility to hepatitis -C virus (HCV) infection. The cell cultures were inoculated with a serum containin HCV and then examined for the presence of viral sequences by cDNA/PCR. In murine retrovirus-infected MOLT-4 (MOLT-4 Ma) cells, intracellular minus-strand viral RNA, a putative replication intermediate, was first detected 3 days after inoculation, and the maximum signal was seen on day 7. When the cells were continuously subcultured in fresh medium, HCV sequences were intermittently detected in cells over a period of 3 weeks. In MOLT-4 cells free of retroviruses, replication of minus-strand HCV RNA appeared less efficient than in MOLT-4 Ma cells. The presence of minus-strand viral RNA in MOLT-4 Ma cells inoculated with HCV was confirmed by in situ hybridization with a strand-specific RNA probe. Immunofluorescence tests with antibodies specific for HCV core and NS4 antigeus showed that MOLT-4 Ma cells were positive for viral antigen 7 days after inoculation. Thus, it appears likely that the HCV genome can replicate in the human T-cell line MOLT-4.

The buoyant density of hepatitis C virus (HCV), with high in vivo infectivity (strain H) or low in vivo infectivity (strain F), was determined by sucrose gradient equilibrium centrifugation. Viral RNA of strain H was detected in fractions with densities of <1.09 g/ml (principally-1.06 g/ml), while that of strain F was found in fractions with densities of-1.06 and-1.17 g/ml. The observed difference was confirmed by differential flotation centrifugation; in NaCl solution with a density of 1.063 g/ml, most of the HCV RNA of strain H was detected in the top fraction, while that of strain F appeared in the bottom. The same relationship between buoyant density and infectivity was observed in flotation centrifugation experiments with other HCV strains. In immunoprecipitation experiments with anti-human immunoglobulin, HCV (as measured by HCV RNA) was precipitated from the samples with low infectivity and high density but not from those with high infectivity and low density. Examination of serial sera from a chimpanzee infected with HCV revealed parallel changes in the buoyant density and immunoprecipitability of HCV-associated RNA during the course of infection. These data suggest that HCV is bound to anti-HCV antibodies as antigen-antibody complexes in chronic hepatitis C.

Split reporter proteins capable of self-association and reactivation have applications in biomedical research, but designing these proteins, especially the selection of appropriate split points, has been somewhat arbitrary. We describe a new methodology to facilitate generating split proteins using split GFP as a self-association module. We first inserted the entire GFP module at one of several candidate split points in the protein of interest, and chose clones that retained the GFP signal and high activity relative to the original protein. Once such chimeric clones were identified, a final pair of split proteins was generated by splitting the GFP-inserted chimera within the GFP domain. Applying this strategy to Renilla reniformis luciferase, we identified a new split point that gave 10 times more activity than the previous split point. The process of membrane fusion was monitored with high sensitivity using a new pair of split reporter proteins. We also successfully identified new split points for HaloTag protein and firefly luciferase, generating pairs of self-associating split proteins that recovered the functions of both GFP and the original protein. This simple method of screening will facilitate the designing of split proteins that are capable of self-association through the split GFP domains.

CD26 is a leukocyte-activation antigen that is expressed on T lymphocytes and macrophages and possesses dipeptidyl peptidase IV (DPPIV) activity, whose natural substrates have not been identified yet. CXC chemokines, stromal cell-derived factor 1␣ (SDF-1␣) and 1␤ (SDF-1␤), sharing the receptor CXCR-4, are highly efficacious chemoattractants for resting lymphocytes and CD34 ؉ progenitor cells, and they efficiently block the CXCR-4-mediated entry into cells of T cell line tropic strains of HIV type 1 (HIV-1). Here we show that both the chemotactic and antiviral activities of these chemokines are abrogated by DPPIV-mediated specific removal of the N-terminal dipeptide, not only when the chemokines are produced in transformed mouse L cell line to express human CD26 but also when they were exposed to a human T cell line (H9) physiologically expressing CD26. Mutagenesis of SDF-1␣ confirmed the critical requirement of the N-terminal dipeptide for its chemotactic and antiviral activities. These data suggest that CD26-mediated cleavage of SDF-1␣ and SDF-1␤ likely occurs in human bodies and promotes HIV-1 replication and disease progression. They may also explain why memory function of CD4 ؉ cells is preferentially lost in HIV-1 infection. Furthermore, CD26 would modulate various other biological processes in which SDF-1␣ and SDF-1␤ are involved.