[PMC free article] [PubMed] [Google Scholar] 16
[PMC free article] [PubMed] [Google Scholar] 16. by simple virus-cell mixing. Following subsequent incubation at 37C for 5 h to allow membrane fusion and uncoating to occur, the number of reverse transcripts per target cell was similarly enhanced. Indeed, by culturing spinoculated samples for 24 h, 100% of the prospective cells were reproducibly shown to be productively infected, as judged from the manifestation of p24forces employed in this procedure were found to be capable of sedimenting viral particles and because CD4-specific antibodies were effective at obstructing disease binding, Verteporfin we propose that spinoculation works by depositing virions within the surfaces of target cells and that diffusion is the major rate-limiting step for viral adsorption under routine in vitro pulsing conditions. Thus, techniques that accelerate the binding of viruses to target cells not only promise to facilitate the experimental investigation of postentry methods of HIV-1 illness but should also assist to enhance the effectiveness of virus-based genetic therapies. The infectious existence cycle of human being immunodeficiency disease type-1 (HIV-1) is initiated when virions bind to vulnerable target cells via the viral surface glycoprotein gp120/41from the core, are presumably essential for formation of the viral nucleoprotein complex that mediates reverse transcription, transport to the nucleus, and integration of viral cDNA into the sponsor genome to establish the provirus (13). Although these complexes have been described by numerous terms, we will refer to them broadly as postentry nucleoprotein, or reverse transcription (RT), complexes. Once the provirus is made, it serves as the template for subsequent disease gene manifestation, genome synthesis, and progeny virion production. Importantly, the relative efficiencies of these defined methods in HIV-1 replication have not been identified to a high degree of accuracy. As a consequence, the underlying reasons for the low infectivity-to-particle ratios that are usually assigned to HIV-1 (for example, 1 in 3,500 to 60,000 [26, 36]) are not well understood. In an effort to address these points, we have been exploiting kinetic (real-time) PCR and RT-PCR to quantitate viral nucleic acids during the progressive phases of single-cycle HIV-1 illness. Early on in these studies, we found that our illness efficiencies were considerably enhanced by spinoculation, or centrifugal illness, techniques. Although such protocols have been used in medical microbiology since the 1950s (18) to enhance illness Rabbit polyclonal to AKR1A1 by a number of difficult-to-culture pathogens (24), including HIV-1 (20, 37) and additional retroviruses (3, 5), the mechanism whereby centrifugation prospects to an enhanced level of illness remains controversial (24). Specifically, some groups possess proposed that centrifugation enhances cellular susceptibility (22, 23) or viral fusion (43), whereas others have suggested that improved viral deposition is the most important aspect of centrifugal enhancement of illness (21, 34). With these uncertainties in mind, we decided to lengthen our quantitative analyses of disease binding, entry, reverse transcription, and particle production to investigate how spinoculation works. In summary, we demonstrate that centrifugal inoculation raises illness of T cells by HIV-1 mostly by depositing large numbers of virions on target cell surfaces in a CD4-dependent manner. MATERIALS AND METHODS Cell lines and viruses. The CD4-positive T-cell lines CEM-SS, HUT 78, and SupT1 were maintained as explained previously (16, 33, 40, 41). Wild-type HIV-1IIIB disease stocks were initially generated by transfection of 293T cells with the pIIIB infectious molecular clone (15), whereas the G glycoprotein of vesicular stomatitis disease (VSV G)-pseudotyped stocks were produced by cotransfection with pIIIB/(39) and pHIT/G (14). At 24 h, virus-containing supernatants were centrifuged at 500 for 10 min, treated with 30 g of DNase I (Roche Biochemical)/ml for 30 min at space temperature in the presence of 10 mM Verteporfin MgCl2, filtered through 0.45-m-pore-size filters, and stored in aliquots at ?80C. CEM-SS cells were Verteporfin then infected with wild-type disease, and high-titer DNaseI-treated stocks were prepared as explained above at the time of peak disease production. Importantly, we used HIV-1IIIB stocks that had been approved through CEM-SS cells in an effort to minimize the presence of.