Superinfection exclusion in BHK-21 cells persistently infected with Junín virus.

We characterized a persistently Junín virus (JUNV)-infected BHK-21 cell line obtained by experimental infection with the XJCl3 strain. This cell line, named K3, produced low levels of virus in supernatants which were not influenced by the presence of defective interfering (DI) particles after the first year of infection. K3 cells were able to exclude superinfection of the homologous JUNV and the antigenically related Tacaribe virus (TCRV), whereas the non-related arenaviruses lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PICV) could replicate normally. Although superinfecting virus binding and internalization to persistently infected cells were slightly reduced, earlier biosynthesis of antigenomic RNA was observed in comparison with BHK-21 cells. Despite the fact that superinfection did not increase the number of cells expressing viral antigens, de novo synthesis of superinfecting virus proteins was detected. The virus produced by JUNV-superinfected K3 cells remained mostly cell-associated in the form of particles tethered to the plasma membrane and aberrant tubular structures. JUNV restriction was correlated with an overexpression of cellular protein TSG101 in K3 cells, which has been pointed out as involved in the budding of several RNA viruses. This correlation was also observed in a cell clone isolated from K3. Reduction of TSG101 expression favoured the release of infectious virus to the supernatant of JUNV-superinfected K3 cells. Our data suggest that overexpression of TSG101 in K3 cells is a novel mechanism that may contribute, along with a diminished synthesis of superinfecting virus proteins, to explain superinfection exclusion in persistently arenavirus-infected cells.

Defective human T-cell lymphotropic virus type I (HTLV-I) provirus in seronegative tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) patients.

Infection with human T-cell lymphotropic virus type I (HTLV-I) have been associated with the development of the tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). We studied the presence of HTLV-I provirus in peripheral blood mononuclear cells (PBMC) from 72 Chilean patients with progressive spastic paraparesis by polymerase chain reaction: 32 seropositive and 40 seronegative cases. We amplified different genomic regions of HTLV-I using primers of 5' ltr, tax, env/tax, pX, pol and env genes. These genes were detected from all seropositive patients. The seronegative patients were negative with 5' ltr, pol, env, and pX primers. However, amplified product of tax and env/tax genes was detected from 16 and four seronegative patients, respectively. Three of them were positive with both genetic regions. The results of this study show that the complete HTLV-I provirus is found in 100% of seropositive cases. In seronegative cases, clinically very similar of seropositive cases, was found only tax gene in 42.5% (17/40) of patients. These results suggest the presence of a defective HTLV-I provirus in some seronegative patients with progressive spastic paraparesis, and suggest a pathogenic role of this truncate provirus for a group of TSP/HAM.

Defective human T-cell lymphotropic virus type I (HTLV-I) provirus in 10 Chilean seronegative patients with tropical spastic paraparesis or HTLV-I-associated myelopathy.

We studied the presence of tax and ltr genes from human T-cell lymphotropic virus type I (HTLV-I) provirus in the peripheral blood mononuclear cells from 15 seronegative patients with tropical spastic paraparesis or HTLV-I-associated myelopathy by PCR. Only a region of the tax gene from 10 patients was amplified. The nucleotide homologies of six Chilean isolates to the ATK-1 clone ranged between 98.7 and 99.4%.

Properties of virus-like particles produced by SIV-chronically infected human cell clones.

SIVsm chronically infected cultures were obtained after infection of CEMX174 cells with either SIVsmH3 or SIVsmE660. These phenotypically CD4 cells, formed syncytia but only when cocultivated with CD4+ cells. Single cell clones were derived from these cultures and examined for the production of virus-specific proteins. The majority of the clones expressed SIV p27 antigen and low levels of virus reverse transcriptase activity. Western blot analysis, performed with either monoclonal or polyclonal sera, showed that a chronically infected clone (B7) produced particles which contained envelope (gp135 and gp43), gag precursors and gag proteins (p27, p16 and p8). However, these particles (SIVsmB7) lacked detectable levels of vpx and of integrase, and contained several fusion proteins which expressed viral protease antigens. This defective virus failed to infect established CD4+ cell lines, as well as primary cultures of macrophages and of peripheral blood lymphocytes, obtained both from humans and from rhesus macaques. Lack of infection correlated with lack of viral DNA detection by PCR amplification of genomic DNA extracted from these cell cultures. In addition, SIVsmB7 virus lacked infectivity in vivo. Rhesus macaques inoculated with high concentrations of SIVsmB7 showed no viremia and their PBMC were PCR negative. Thus, B7 cells produced stable, non-infectious virus mutants, which contained env and gag proteins, but lacked detectable amounts of vpx and of enzymes required for virus replication. Due to the high constitutive expression of this virus-like particle, we are now testing this preparation as a vaccine.