Characterization of a human immunodeficiency virus type 1 pre-integration complex in which the majority of the cDNA is resistant to DNase I digestion

The human immunodeficiency virus type 1 (HIV-1) pre-integration complex (PIC) is a cytoplasmic nucleoprotein structure derived from the core of the virion and is responsible for reverse transcription of viral RNA to cDNA, transport to the nucleus and integration of the cDNA into the genome of the infected target cell. Others have shown by Mu phage-mediated PCR footprinting that only the LTRs of the cDNA of PICs isolated early in infection are protected by bound protein, while the rest of the genome is susceptible to nuclease attack. Here, using DNase I footprinting, we confirmed that the majority of the cDNA of PICs isolated at 8·5 h after infection with cell-free virus was sensitive to digestion with DNase I and that only part of the LTRs (approximately 6% of the total cDNA) was protected. However, PICs isolated 90 min later (at 10 h post-infection) were very different in that the majority (approximately 90%) of cDNA was protected from nuclease degradation. These late PICs were integration active in vitro. We conclude that HIV-1 has at least two types of PIC, an early PIC characterized by protein bound only at the LTRs, and a late, and possibly more mature form, in which protein is bound along the length of the cDNA.

Defective presentation to class I-restricted cytotoxic T lymphocytes in vaccinia-infected cells is overcome by enhanced degradation of antigen.

Vaccinia infection interferes with the presentation of influenza Haemagglutinin (HA) and Nucleoprotein (NP) to class I-restricted CTL. The inhibitory effect is selective for certain epitopes, and is more profound during the late phase of infection. For influenza A/NT/60/68 NP, the block is present during both early and late phases of infection, and is selective for the COOH-terminal epitope defined by peptide 366-379, having no detectable effect on the presentation of the NH2-terminal epitope 50-63. The presentation of HA is inhibited only during the late phase of vaccinia infection. For both proteins, presentation is partially (NP) or completely (HA) restored by expression of rapidly degraded protein fragments in the vaccinia infected target cell. For HA, deletion of the NH2-terminal signal sequence completely overcomes the block. For NP, either a large NH2-terminal deletion or the construction of a rapidly degraded ubiquitin-NP fusion protein partially restores presentation. These results illustrate the relationship between degradation of viral proteins in the cytoplasm of an infected cell and recognition of epitopes at the cell surface by class I-restricted T cells.