A single cycle of viral replication is the time required for a virus to enter the host cell, replicate its genome, and produce infectious progeny virions. The primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), require on average 24 h to complete one cycle of replication. We have now developed and characterized a reporter assay system in CEMx174 cells for the quantitative measurement of HIV/SIV infection within a single replication cycle. The SIV(mac)239 LTR (-225 --> +149) was cloned upstream of the firefly luciferase reporter gene and this reporter plasmid is maintained in CEMx174 cells under stable selection. This cell line, designated LuSIV, is highly sensitive to infection by primary and laboratory strains of HIV/SIV, resulting in Tat-mediated expression of luciferase, which correlates with viral infectivity. Furthermore, manipulation of LuSIV cells for the detection of luciferase activity is easy to perform and requires a minimal amount of time as compared to current HIV/SIV detection systems. The LuSIV system is a powerful tool for the analysis of HIV/SIV infection that provides a unique assay system that can detect virus replication prior to 24 h and does not require virus to spread from cell to cell. Thus these cells can be used for the study of replication-deficient viruses and the high throughput screening of antivirals, or other inhibitors of infection.

HIV-1 incorporates a variety of host membrane proteins during budding. We have previously shown that adhesion molecules are acquired by the virus in their activated or functional states. Our studies and those of others indicate that adhesion molecules can have profound effects on virus infectivity and its resistance to neutralization by antiviral antibodies. In this study we have examined the effect on infectivity of immobilization or margination of HIV-1 through acquired integrins LFA-1 and VLA-4 onto nonsusceptible cells and solid-phase adhesion ligands (ICAM-1 and VCAM-1, respectively). LFA-1- and VLA-4-mediated HIV-1 binding was supported by ICAM-1 and VCAM-1 immunoglobulin Fc chimeras, respectively. Integrin-mediated HIV-1 binding was also supported by 293 cells transfected with ICAM-1. In both cases the specificity of binding was confirmed with the appropriate blocking monoclonal antibodies or soluble adhesion ligands. We used a sensitive single-cycle infection assay based on a cell line expressing an LTR-luciferase cDNA construct to compare the infectivity of bound virus with that of free virus. Our results show that the binding of HIV-1 to nonsusceptible cells or immobilized adhesion ligands through acquired integrins can increase its infectivity by as much as two orders of magnitude. These results have implications for in vivo dissemination and transmission of HIV-1 and may also explain the high level of virus replication seen in solid lymphoid organs.

The N terminus of the capsid protein (CA) undergoes a considerable conformational change when the human immunodeficiency virus (HIV) protease cleaves it free from the Pr55Gag polyprotein. This rearrangement is thought to facilitate the establishment of specific CA-CA interactions that are required for the formation of the mature viral core. Substitution of amino acids that are critical for this refolding of the N terminus is generally detrimental to virus replication and mature virion core morphology. Here, we identify a conserved threonine in simian immunodeficiency virus (SIV) CA, T ( As hydrogen bonding between these two residues is present in HIV CA as well, this interaction presents a potential target for antiviral drug design.