Integration is essential for efficient gene expression of human immunodeficiency virus type 1

Abstract

A mutant of human immunodeficiency virus type 1 which carries a frameshift insertion in the integrase/endonuclease region of pol gene was constructed in vitro. Upon transfection into cells, although this mutant exhibited a normal phenotype with respect to expression of gag, pol, and env genes and to generation of progeny virions, no replication-competent virus in CD4-positive cells emerged. An assay for the single-step replication of a defective viral genome dependent on trans complementation by rev protein was established and used to monitor the early phase of viral infection process. Viral clones with a mutation in the vif, vpr, or vpu gene displayed no abnormality in the early phase. In contrast, the integrase mutant did not direct a marker gene expression after infection. Together with an observation that the mutant lacked the ability to integrate, these results indicated that the integration was required for efficient viral gene expression and productive infection of human immunodeficiency virus type 1.

Human Immunodeficiency Virus Vpx Is Required for the Early Phase of Replication in Peripheral Blood Mononuclear Cells

“…To determine the infection stage in PBMC when the Vpx protein is required, we have developed a system, designated a single-round replication assay, which is similar to that previously described by us for HIV-1 study (24,25). As shown in Fig (24,25). Early phase of viral replication can be monitored by the highly sensitive CAT assay (11).…”

“…system utilized replication-defective rev-minus proviral clones carrying CAT gene in the nef gene ( Fig. 1 b) for quantitative analysis of early phase infection process (from adsorption to integration) (24,25). For quantitative estimation of late infection process (from transcription to virion formation and release) in this system, PBMCs were transfected with regular proviral clones (Fig.…”

“…Using isogenic wild-type and mutant viruses, we determined their relative replication potential in established lymphoid cell lines not examined in previous studies and in primary human blood cells. We also utilized a newly established singleround replication assay system (24,25)…”

High‐level expression of active HIV‐1 integrase from a synthetic gene in human cells

“…The only viral enzyme required for integration is integrase (IN), encoded by the 3Ј fragment of the retroviral pol gene. Since integration is an essential step in the replication cycle of HIV (1,2) and no human counterpart is known to exist, there is great interest in developing inhibitors of HIV integrase (3,4).…”

“…Although the overall conformation of functional integrase remains unknown, complementation observed between proteins containing mutations in 1 These authors contributed equally to the work. 2 Correspondence: Rega Institute for Medical Research, K.U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.…”