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This study provides the first direct evidence that HIV-1-associated cholesterol is important for the maintenance of virion structure and infectivity. While the buoyant density of cholesterol-defective HIV-1 can be restored by a cholesterol structural analogue, cholestenone, the requirement for cholesterol is essential for HIV-1 infectivity.

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Human immunodeficiency virus type 1 (HIV-1) contains two copies of genomic RNA that are noncovalently linked via a palindrome sequence within the dimer initiation site (DIS) stem-loop. In contrast to the current paradigm that the DIS stem or stem-loop is critical for HIV-1 infectivity, which arose from studies using T-cell lines, we demonstrate here that HIV-1 mutants with deletions in the DIS stem-loop are replication competent in peripheral blood mononuclear cells (PBMCs). The DIS mutants contained either the wild-type (5GCGC GC3) or an arbitrary (5ACGCGT3) palindrome sequence in place of the 39-nucleotide DIS stem-loop (NL CGCGCG and NL ACGCGT ). These DIS mutants were replication defective in SupT1 cells, concurring with the current model in which DIS mutants are replication defective in T-cell lines. All of the HIV-1 DIS mutants were replication competent in PBMCs over a 40-day infection period and had retained their respective DIS mutations at 40 days postinfection. Although the stability of the virion RNA dimer was not affected by our DIS mutations, the RNA dimers exhibited a diffuse migration profile when compared to the wild type. No defect in protein processing of the Gag and GagProPol precursor proteins was found in the DIS mutants. Our data provide direct evidence that the DIS stem-loop is dispensable for viral replication in PBMCs and that the requirement of the DIS stem-loop in HIV-1 replication is cell type dependent.All retroviruses, including human immunodeficiency virus type 1 (HIV-1), contain two copies of virion genomic RNA (for a review, see references 6, 27, and 38). These virion dimeric RNAs are noncovalently linked at the 5Ј end of the RNA genome and undergo rearrangement to form more-stable RNA dimers during the maturation of virion proteins (11,12,35,45).The packaging of the HIV-1 RNA genome is mediated by the four stem-loop structures found near the 5Ј end of the RNA genome (6). These stem-loop structures are also referred to as the dimer initiation site (DIS) stem-loop, splice donor (SD) stem-loop, packaging (⌿) stem-loop, and Gag initiation stem-loop (1). A highly conserved palindrome sequence (5ЈG CGCGC3Ј, also known as the DIS and the kissing-loop) within the DIS stem-loop is important for the formation of viral RNA dimers in vitro (20, 44). Other variants of palindrome DIS-loop sequences (5ЈGTGCAC3Ј) and (5ЈTGCGCA3Ј) have also been found in a minor proportion of HIV-1 subtypes (46). The fact that the DIS stem-loop forms part of the HIV-1 genomic RNA packaging sequence may, in part, explain the strong correlation between genomic RNA dimerization and virion RNA packaging during HIV-1 assembly (1).The formation of a dimeric RNA genome in infectious retroviruses can be roughly divided into three different steps. These are (i) the initiation of genomic RNA dimer formation, (ii) the conformational rearrangement of the dimeric RNA (11,12,35,36,41,45), and (iii) the stabilization of genomic RNA dimers (11,12,35,36,45). Steps ii and iii are often collectively referred to as the maturation ...