Specific binding of HIV-1 viral protein NCp7 to a unique 35-base RNA stem-loop SL1 is critical for formation and packaging of the genomic RNA dimer found within HIV-1 virions. NCp7 binding stimulates refolding of SL1 from a metastable kissing dimer (KD) into thermodynamically stable linear dimer (LD). Using UV melting, gel electrophoresis and heteronuclear NMR, we investigated effects of various site-specific mutations within the full-length SL1 on temperature- or NCp7-induced refolding in vitro. Refolding involved intramolecular melting of SL1 stems but not dissociation of the intermolecular KD interface. Refolding required only two NCp7 molecules per KD but was limited by the amount of NCp7 present, implying that the protein does not catalytically promote refolding. Efficient refolding depended strictly on the presence and, to a lesser degree, on sequence of a highly conserved G-rich internal loop that normally limits thermal stability of the SL1 stem. Adding two base pairs to the lower stem created a hyperstable SL1 mutant that failed to refold, even when bound by NCp7 at high stoichiometries. NMR analysis of these kinetically trapped mutant RNA–protein complexes indicated that NCp7 initiates refolding by dissociating base pairs in the upper stem of SL1. This study illuminates structural transitions critical for HIV-1 assembly and replication.

Gene therapy with hematopoietic stem and progenitor cells is a promising approach to engineering immunity to human immunodeficiency virus (HIV) that may lead to a functional cure for acquired immunodeficiency syndrome (AIDS). In support of this approach, we created lentiviral vectors with an engineered polycistronic platform derived from the endogenous MCM7 gene to express a diverse set of small antiviral RNAs and a drug resistance MGMTP140K marker. Multiple strategies for simultaneous expression of up to five RNA transgenes were tested. The placement and orientation of each transgene and its promoter were important determinants for optimal gene expression. Antiviral RNA expression from the MCM7 platform with a U1 promoter was sufficient to provide protection from R5-tropic HIV in macrophages and resulted in reduced hematopoietic toxicity compared with constructs expressing RNA from independent RNA polymerase III promoters. The addition of an HIV entry inhibitor and nucleolar TAR RNA decoy did not enhance antiviral potency over constructs that targeted only viral RNA transcripts. We also demonstrated selective enrichment of gene-modified cells in vivo using a humanized mouse model. The use of these less toxic, potent anti-HIV vectors expressing a drug selection marker is likely to enhance the in vivo efficacy of our stem cell gene therapy approach in treating HIV/AIDS.

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The type 1 human immunodeficiency virus (HIV-1), like all retroviruses, contains two copies of the RNA genome as a dimer. A dimer initially forms via a self-complementary sequence in the dimer initiation site (DIS) of the genomic RNA, but that dimer is converted to a mature dimer in a process generally promoted by the viral nucleocapsid (NC) protein. Formation of the mature dimer is correlated with infectivity. Study of genomic dimerization has been facilitated by discovery of short RNA transcripts containing the DIS stem-loop 1 (SL1), which can dimerize spontaneously without any protein factors in vitro as well as via the NC protein. On the basis of the palindromic nature of the apical loop of SL1, a kissing loop model has been proposed. First, a metastable kissing dimer is formed via a loop-loop interaction and then converted into a more stable extended dimer by the NC protein. This dimerization process in vitro is believed to mimic the in vivo RNA maturation. During experimental screening of potential inhibitors, we discovered a small molecule, Lys-Ala-7-amido-4-methylcoumarin (KA-AMC), which facilitates the in vitro conversion from kissing dimer to extended dimer. Here we report the structure-activity relationship for KA-AMC for promoting dimer maturation. Guanidino groups and increasing positive charge on the side chain enhance activity. For activity, the charged side chain is preferred on the benzene ring, and O 1 in the coumarin scaffold is essential. NMR studies show that the coumarin derivatives stack with aromatic bases of the RNA. The coumarin derivatives may aid in the investigation of some aspects of dimer maturation and serve as a scaffold for design of maturation inhibitors or of activators of premature maturation, either of which can lead to a potential HIV therapeutic.

HIV-1 causes AIDS, a syndrome that affects millions of people globally. Existing HAART is efficient in slowing down disease progression but cannot eradicate the virus. Furthermore the severity of the side effects and the emergence of drug-resistant mutants call for better therapy. Gene therapy serves as an attractive alternative as it reconstitutes the immune system with HIV-resistant cells and could thereby provide a potential cure. The feasibility of this approach was first demonstrated with the ‘Berlin patient’, who was functionally cured from HIV/AIDS with undetectable HIV-1 viral load after transplantation of bone marrow harboring a naturally occurring CCR5 mutation that blocks viral entry. Here, we give an overview of the current status of HIV gene therapy and remaining challenges and obstacles.