Cyclophilins are a family of proteins that bind the immunosuppressant cyclosporin A, possess peptidyl-prolyl cis-trans isomerase activity, and assist in the folding of proteins. Human cyclophilins A and B are host cell proteins that bind specifically to the HIV-1 Gag polyprotein p55gag in vitro. Here we report that viral particles formed by p55gag, in contrast to particles formed by the Gag polyproteins of other retroviruses, contain significant amounts of cyclophilin A. Sequences in the capsid domain of p55gag are both required and sufficient for the virion-association of cyclophilin A. The association of cyclophilin A with HIV-1 virions was inhibited in a dose-dependent manner by cyclosporin A as well as by SDZ NIM811 ([Melle-4]cyclosporin), a non-immunosuppressive analogue of cyclosporin A. Drug-induced reductions in virion-associated cyclophilin A levels were accompanied by reductions in virion infectivity, indicating that the association is functionally relevant. Moreover, SDZ NIM811 inhibited the replication of HIV-1 but was inactive against SIVMAC, a primate immunodeficiency virus closely related to HIV-1, which does not incorporate cyclophilin A.

In vivo transduction of nondividing cells by human immunodeficiency virus type 1 (HIV-1)-based vectors results in transgene expression that is stable over several months. However, the use of HIV-1 vectors raises concerns about their safety. Here we describe a self-inactivating HIV-1 vector with a 400-nucleotide deletion in the 3′ long terminal repeat (LTR). The deletion, which includes the TATA box, abolished the LTR promoter activity but did not affect vector titers or transgene expression in vitro. The self-inactivating vector transduced neurons in vivo as efficiently as a vector with full-length LTRs. The inactivation design achieved in this work improves significantly the biosafety of HIV-derived vectors, as it reduces the likelihood that replication-competent retroviruses will originate in the vector producer and target cells, and hampers recombination with wild-type HIV in an infected host. Moreover, it improves the potential performance of the vector by removing LTR sequences previously associated with transcriptional interference and suppression in vivo and by allowing the construction of more-stringent tissue-specific or regulatable vectors.

Matrix (MA), a major structural protein of retroviruses, is thought to play a critical role in several steps of the HIV-1 replication cycle, including the plasma membrane targeting of Gag, the incorporation of envelope (Env) glycoproteins into nascent particles, and the nuclear import of the viral genome in nondividing cells. We now show that the entire MA protein is dispensable for the incorporation of HIV-1 Env glycoproteins with a shortened cytoplasmic domain. Furthermore, efficient HIV-1 replication in the absence of up to 90% of MA was observed in a cell line in which the cytoplasmic domain of Env is not required. Additional compensatory changes in Gag permitted efficient virus replication even if all of MA was replaced by a heterologous membrane targeting signal. Viruses which lacked the globular domain of MA but retained its N-terminal myristyl anchor exhibited an increased ability to form both extracellular and intracellular virus particles, consistent with a myristyl switch model of Gag membrane targeting. Pseudotyped HIV-1 particles that lacked the structurally conserved globular head of MA efficiently infected macrophages, indicating that MA is dispensable for nuclear import in terminally differentiated cells.