Andrew C. S. Saphire

Syndecan captures, protects, and transmits HIV to T lymphocytes

This study demonstrates that syndecan functions as an in trans HIV receptor. We show that syndecan, when expressed in nonpermissive cells, becomes the major mediator for HIV adsorption. This adsorption is mediated by the binding of gp120 to the heparan sulfate chains of syndecan. Although syndecan does not substitute for HIV entry receptors, it enhances the in trans infectivity of a broad range of primate lentiviruses including primary viruses produced from PBMCs. Furthermore, syndecan preserves virus infectivity for a week, whereas unbound virus loses its infectivity in less than a day. Moreover, we obtain evidence suggesting that the vast syndecan-rich endothelial lining of the vasculature can provide a microenvironment which boosts HIV replication in T cells.

Host cyclophilin A mediates HIV‐1 attachment to target cells via heparans

The present study proposes a novel mode of action for cyclophilin A (CypA) in the HIV‐1 life cycle. We demonstrate that CypA‐deficient viruses do not replicate because they fail to attach to target cells. We show that CypA is exposed at the viral membrane and mediates HIV‐1 attachment. We identify heparan as the exclusive cellular binding partner for CypA. Furthermore, CypA binds directly to heparan via a domain rich in basic residues similar to known heparin‐binding motifs. This interaction between exposed CypA and cell surface heparans represents the initial step of HIV‐1 attachment and is a necessary precursor to gp120‐binding to CD4. In conclusion, HIV‐1 attachment to target cells is a multi‐step process that requires an initial CypA–heparan interaction followed by the gp120–CD4 interaction.

Nuclear Import of Adenovirus DNA in Vitro Involves the Nuclear Protein Import Pathway and hsc70

Adenovirus, a respiratory virus with a double-stranded DNA genome, replicates in the nuclei of mammalian cells. We have developed a cytosol-dependent in vitro assay utilizing adenovirus nucleocapsids to examine the requirements for adenovirus docking to the nuclear pore complex and for DNA import into the nucleus. Our assay reveals that adenovirus DNA import is blocked by a competitive excess of classical protein nuclear localization sequences and other inhibitors of nuclear protein import and indicates that this process is dependent on hsc70. Previous work revealed that the hexon (coat) protein of adenovirus is the only major protein on the surface of the adenovirus nucleocapsid that docks at the nuclear pore complex. This, together with our finding that in vitro nuclear import of hexon is inhibited by an excess of classical nuclear localization sequences, suggests a role for the hexon protein in adenovirus DNA import. However, recombinant transport factors that are sufficient for hexon import in permeabilized cells do not support DNA import, indicating that there are other as yet unidentified factors required for this process.

Host Cell Interactome of HIV-1 Rev Includes RNA Helicases Involved in Multiple Facets of Virus Production

The HIV-1 Rev protein plays a key role in the late phase of virus replication. It binds to the Rev Response Element found in underspliced HIV mRNAs, and drives their nuclear export by the CRM1 receptor pathway. Moreover, mounting evidence suggests that Rev has additional functions in viral replication. Here we employed proteomics and statistical analysis to identify candidate host cell factors that interact with Rev. For this we studied Rev complexes assembled in vitro with nuclear or cytosolic extracts under conditions emulating various intracellular environments of Rev. We ranked the protein-protein interactions by combining several statistical features derived from pairwise comparison of conditions in which the abundance of the binding partners changed. As a validation set, we selected the eight DEAD/H box proteins of the RNA helicase family from the top-ranking 5% of the proteins. These proteins all associate with ectopically expressed Rev in immunoprecipitates of cultured cells. From gene knockdown approaches, our work in combination with previous studies indicates that six of the eight DEAD/H proteins are linked to HIV production in our cell model. In a more detailed analysis of infected cells where either DDX3X, DDX5, DDX17, or DDX21 was silenced, we observed distinctive phenotypes for multiple replication features, variously involving virus particle release, the levels of unspliced and spliced HIV mRNAs, and the nuclear and cytoplasmic concentrations of these transcripts. Altogether the work indicates that our top-scoring data set is enriched in Rev-interacting proteins relevant to HIV replication. Our more detailed analysis of several Rev-interacting DEAD proteins suggests a complex set of functions for the helicases in regulation of HIV mRNAs. The strategy used here for identifying Rev interaction partners should prove effective for analyzing other viral and cellular proteins.

DDX1 is an RNA-dependent ATPase Involved in HIV-1 Rev Function and Virus Replication

Abstract The human immunodeficiency virus type 1 (HIV-1) Rev protein is essential for the virus because it promotes nuclear export of alternatively processed mRNAs, and Rev is also linked to translation of viral mRNAs and genome encapsidation. Previously, the human DEAD-box helicase DDX1 was suggested to be involved in Rev functions, but this relationship is not well understood. Biochemical studies of DDX1 and its interactions with Rev and model RNA oligonucleotides were carried out to investigate the molecular basis for association of these components. A combination of gel-filtration chromatography and circular dichroism spectroscopy demonstrated that recombinant DDX1 expressed in Escherichia coli is a well-behaved folded protein. Binding assays using fluorescently labeled Rev and cell-based immunoprecipitation analysis confirmed a specific RNA-independent DDX1–Rev interaction. Additionally, DDX1 was shown to be an RNA-activated ATPase, wherein Rev-bound RNA was equally effective at stimulating ATPase activity as protein-free RNA. Gel mobility shift assays further demonstrated that DDX1 forms complexes with Rev-bound RNA. RNA silencing of DDX1 provided strong evidence that DDX1 is required for both Rev activity and HIV production from infected cells. Collectively, these studies demonstrate a clear link between DDX1 and HIV-1 Rev in cell-based assays of HIV-1 production and provide the first demonstration that recombinant DDX1 binds Rev and RNA and has RNA-dependent catalytic activity.

Human immunodeficiency virus type 1 hijacks host cyclophilin A for its attachment to target cells

Our laboratory has identified a new facet of human immunodeficiency virus type 1 (HIV-1) entry. We demonstrated that the incorporation of host cyclophilin A (CypA) into nascent viruses is absolutely required for HIV-1 attachment to target cells. Although CypA is initially incorporated into the interior of the virus, we found that during maturation CypA relocatesto the viral surface. Our work indicates that exposed CypA mediates HIV-1 attachment to target cells via heparans. We believe that this interaction between CypA and heparan represents the initial step in HIV-1 entry.

Cyclophilin A Plays Distinct Roles in Human Immunodeficiency Virus Type 1 Entry and Postentry Events, as Revealed by Spinoculation

ABSTRACT Cyclophilin A (CypA) is necessary for effective human immunodeficiency virus type 1 (HIV-1) replication. However, the functions of CypA and the precise steps at which CypA acts in the HIV-1 life cycle remain to be determined. By using a methodology that bypasses the need for attachment factors—spinoculation—we present evidence that CypA participates in both entry and postentry events.

All Four Homochiral Enantiomers of a Nuclear Localization Sequence Derived from c-Myc Serve as Functional Import Signals

The information that targets a protein to the nucleus often consists of a short cluster of basic amino acids called a nuclear localization sequence (NLS). Since a wide range of sequences rich in basic amino acid residues function as NLSs, we postulated that an NLS-like sequence composed exclusively of d-amino acids might have biological activity. We synthesized peptides corresponding to the c-Myc NLS composed of either all l ord-amino acids, both in the forward and reverse order. We tested these peptides for nuclear import activity in a digitonin-permeabilized cell assay. All four peptide-bovine serum albumin conjugates localized to the nucleus with similar efficiency, and each conjugate competed for import with an SV40 large T antigen-derived NLS conjugate. Cross-linking experiments with free NLS peptides in HeLa cytosol indicated that each peptide bound to a protein that migrated at the molecular weight of importin α. Recombinant importin α, importin β, Ran, and NTF2 alone were sufficient to support the import of both l-form and d-form conjugates in permeabilized cells. This indicates that bothd- and l-form NLS peptides use the same import machinery. Although the free d-forms of the NLS were proteolytically resistant in cytosol, the l-forms were rapidly degraded. To our knowledge, this is the first example of an intracellular pathway in which the receptor is insensitive to the chirality of the ligand.