Unusual structure of the human immunodeficiency virus type 1 trans-activation response element.

The trans-activation response element (TAR) of human immunodeficiency virus type 1 is a structured RNA consisting of the first 60 nucleotides of all human immunodeficiency virus type 1 RNAs. Computer analyses and limited structural analyses indicated that TAR consists of a stem-bulge-loop structure. Mutational analyses showed that sequences in the bulge are required for Tat binding, whereas sequences in both the bulge and the loop are required for trans activation. In this study, we probed the structures of TAR and various mutants of TAR with chemical probes and RNases and used these methods to footprint a Tat peptide on TAR. Our data show that the structure of wild-type TAR is different from previously published models. The bulge, a Tat-binding site, consists of four nucleotides. The loop is structured, rather than simply single stranded, in a fashion reminiscent of the structures of the tetraloop 5'-UUCG-3' and the GNRA loop (C. Cheong, G. Varani, and I. Tinoco, Jr., Nature [London] 346:680-682, 1990; H.A. Heus and A. Pardi, Science 253:191-193, 1991). RNA footprint data indicate that three bases in the bulge are protected and suggest that a conformational change occurs upon Tat binding.     

A human chromosome 12-associated 83-kilodalton cellular protein specifically binds to the loop region of human immunodeficiency virus type 1 trans-activation response element RNA.

trans activation of human immunodeficiency virus type 1 (HIV-1) involves the viral trans-activator protein (Tat) and a cellular factor(s) encoded on human chromosome 12 (HuChr12) that targets the trans-activation response element (TAR) in the viral long terminal repeat. Because nascent TAR RNA is predicted to form a secondary structure that specifically binds cellular proteins, we investigated the composition of the TAR RNA-protein complex for HuChr12-specific proteins. UV cross-linking of TAR RNA-nuclear protein complexes formed in vitro identified an 83-kDa protein in human cells and in a human-hamster hybrid cell containing only HuChr12. The 83-kDa TAR RNA-binding protein was absent in the parental hamster cells. TAR RNA mutations that inhibited binding of the 83-kDa protein in vitro also inhibited HuChr12-dependent Tat trans activation. These TAR mutations changed the native sequence or secondary structure of the TAR loop. The TAR RNA binding activity of the 83-kDa protein also correlated with a HuChr12-dependent increase in steady-state HIV-1 RNA expression during Tat trans activation. Our results suggest that either a species-specific 83-kDa TAR RNA loop-binding protein is directly encoded on HuChr12 or a HuChr12 protein(s) induces the expression of an 83-kDa TAR-binding protein in nonprimate cells.     

Inhibition of human immunodeficiency virus type 1 Rev-Rev-response element complex formation by complementary oligonucleotides.

Complementary 18-mer oligodeoxynucleotides (oligonucleotides) specifically inhibited the formation of human immunodeficiency virus Rev-Rev-response element (RRE) complexes. Inhibition of Rev-RRE binding required blockage of G-7819 to G-7820 in band shift assays. Structural studies revealed both local and distal effects. RRE structure was also disrupted by oligonucleotides targeted to other minor stems, by altering RNA renaturation conditions, or by reducing Rev concentrations--indicating a dynamic RRE structure and involvement of a minor RRE stem in the maturation of initial Rev-RRE complexes. Thus, complementary oligonucleotides alter RRE structure and may prove useful for the design of therapeutic anti-RRE oligonucleotides.     

Packaging of human immunodeficiency virus type 1 RNA requires cis-acting sequences outside the 5' leader region.

cis elements required for the encapsidation of human immunodeficiency virus type 1 (HIV-1) RNA have been investigated by using a replication-competent helper virus to package a series of HIV-1-based vectors which had been stably transfected into human CD4 T-cell lines. A previously identified packaging signal in the 5' leader region was not sufficient for the encapsidation of small vectors containing heterologous genes. In contrast, vectors containing additional gag and env sequences were packaged with high efficiency and transduced into CD4-expressing target cells with titers exceeding 10(4) CFU/ml. The presence of gag sequences did not enhance vector packaging efficiency. A 1.1-kb env gene fragment encompassing the Rev-responsive element was absolutely required for the expression and encapsidation of vectors containing cis-acting repressive sequences and appeared also to contain an important packaging signal. Vectors as small as 2.6 kb were successfully packaged in this system. The presence of abundant, packageable vector RNA did not appear to interfere with encapsidation of the wild-type HIV-1 genome, suggesting that HIV-1 RNA packaging capacity is not saturated during acute infection.     

Minimal Rev-response element for type 1 human immunodeficiency virus.

Rev protein regulates nuclear export of viral mRNAs that contain a 240-base RNA sequence termed the Rev-response element (RRE). We demonstrate that an 88-base truncated RRE encompassing a known Rev binding site can mediate Rev responsiveness in vivo. Two tandem copies of this mutant function as efficiently as the full-length RRE.     

Novel human endogenous sequences related to human immunodeficiency virus type 1.

Endogenous retrovirus-related sequences exist within the normal genomic DNA of all eukaryotes, and these endogenous sequences have been shown to be important to the nature and biology of related exogenous retroviruses and may also play a role in cellular functions. To date, no endogenous sequences related to human immunodeficiency virus type 1 (HIV-1) have been reported. Herein we describe the first report of the presence of nucleotide sequences related to HIV-1 in human, chimpanzee, and rhesus monkey DNAs from normal uninfected individuals. We also present the isolation and characterization of two of these endogenous HIV-1-related sequences, EHS-1 and EHS-2. With use of low-stringency Southern blot hybridization, complex banding patterns were detected in human DNA with 5' and 3' HIV-1-derived probes. When an HIV-1 env region probe was used, we detected a less complex, conserved banding pattern in human DNA as well as a related but distinct banding pattern in chimpanzee and rhesus monkey DNAs. EHS-1 and -2 were cloned from normal human genomic DNA libraries by using the env region probe. Clone EHS-1 shows sequence similarity with the domain of the envelope cellular protease cleavage site of HIV-1, while EHS-2 has sequence similarity to the overlapping reading frame for Rev and gp41. Stringent hybridization of EHS-1 back to primate genomic DNA indicates two distinct EHS-1 loci in normal human DNA, an identical band pattern in chimpanzee DNA, and a single locus in rhesus monkey DNA. Likewise, EHS-2 is present as a single highly conserved locus in all three species. An oligonucleotide derived from EHS-2 across a region of near identity to HIV-1 detects a complex banding pattern in all primates tested similar to that seen with the 3' HIV-1 probe. These data suggest that most of the HIV-1-related sequences identified in primate DNA share a common core of nucleic acid sequence found in both EHS-2 and rev and that some of these HIV-1-related sequences have additional larger regions of sequence similarity to HIV-1.     

Inhibition of human immunodeficiency virus type 1 Env-mediated fusion by DC-SIGN

DC-SIGN, a lectin expressed on dendritic cell and macrophage subsets, binds to human immunodeficiency virus Env glycoproteins, allowing capture of viral particles. Captured virions either infect target cells or are efficiently transmitted to lymphocytes. Cellular mechanisms underlying the effects of DC-SIGN remain poorly understood. Here we have analyzed the effects of DC-SIGN on viral entry and on syncytium formation induced by Env glycoproteins. The lectin enhanced susceptibility to viral infection and dramatically increased virion internalization. Captured virions accumulated in the vesicular pathway, and their access to the cytosol was altered. Strikingly, the presence of DC-SIGN on target cells inhibited their ability to form syncytia with Env-expressing cells. However, increasing CD4 surface levels on target cells alleviated this inhibitory effect of DC-SIGN. Moreover, the potency of the viral fusion inhibitor T-20 was not affected in DC-SIGN-expressing cells. Altogether, our results indicate that DC-SIGN exerts subtle and complex effects during early steps of HIV type 1 replication. DC-SIGN facilitates capture and accumulation of viral particles in a vesicular compartment and inhibits viral fusion. Competition between CD4 and DC-SIGN for Env binding likely affects virus access to the cytosol and syncytium formation.     

Inhibition of human immunodeficiency virus type 1-induced cell fusion by recombinant human interferons.

Pretreatment of HeLa T4 cells with recombinant alpha, beta, or gamma interferon (IFN) was found to significantly inhibit syncytium formation induced by the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein. All three IFNs were found to be potent inhibitors of fusion in a system in which Spodoptera frugiperda cells, infected with a baculovirus recombinant expressing the HIV-1 envelope protein, were cocultivated with HeLa T4 cells. In addition, these IFNs were also found to block HeLa T4 cell fusion induced by the HIV-1 envelope proteins expressed from a vaccinia virus recombinant. Furthermore, the IFNs inhibited cell fusion between HIV-1 envelope glycoprotein-expressing cells and either immortalized or fresh CD4+ lymphocytes pretreated with the IFNs. These results suggest that further testing of human IFNs for therapy of HIV-1 infection will be of interest.     

cis-acting sequences involved in human immunodeficiency virus type 1 RNA packaging.

We have previously described a series of human immunodeficiency virus type 1-based vectors in which efficient RNA encapsidation appeared to correlate with the presence of a 1.1-kb env gene fragment encompassing the Rev-responsive element (RRE). In this report, we explore in detail the role of the RRE and flanking env sequences in vector expression and RNA encapsidation. The analysis of a new series of vectors containing deletions within the env fragment failed to identify a discrete packaging signal, although the loss of certain sequences reduced packaging efficiency three- to fourfold. Complete removal of the env fragment resulted in a 100-fold decrease in the vector transduction titer but did not abolish RNA encapsidation. We conclude that the RRE and 3' env sequences are not essential for human immunodeficiency virus type 1 vector encapsidation but may be important in vectors in which a heterologous gene has been placed adjacent to the 5' packaging signal, potentially disrupting its structure.     

Inhibition of human immunodeficiency virus type 1 multiplication by antisense and sense RNA expression.

Human immunodeficiency virus type 1 (HIV-1) primarily infects CD4+ lymphocytes and macrophages and causes AIDS in humans. Retroviral vectors allowing neomycin phosphotransferase (npt) gene expression were engineered to express 5' sequences of HIV-1 RNA in the antisense or sense orientation and used to transform the human CD4+ lymphocyte-derived MT4 cell line. Cells expressing antisense or sense RNA to the HIV-1 tat mRNA leader sequence, as part of the 3' untranslated region of the npt mRNA, remained sensitive to HIV-1 infection. In contrast, resistance to HIV-1 infection was observed in cells expressing antisense RNA to the HIV-1 primer-binding site or to the region 5' to the primer-binding site as part of the 3' region of the npt mRNA. Cells expressing the tat mRNA leader sequence in the sense orientation as a precise replacement of the 5' untranslated region of npt mRNA were also resistant to HIV-1. These results indicate that sense and antisense approaches can be used to interfere with HIV-1 multiplication.