Differential growth kinetics are exhibited by human immunodeficiency virus type 1 TAR mutants.

The human immunodeficiency virus type 1 (HIV-1) TAR element is critical for the activation of gene expression by the transactivator protein, Tat. Mutagenesis has demonstrated that a stable stem-loop RNA structure containing both loop and bulge structures transcribed from TAR is the major target for tat activation. Though transient assays have defined elements critical for TAR function, no studies have yet determined the role of TAR in viral replication because of the inability to generate viral stocks containing mutations in TAR. In the current study, we developed a strategy which enabled us to generate stable 293 cell lines which were capable of producing high titers of different viruses containing TAR mutations. Viruses generated from these cell lines were used to infect both T-lymphocyte cell lines and peripheral blood mononuclear cells. Viruses containing TAR mutations in either the upper stem, the bulge, or the loop exhibited dramatically decreased HIV-1 gene expression and replication in all cell lines tested. However, we were able to isolate lymphoid cell lines which stably expressed gene products from each of these TAR mutant viruses. Though the amounts of virus in these cell lines were roughly equivalent, cells containing TAR mutant viruses were extremely defective for gene expression compared with cell lines containing wild-type virus. The magnitude of this decrease in viral gene expression was much greater than previously seen in transient expression assays using HIV-1 long terminal repeat chloramphenicol acetyltransferase gene constructs. In contrast to the defects in viral growth found in T-lymphocyte cell lines, several of the viruses containing TAR mutations were much less defective for gene expression and replication in activated peripheral blood mononuclear cells. These results indicate that maintenance of the TAR element is critical for viral gene expression and replication in all cell lines tested, though the cell type which is infected is also a major determinant of the replication properties of TAR mutant viruses.     

Inhibition of HIV-LTR gene expression by oligonucleotides targeted to the TAR element.

All human immunodeficiency virus mRNAs contain a sequence known as TAR (trans-activating responsive sequence). The TAR element forms a stable RNA stem-loop structure which binds the HIV tat (trans-activator) protein and mediates increased viral gene expression. In principle, molecules which bind to the TAR RNA structure would inhibit trans-activation by perturbing the native RNA secondary structure. We have constructed a series of phosphodiester and phosphorothioate antisense oligonucleotides which specifically bind to the HIV TAR element. Specific binding to the TAR element was demonstrated in vitro with enzymatically synthesized TAR RNA. The TAR-directed phosphorothioates inhibited trans-activation in a sequence-dependent fashion in a cell culture model using an HIV LTR/human placental alkaline phosphatase gene fusion and tat protein supplied in trans. The molecules also inhibited HIV replication in both acute and chronically infected viral assays, but without sequence specificity. We have constructed a series of vectors consisting of the MMTV promoter and 5'-untranslated region of four different mRNAs, including the TAR region, to study the effect of TAR on gene expression in heterologous systems. The results suggest that, in the absence of the HIV LTR, the TAR element has a repressive effect on gene expression, which is relieved by tat.     

Minus-strand copies of replicating coronavirus mRNAs contain antileaders.

The 5' leader sequence on mRNAs of the porcine transmissible gastroenteritis coronavirus was determined and found to be 90 nucleotides in length. An oligodeoxynucleotide with a sequence from within the leader was used as a probe in Northern analysis on RNA from infected cells, and an antileader (a minus-strand copy of the leader sequence) was shown to be present on all mRNA minus-strand species. RNase protection analysis showed the antileader to be approximately the same length as the leader. The kinetics of antileader appearance was the same as that for the appearance of minus-strand RNA species. This, along with a demonstration that viral mRNAs become packaged, gives further support to the idea that coronavirus mRNAs can undergo replication via subgenomic mRNA-length replicative intermediates, and that input mRNAs from infecting virions may serve as initial templates for their own replication. In this sense, then, coronaviruses behave in part like RNA viruses with segmented genomes.     

The integrity of the stem structure of human immunodeficiency virus type 1 Tat-responsive sequence of RNA is required for interaction with the interferon-induced 68,000-Mr protein kinase.

A number of eucaryotic viruses have devised strategies to minimize the deleterious effects on protein synthesis caused by activation of the interferon-induced, double-stranded-RNA-activated protein kinase, P68. In a recent report, we described the down regulation of the P68 protein kinase in cells infected by human immunodeficiency virus type 1 (HIV-1) (S. Roy, M. G. Katze, N. T. Parkin, I. Edery, A. G. Hovanessian, and N. Sonenberg, Science 247:1216-1219, (1990). We now present evidence that such a decrease in amounts of P68 could be essential for HIV-1 replication because of the presence of the Tat-responsive sequence (TAR sequence) present in the 5' untranslated region of HIV-1 mRNAs, which activates the P68 kinase. We found that poly(A)+ mRNAs prepared from HIV-1-infected cells efficiently activated the protein kinase as did mRNAs from stably transformed cell lines constitutively expressing the TAR region. Furthermore, we found that TAR-containing RNAs complexed with purified P68 protein kinase in vitro by two independent assays and could be cross-linked to P68 kinase present in a HeLa cell extract. Experiments using in vitro-synthesized wild-type and mutant TAR RNAs revealed that both the efficient binding to and the activation of P68 kinase were dependent on the TAR RNA stem structure. The TAR-P68 complex could be competed out by a synthetic RNA that bound to and activated the protein kinase but not by a synthetic RNA that bound with low affinity and did not activate P68. The possible biological consequences of a P68-TAR interaction that may include the switch from latent to active virus replication are discussed.     

Simian immunodeficiency virus displays complex patterns of RNA splicing.

The human and simian immunodeficiency viruses encode at least six gene products that apparently serve regulatory functions. To evaluate the regulation of simian immunodeficiency virus gene expression at the level of RNA splicing, we used the polymerase chain reaction to amplify and clone cDNAs corresponding to a large array of mRNAs from infected cells. We identified mRNAs that used splice acceptor sites upstream of the initiator codons for tat, rev, vpr, nef, vif, and vpx, suggesting that these proteins may be expressed from different mRNAs. We also provide hybridization data suggesting that the same splice acceptor site may be used for both rev and env mRNAs. Furthermore, we isolated both tat and rev cDNAs that utilized three alternative splice acceptor sites at the start of coding exon 2, indicating that different versions of these proteins may be encoded. Finally, approximately 10 to 20% of simian immunodeficiency virus mRNAs spliced an intron from their untranslated 5' ends, and sequences contained within this intron constituted a portion of the tat-responsive TAR element. Thus, alternative pre-mRNA splicing adds a level of complexity to simian immunodeficiency virus expression, which may affect several levels of gene regulation.     

TAR-independent replication of human immunodeficiency virus type 1 in glial cells.

The molecular mechanisms involved in the replication of human immunodeficiency virus type 1 (HIV-1) may differ in various cell types and with various exogenous stimuli. Astrocytic glial cells, which can support HIV-1 replication in cell cultures and may be infected in vivo, are demonstrated to provide a cellular milieu in which TAR mutant HIV-1 viruses may replicate. Using transfections of various TAR mutant HIV-1 proviral constructs, we demonstrate TAR-independent replication in unstimulated astrocytic cells. We further demonstrate, using viral constructs with mutations in the tat gene and in the nuclear factor kappa B (NF-kappa B)-binding sites (enhancer) of the HIV-1 long terminal repeat, that TAR-independent HIV-1 replication in astrocytic cells requires both intact NF-kappa B moiety-binding motifs in the HIV-1 long terminal repeat and Tat expression. We measured HIV-1 p24 antigen production, syncytium formation, and levels and patterns of viral RNA expression by Northern (RNA) blotting to characterize TAR-independent HIV-1 expression in astrocytic glial cells. This alternative regulatory pathway of TAR-independent, Tat-responsive viral production may be important in certain cell types for therapies which seek to perturb Tat-TAR binding as a strategy to interrupt the viral lytic cycle.     

Early infection of the central nervous system by the GDVII and DA strains of Theiler's virus.

The DA strain of Theiler's virus, a murine picornavirus, causes a persistent infection of glial cells of the white matter of the spinal cord, associated with chronic inflammation and primary demyelination. The GDVII strain causes an acute fatal grey matter encephalomyelitis. We characterized the target cells of GDVII and DA viruses 4 days following intracerebral inoculation, and we compared the levels of viral RNA within these cells. GDVII virus infected approximately 10 times more cells than DA virus. Whereas GDVII virus infected neurons exclusively, DA virus infected also astrocytes and possible macrophage-microglial cells. The levels of viral RNA in neurons infected with GDVII and DA viruses were of the same order. These results show that DA virus infects glial cells already at the beginning of the disease and that the more efficient spread of GDVII virus is probably not due to a higher level of RNA replication per cell.