Inhibition of entry of HIV into cells by poly(A).poly(U)

Polyadenylic-polyuridylic acid referred to as poly(A).poly(U), is a synthetic double-stranded RNA that has been shown to manifest both antitumoral and immunodulatory activities. Previously, we have reported that poly(A).poly(U) inhibits HIV infection in cell cultures. Here we provide direct evidence to demonstrate that the inhibitory action of poly(A).poly(U) is through its capacity to prevent entry of HIV particles into CD4-positive T lymphocytes. Such inhibition of HIV entry is also observed in the case of other polyanions such as heparin, dextran sulfate, and poly(I).poly(C). The mechanism of inhibition appears to occur postbinding of HIV particles to the CD4 receptor molecules, because the binding of the external envelope glycoprotein of HIV-1 (gp120) is not affected significantly in the presence of poly(A).poly(U) or other polyanions. These results confirm the potential of poly(A).poly(U) as an antiviral drug against HIV infection.     

Double-stranded (ds) RNA binding and not dimerization correlates with the activation of the dsRNA-dependent protein kinase (PKR)

Upon binding to double-stranded (ds) RNA, the dsRNA-dependent protein kinase (PKR) sequentially undergoes autophosphorylation and activation. Activated PKR may exist as a dimer and phosphorylates the eukaryotic translation initiation factor 2 alpha subunit (cIF-2 alpha) to inhibit polypeptide chain initiation. Transfection of COS-1 cells with a plasmid cDNA expression vector encoding a marker gene, activates endogenous PKR, and selectively inhibits translation of the marker mRNA, dihydrofolate reductase (DHFR). This system was used to study the dsRNA binding and dimerization requirements for over-expressed PKR mutants and subdomains to affect DHFR translation. DHFR translation was rescued by expression of either an ATP hydrolysis defective mutant PKR K296P, the amino-terminal 1-243 fragment containing two dsRNA binding motifs, or the isolated first RNA binding motif (amino acids 1-123). Mutation of K64E within the dsRNA binding motif 1 destroyed dsRNA binding and the ability to rescue DHFR translation. Immunoprecipitation of T7 epitope-tagged PKR derivatives from cell lysates detected interaction between intact PKR and the amino-terminal 1-243 fragment as well as a 1-243 fragment harboring the K64E mutation. Expression of adenovirus VAI RNA, a potent inhibitor of PKR activity, did not disrupt this interaction. In contrast, intact PKR did not interact with fragments containing the first dsRNA binding motif (1-123), the second dsRNA binding motif (98-243), or the isolated PKR kinase catalytic domain (228-551). These results demonstrate that the translational stimulation mediated by the dominant negative PKR mutant does not require dimerization, but requires the ability to bind dsRNA and indicate these mutants act by competition for binding to activators.     

Poly (A)-rich ribonucleoprotein complexes from HeLa cell messenger RNA

Polyribosomal messenger RNA from HeLa cells contain 3'-OH-terminal polyadenylate sequences approximately 133 nucleotides in length (weight average). When analyzed at the ribonucleoprotein level of organization these poly(A)-rich sequences are found to contain tightly bound proteins. These proteins remain associated with the poly(A)-rich RNA during affinity chromatography of RNase A and T1-digested polyribosomes on poly(U)-Sepharose in 0.5 M NaCl, and co-elute from the column with the RNA at 50% formamide. Controls establish that the co-purification of the proteins with poly(A) on poly(U)-Sepharose requires the molecular integrity of the poly(A). Polyacrylamide gel electrophoresis resolves the poly(A)-specific proteins into two components of 74,000 and 62,000 molecular weight. The larger protein is the same size as that previously reported to be associated with poly(A)-rich sequences in HeLa heterogeneous nuclear RNA (Kish, V.M., and Pederson, T. (1975), J. Mol. Biol. 95, 227-238). It is concluded that both HeLa nuclear and polyribosomal poly(A) sequences have a protein (62,000 molecular weight) associated with poly(A) appears to be confined only to messenger RNA.     

Interaction of poly(rC) binding protein 2 with the 5' noncoding region of hepatitis A virus RNA and its effects on translation

Utilization of internal ribosome entry segment (IRES) structures in the 5' noncoding region (5'NCR) of picornavirus RNAs for initiation of translation requires a number of host cell factors whose distribution may vary in different cells and whose requirement may vary for different picornaviruses. We have examined the requirement of the cellular protein poly(rC) binding protein 2 (PCBP2) for hepatitis A virus (HAV) RNA translation. PCBP2 has recently been identified as a factor required for translation and replication of poliovirus (PV) RNA. PCBP2 was shown to be present in FRhK-4 cells, which are permissive for growth of HAV, as it is in HeLa cells, which support translation of HAV RNA but which have not been reported to host replication of the virus. Competition RNA mobility shift assays showed that the 5'NCR of HAV RNA competed for binding of PCBP2 with a probe representing stem-loop IV of the PV 5'NCR. The binding site on HAV RNA was mapped to nucleotides 1 to 157, which includes a pyrimidine-rich sequence. HeLa cell extracts that had been depleted of PCBP2 by passage over a PV stem-loop IV RNA affinity column supported only low levels of HAV RNA translation. Translation activity was restored upon addition of recombinant PCBP2 to the depleted extract. Removal of the 5'-terminal 138 nucleotides of the HAV RNA, or removal of the entire IRES, eliminated the dependence of HAV RNA translation on PCBP2.     

Activation of the double-stranded RNA-regulated protein kinase by depletion of endoplasmic reticular calcium stores

Perturbants of the endoplasmic reticulum (ER), including Ca(2+)-mobilizing agents, provoke a rapid suppression of translational initiation in conjunction with an increased phosphorylation of the alpha-subunit of eukaryotic initiation factor (eIF)-2. Depletion of ER Ca2+ stores was found to signal the activation of a specific eIF-2 alpha kinase. Analysis of extracts derived from cultured cells that had been pretreated with Ca2+ ionophore A23187 or thapsigargin revealed a 2-3-fold increase in eIF-2 alpha kinase activity without detectable changes in eIF-2 alpha phosphatase activity. A peptide of 65-68 kDa, which was phosphorylated concurrently with eIF-2 alpha in extracts of pretreated cells, was identified as the interferon-inducible, double-stranded RNA (dsRNA)-regulated protein kinase (PKR). Depletion of ER Ca2+ stores did not alter the PKR contents of extracts. When incubated with reovirus dsRNA, extracts derived from cells with depleted ER Ca2+ stores displayed greater degrees of phosphorylation of PKR and of eIF-2 alpha than did control extracts. The enhanced dsRNA-dependent phosphorylation of PKR was observed regardless of prior induction of the kinase with interferon. Lower concentrations of dsRNA were required for maximal phosphorylation of PKR in extracts of treated as compared to control preparations. These findings suggest that PKR mediates the translational suppression occurring in response to perturbation of ER Ca2+ homeostasis.     

Poly(A) tail shortening by a mammalian poly(A)-specific 3'-exoribonuclease

3'-Exonucleolytic removal of the poly(A) tail is the first and often rate-limiting step in the decay of many eucaryotic mRNAs. In a cytoplasmic extract from HeLa cells, the poly(A) tail of mRNA was degraded from the 3'-end. In agreement with earlier in vivo observations, prominent decay intermediates differed in length by about 30 nucleotides. The Mg2+-dependent, poly(A)-specific 3'-exoribonuclease responsible for this poly(A) shortening activity was purified from calf thymus. A polypeptide of 74 kDa copurified with the activity. The deadenylating nuclease (DAN) required a free 3'-OH group, released solely 5'-AMP, degraded RNA in a distributive fashion, and preferred poly(A) as a substrate. At low salt concentration, the activity of purified DAN was strongly dependent on spermidine or other, yet unidentified factors. Under these reaction conditions, DAN was also stimulated by the cytoplasmic poly(A)-binding protein I (PAB I). At physiological salt concentration, the stimulatory effect of spermidine was weak and PAB I was inhibitory. At either salt concentration DAN and PAB I reconstituted poly(A) shortening with the same pattern of intermediates seen in cytoplasmic extract. The properties of DAN suggest that the enzyme might be involved in the deadenylation of mRNA in vivo.     

Human immunodeficiency virus 1 (HIV-1)-specific reverse transcriptase (RT) inhibitors may suppress the replication of specific drug-resistant (E138K)RT HIV-1 mutants or select for highly resistant (Y181C-->C181I)RT HIV-1 mutants

Mutant HIV-1 that expresses a Glu138-->Lys substitution in its RT [(E138K)RT] is resistant to the HIV-1-specific RT inhibitor 2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"- oxathiole-2",2"-dioxide)pyrimidine (TSAO). However, cell cultures infected with this mutant were completely protected against virus-mediated destruction by micromolar concentrations of the HIV-1-specific RT inhibitors tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO), nevirapine, and bis(heteroaryl)piperazine (BHAP). In contrast, cells infected with a virus mutant that expresses a Tyr181-->Cys substitution in its RT [(Y181C)RT] were not protected by nevirapine and TIBO and were only temporarily protected by BHAP. HIV-1 mutant that emerged under the latter conditions contained a Cys181-->Ile substitution in their RT [(LC181I)RT]. This mutant proved highly resistant to all HIV-1-specific RT inhibitors tested, except for several 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) derivatives. When recombinant (C181I)RT was evaluated for susceptibility to the HIV-1-specific RT inhibitors, it was resistant to all inhibitors except the HEPT compounds. Since a (Y181F)RT HIV mutant strain was isolated from cells infected with (Y181C)RT HIV-1 and treated with BHAP, we postulate that the Ile codon was derived from a Cys-->Phe transversion mutation (TGT-->TTT), followed by a Phe-->Ile transversion mutation (TTT-->ATT).     

A review of the neurobehavioral deficits in children with fetal alcohol syndrome or prenatal exposure to alcohol

HeLa cells and HeLa cells expressing the HIV-1 regulatory protein Rev were immunostained for Rev and pre-mRNA processing factors and examined histographically by confocal laser scanning microscopy. Following short pulse-labelling with bromouridine tri-phosphate nascent RNA gave a granular nucleoplasmic staining increasing somewhat towards the periphery as did also the heterogeneous ribonucleoproteins (hnRNPs) A1 and particularly C1/C2, a distribution pattern which has not been described. The sm-antigen of the small ribonucleoprotein particle (snRNP) proteins U1, U2, U4/U6 and U5 stained the nucleoplasm diffusely in addition to speckles which co-localised with speckles of the non-snRNP splicing factor SC-35. Brominated RNA and the hnRNPs A1 and C1/C2 were to varying degrees excluded from the speckles. Rev concentrated in the nucleolus and often as a perinucleolar ring/zone. Rev also stained the nucleoplasm and cytoplasm without co-localising with the above-mentioned proteins or brominated RNA and was not enriched or excluded in SC-35 speckles. The nucleolar proteins B23 and C23, like Rev, gave primarily a perinucleolar ring and stained the nucleoplasm but did not otherwise co-localise with Rev or with nuclear proteins. Histographic recording of immunofluorescence images proved to be a valuable tool in the study of localisation of HIV-1 Rev and cellular components and of possible co-localisations. A parallel comparison of the subcellular patterns of pre-mRNA processing factors versus major nucleolar antigens is new and suggests that the factors are not strictly separated in the nucleoplasm.     

The 2-5A system in viral infection and apoptosis

The 2-5A system is an established endogenous antiviral pathway. Interferon treatment of cells leads to an increase in basal, but latent, levels of 2-5A-dependent RNase (RNase L) and the family of 2'-5' oligoadenylate synthetases (OAS). Double-stranded RNA, thought to be derived from viral replication intermediates, activates OAS. Activated OAS converts ATP into unusual short 2'-5' linked oligoadenylates called 2-5A [ppp5'(A2'p5')2A]. The 2-5A binds to and activates RNase L which cleaves single stranded RNA with moderate specificity for sites 3' of UpUp and UpAp sequences, and thus leads to degradation of cellular rRNA. During apoptosis, generalized cellular RNA degradation, distinct from the differential expression of mRNA species that may regulate specific gene expression during apoptosis, has been observed. The mechanism of RNA breakdown during apoptosis has been commonly considered a non-specific event that reflects the generalized shut down of translation and homeostatic regulation during cell death. Due to the similar RNA degradation that occurs during both apoptosis and viral infection we investigated the potential role of RNase L in apoptosis. To investigate whether RNase L activity could lead to apoptosis, NIH3T3 cells were transfected with a lac-inducible vector containing the human RNase L gene. Treatment of these cells with isopropylthiogalactoside (IPTG) caused loss of cell viability that was confirmed as an apoptotic cell death by morphological and biochemical criteria. Similarly, specific allosteric activation of endogenous RNase L by introduction of 2-5A directly into L929 cells also induced apoptosis. In L929 cells poly(I).poly(C) treatment in combination with interferon caused an increase in apoptosis whereas neither interferon or double stranded RNA alone altered cell viability. Therefore, increased expression or activation of RNase L causes apoptosis. Inhibition of RNase L, specifically with a dominant negative mutant, suppressed poly(I)Ypoly(C)-induced apoptosis in interferon-primed fibroblasts. Poliovirus, a picornovirus with a single-stranded RNA genome, causes apoptosis of HeLa cells. Expression of the dominant negative inhibitor of RNase L in HeLa prevented virus-induced apoptosis and maintained cell viability. Thus, reduction or inhibition of RNase L activity prevents apoptosis. Both apoptosis and the 2-5A system can provide defense against viral infection in multicellular organisms by preventing production and therefore spread of progeny virus. RNase L appears to function in both mechanisms, therefore, initiation of apoptosis may be one mechanism for the antiviral activity of the 2-5A system.     

Poly(2-aminoadenylic acid): interaction with poly(uridylic acid)

Poly(2-aminoadenylic acid) forms both double and triple helices with poly(uridylic acid) [poly(U)]. The 2-amino group forms a third hydrogen bond, elevating the 2 leads to 1 transition temperature by 33 degrees C. The third strand, however, has about the same stability as poly(A)-2poly(U), as measured by Tm 3 leads to 2. This selective stabilization of the two-stranded helix results in a much greater resolution of the differnt thermal transitions than that observed in analogous polynucleotide systems. In contrast to other A, U systems 3 leads to 1 and 2 leads to 3 transitions are not observed under any conditions, and the triple helix always undergoes a 3 leads to 2 transition even at very high ionic strength. A 1:1 mixture of poly(2NH2A) and poly(U) exhibits no transient formation of 1:2 complex, unlike similar mixtures of poly(A) with poly(U) and poly(T). This difference is evidently due to a more rapid displacement reaction: [poly(2NH2A) + poly(2NH2A)-2poly(U) leads to 2 poly(2NH2A)-poly(U)] With poly(2NH2A) than with poly(A). We describe a method for establishing the combining ratios of polynucleotide complexes which used a computer to calculate the angles of intersection of mixing curves as explicit and continuous functions of the wavelength. The wavelength dispersions of the angles of intersection determine optimum wavelengths for establishing stoichiometry and can also provide reliable negative evidence that presumably plausible complexes are not formed. Analogous computer procedures have been developed to determine wavelengths which are selective for the formation of both 1:1 and 1:2 complexes. Infrared spectra of the 1:1 and 1:2 complexes resemble those of other A, U homoribopolynucleotide helices in having two and three strong bands, respectively, in the region of carbonyl stretching vibrations. CD spectra of the two complexes are unusual in having negative first extrema of moderate intensity. We attribute these extrema to intrastrand interactions of strong, well-resolved transitions at 278 nm (B2u) of the 2-aminoadenine residues. The CD spectra are correlated with those of other polynucleotide helices.     

Virus-induced cell death in plants expressing the mammalian 2',5' oligoadenylate system

The major components of the 2-5A system, responsible for the mammalian interferon-induced antiviral response, are the 2',5' oligoadenylate synthetase (2-5Aase) and 2',5' oligoadenylate (2-5A) dependent ribonuclease (RNase L). Transgenic tobacco plants expressing these two enzyme activities were produced by crossing the transgenic plants expressing RNase L with those expressing 2-5Aase. The double transgenic plants showed complete resistance against cucumber mosaic virus (CMV), infection with necrotic spots only forming on the virus-inoculated leaf. On the other hand, although plants inoculated with potato virus Y (PVY) formed necrotic spots on the inoculated leaf and virus amplification could not be detected, all plants died within 20 days of inoculation. The transgenic tobacco plants expressing either 2-5Aase or RNase L activity showed typical disease symptoms with CMV- or PVY-inoculation. These results suggest that the introduced 2-5A system is activated in tobacco cells by dsRNA, the replicating intermediates of RNA viruses, leading to death of the host cells, which has not been observed in mammalian cells.     

Different requirements for membrane fusion mediated by the envelopes of human immunodeficiency virus types 1 and 2

CD4+ cells derived from the human cell lines U87MG and SCL1 cannot be infected by human immunodeficiency virus type 1 (HIV-1) or fuse with cells expressing the HIV-1 envelope. This block was complemented in heterokaryons with HeLa cells and probably reflects the absence of cellular factors necessary for membrane fusion. Since U87MG cells expressing CD4 are permissive to HIV-2, distinct cellular factors could be required for fusion mediated by two related human retroviruses.     

The 2-5A system: modulation of viral and cellular processes through acceleration of RNA degradation

The 2-5A system is an RNA degradation pathway that can be induced by the interferons (IFNs). Treatment of cells with IFN activates genes encoding several double-stranded RNA (dsRNA)-dependent synthetases. These enzymes generate 5'-triphosphorylated, 2',5'-phosphodiester-linked oligoadenylates (2-5A) from ATP. The effects of 2-5A in cells are transient since 2-5A is unstable in cells due to the activities of phosphodiesterase and phosphatase. 2-5A activates the endoribonuclease 2-5A-dependent RNase L, causing degradation of single-stranded RNA with moderate specificity. The human 2-5A-dependent RNase is an 83.5 kDa polypeptide that has little, if any, RNase activity, unless 2-5A is present. 2-5A binding to RNase L switches the enzyme from its off-state to its on-state. At least three 2',5'-linked oligoadenylates and a single 5'-phosphoryl group are required for maximal activation of the RNase. Even though the constitutive presence of 2-5A-dependent RNase is observed in nearly all mammalian cell types, cellular amounts of 2-5A-dependent mRNA and activity can increase after IFN treatment. One well-established role of the 2-5A system is as a host defense against some types of viruses. Since virus infection of cells results in the production and secretion of IFNs, and since dsRNA is both a frequent product of virus infection and an activator of 2-5A synthesis, the replication of encephalomyocarditis virus, which produces dsRNA during its life cycle, is greatly suppressed in IFN-treated cells as a direct result of RNA decay by the activated 2-5A-dependent RNase. This review covers the organic chemistry, enzymology, and molecular biology of 2-5A and its associated enzymes. Additional possible biological roles of the 2-5A system, such as in cell growth and differentiation, human immunodeficiency virus replication, heat shock, atherosclerotic plaque, pathogenesis of Type I diabetes, and apoptosis, are presented.