The molecular organization of the influenza virus surface. Studies using photoreactive and fluorescent labeled phospholipid probes.

The membrane structures of remantadin-sensitive and remantadin-resistant influenza virus strains were studied using a photoreactive fatty acid as well as analogues of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, carrying a fluorescent or photoreactive reporter group at the end of one of the aliphatic chains. The results obtained demonstrated for the first time that the phospholipids of the viral membrane form lateral domains differing by the fluidity of their hydrocarbon chains and, probably, by the head-group composition of the lipids. The hemagglutinin small subunit (HA2) was shown to protrude into the apolar region of the phospholipid bilayer, whereas the M1 protein makes contact only with the inner surface. In the remantadin-sensitive virions the heavy hemagglutinin chain (HA1) appears not to be in contact with the lipid bilayer, whereas in the remantadin-resistant strain HA1 has a hydrophobic segment that proved to be inserted into the bilayer.

Action of influenza virus neuraminidase on gangliosides. Haemagglutinin inhibits viral neuraminidase.

The action of partly purified neuraminidase (NA) of influenza A virus, a mixture of detergent solubilized NA and haemagglutinin (HA) and of intact virions on gangliosides GT1b, GD1a, GD1b, GM1 was studied. The viral NA transformed GT1b mainly into GD1b with formation of only minor amounts of GM1. HA was found to inhibit the hydrolysis activity of viral NA. At the same time viral NA transformed GD1a quantitatively into GM1 which was not hydrolyzed by the enzyme. These results suggest that the function of NA is to transfer the 'primary' receptor (such as GT1b) into the proper carbohydrate sequence (GD1b-like) which is proposed to serve as the minimal structure required for influenza virus reception.

HIV-1 matrix protein p17 resides in cell nuclei in association with genomic RNA.

We have shown previously that HIV-1 matrix protein p17 is transported to the nucleus of Jurkat-tat and H9 cells soon after infection. As shown in this combination, gag polyprotein p55 synthesized 48 h after cell infection is cleaved in cytosol rapidly after its synthesis, and nascent p17 enters the nuclei and gradually accumulates there. Uncleaved p55 molecules and intermediate precursors are rapidly transported to the membranes and are also found in nuclei. Mature gag proteins are seen in membranes only after prolonged period of labelling or chase (4 or more hours later). To determine whether the nascent p17 is associated with viral genomic RNA in the nuclei, the cells were fractionated, the viral complexes were immunoprecipitated by monoclonal antibodies (MAbs) against gag proteins, and RNA was extracted and analyzed by slot and blot hybridization. MAb against p17 precipitated all the viral RNA from the nuclei including full-size genomic RNA and essential parts from membranes while MAb against p24 did not precipitate any viral RNA from the nuclei. These data suggest that matrix protein is linked to genomic RNA in the nuclei and raise the possibility that p17 may transfer viral nucleocapsids from the nuclei to plasma membranes, the site of virus assembly.

Highly immunogenic human immunodeficiency viruslike particles are produced by recombinant vaccinia virus-infected cells.

CV-1 cells were infected with two recombinant vaccinia viruses carrying the gag gene with deletion of 231 bp from 3' terminus (strain vC5) and env gene (strain vE234L) of human immunodeficiency virus type 1 (HIV-1). Both recombinant proteins synthesized in the cells (p50gag and gp160/120env) were localized predominantly in cell membranes; however, some amount of p50 was found in cell nuclei. Thin-section immunoelectron microscopy showed accumulation of viruslike particles undistinguished from immature HIV-1 virions in the culture medium of the cells infected with vC5. The similar particles containing gag and env proteins were produced into the culture medium when the cells were coinfected with vC5 and vE234L strains. The particles contained heterogeneous cellular RNA, but no virus-specific RNA as shown by Northern blot hybridization. Immunization of the rabbits with purified viruslike particles produced virus-specific antibodies against gag and env proteins. The titer of antibodies was significantly higher than after immunization with cell lysate or recombinant proteins purified from the infected cells. Highly immunogenic HIV-1-like particles containing gag and env proteins but no virus-specific RNA are good candidates for potential vaccine.

Alphavirus replication in cultured cells and infected animals is inhibited by antiproteinase agents.

The influence of different antiproteinase agents on alphavirus replication was examined. Sindbis virus multicycle replication in cultured cells was suppressed by N-tosyl-phenylalanine chloromethyl ketone (TPCK), an inhibitor of chymotrypsin-like proteinases, and by aprotinin, an inhibitor of a wide spectrum of proteinases. Antiviral activity of TPCK was also demonstrated in Sindbis virus-infected animals. Parenteral injections of TPCK in infected mice reduced virus titers in brain and blood. The possible mechanism(s) of antiviral action of the antiproteinase agents are discussed.

Inhibition of HIV-1 replication by newly developed adamantane-containing polyanionic agents.

Newly developed antiviral compounds consisting of an adamantane derivative chemically linked to a water-soluble polyanionic matrix were shown to inhibit HIV-1 infection in lymphoblastoid cells, HeLa CD4+ beta-galactosidase (MAGI) cells and macrophages. The effect of the compounds was recorded by measuring viral reverse transcriptase activity and p24 by ELISA in culture supernatant and by immunoblotting of cell lysates. In this paper we describe the data obtained with one of the most promising compounds, Amant. Amant was not toxic for the host cells at concentrations as high as 1 mg/ml. The inhibition of HIV-1 replication in MT-4 and MAGI cells was observed when Amant was added either before infection or with the virus (0 h of infection), and was expressed even when the compound added at 0 h was removed 1.5 h after infection. Its inhibitory concentration (IC50) against HIV-1 and HIV-2 replication was 2-6 and 93 microg/ml, respectively. The anti-HIV-1 effect of the compound was gradually decreased when it was added 1 and 2 h post infection, and no inhibition was observed when the compound was added 4 h after infection, suggesting that the compound as a membranotropic drug blocks an early step of replication. It completely prevented the transport of Gag proteins into the nuclei. Pretreatment of the virus with Amant did not reduce its infectious activity. The classical adamantane derivatives amantadine and rimantadine hydrochloride did not inhibit HIV replication.

Nucleotide and oligonucleotide derivatives as enzyme and nucleic acid targeted irreversible inhibitors. Biochemical aspects.

Sequence specific modification of nucleic acids with reactive oligonucleotide derivatives, complementary addressed modification, can provide an efficient approach for specific inactivation of certain cellular nucleic acids. In experiments with ascites tumor Krebs II cells and alkylating oligothymidylate derivatives it was found that alkylating oligonucleotide derivatives enter the living cell and modify complementary sequences in cellular nucleic acids with high efficiency. Complementary addressed modification of poly(A) sequences in cellular RNA with oligothymidylate derivatives was investigated in detail. The results of experiments on alkylation of cellular nucleic acids are consistent with complementary addressed modification of poly(A) sequences in cellular DNA. These results are supported by experiments on modification of chromatin DNA in which it was found that chromatin DNA interacts with oliogothymidylate derivatives more readily than the isolated double stranded DNA. It was found that alkylating oligonucleotide derivatives complementary to a sequence in immunoglobulin mRNA of MOPC 21 cells arrest the cellular immunoglobulin synthesis. Alkylating oligonucleotide derivatives complementary to RNAs of fowl plague virus inhibit virus multiplication in cell culture.

Influenza virus assembly and its defects.

The most abundant protein within the influenza virus particles is membrane protein (M protein) which forms an inner virus membrane under a lipid bilayer and plays the role of mediator during the process of assembly of a virus particle on plasma membranes. Ehrlich ascites tumor cells (EAT) when infected with influenza virus, strain WSN, produced virus-like particles containing greatly reduced amounts of M protein. Such particles were extremely fragile and easily lost hemagglutinins. The loss of this glycoprotein was accompanied by a decrease in infectious activity. SDS-PAGE analysis of RNA duplexes formed after hybridization of intracellular labeled mRNAs and unlabeled virion RNA showed that the mRNA for M protein was synthesized in EAT nearly in the same amounts as in productively infected chicken fibroblasts. Accordingly, M protein was readily revealed when the polypeptides of infected EAT were analyzed by SDS-PAGE. Thus, the reduced amount of M protein in virus particles was likely not due to the decrease in its synthesis but rather to its defective structure or to its defective transport and misintegration into plasma membranes of EAT.

Intracellular localization of recombinant vaccinia virus produced HIV antigens and their use for confirmation of HIV seropositivity.

Recombinant vaccinia strains vC5 and vE234L expressing the rp50 and rgp160/rgp120 recombinant proteins were used in immunoblot and immunofluorescence assays. No false reactions were found, although 30 sera giving false reactivity with gag or env encoded proteins in a commercial immunoblot assay were included into the test panel. We recommend the recombinant protein-based assay for confirmation and discrimination of HIV seropositivity.

Properties of influenzavirus nucleocapsids in nonpermissive cells.

The properties of fowl plague virus (Influenzavirus A) nucleocapsids isolated from the cytoplasm of infected Ehrlich ascites carcinoma cells and chick embryo cells were compared. Nucleocapsids isolated from both systems possessed similar polypeptides (P and NP) but differed in their biophysical characteristics. Nucleocapsids from ascites cells sedimented in velocity sucrose gradients slower (from 25 to 50 S) and the majority of them banded at higher density in CsCl gradients (rho 1.38 as compared to 1.34 g/ml) than nucleocapsids from chick embryo cells. In the electron microscope they appeared as thin threads 3--4 nm in diameter.

Characteristics of Sendai virus RNA transcriptive complex formed in the cytoplasm of infected ascites cells.

Virus-specific structures with sedimentation coefficients of 250-300, 200 and 150 S were isolated from the polysome fraction of Sendai virus-infected Ahrlich ascitic carcinoma cells treated with cycloheximide, at early stages of infection (1.5 to 2 hours after inoculation). All these 3 types of structure contained both parental and newly synthesized viral RNA. RNA extracted from these structures consisted of 2 components sedimenting in sucrose density gradients in the zones of 50-70 and 35-40 S. Both components contained parental and newly synthesized RNA and were partially resistant to ribonuclease. RNA extracted from rapidly sedimenting structures (250-300 S) contained mainly the 50-70 S component; RNA recovered from 200 S structures contained the 35-40 S component. By analogy with reported data, the isolated forms of RNA have been characterized as transcriptive intermediates.

Physicochemical properties of Sendai virus RNA. II. Effect of ionic strength on thermostability of RNA.

The dependence of the melting point (Tm) and width of the melting range (deltaTm) on the ionic strength and pH of the medium was investigated for the double-stranded RNA formed through self-hybridization during the isolation of RNA from Sendai virus. It was shown that Tm is a linear function of the logarithm of the sodium ion concentration in the range of concentrations from 10(-1) to 10(-4) M, with a slope of 11.5 degrees toward the abscissa for each order of magnitude. The width of the melting range increased slightly with a decrease in the ionic strength. A change in the pH of the solutions from 5 to 8 had almost no effect on the melting point or the width of the melting range. The degree of purification of the preparations of RNA and the presence of EDTA in the solutions affected the form of the dependence of the mp on the logarithm of the sodium ion concentration very strongly, especially in the region of low ionic strengths.

HIV-1 assembly and maturation.

HIV-1 particles have been studied by structural and chemical approaches, however, the processes of assembly, budding and maturation are just beginning to be characterized, and molecular details of these processes remain poorly defined. This brief review summarizes some recent findings on the final steps of the HIV-1 life cycle and touches upon some unanswered questions, particularly regarding the processes involved in virus maturation and infectivity.

Nucleoproteins of animal influenza viruses, in contrast to those of human strains, are not cleaved in infected cells.

We previously reported that nucleoproteins (NPs) of human influenza viruses are cleaved in infected cells and, as a result, two forms of NP, uncleaved (mol. wt. 56000) and cleaved (mol. wt. 53000) were accumulated late in infection. Here, we report that NPs of animal influenza viruses of non-human origin (isolated from pigs, equids , seals, whales, birds) exhibited proteolytic resistance in infected cells and did not undergo a change in mol. wt. in the course of infection. The resistance of the animal virus NPs to proteolytic cleavage was shown to be a virus-specific property and not the consequence of a low level of proteolysis in infected cells. Influenza A/H3N2 viruses isolated from swine in Hong Kong in 1976 were found to have a cleavable NP like that of 'human' viruses, supporting the hypothesis concerning the 'human' origin of these strains. The NP of human influenza virus (A/Aichi/2/68) adapted to an animal host (mouse) retained susceptibility to limited intracellular proteolysis. Thus, NP resistance to cleavage seems to be a stable viral characteristic enabling the NP56 ---- NP53 modification to be used as an indication of the origin of influenza viruses.

Unusual features of protein interaction in human immunodeficiency virus (HIV) virions.

The treatment of HIV-1 virions with ionic and nonionic detergents (NP 40, octylglucoside, Na deoxycholate) resulted in an effect unusual for enveloped viruses: instead of solubilization of glycoproteins, the core protein p24 was solubilized while envelope glycoproteins with other structural proteins were found in subviral particles. These data are consistent with a model of HIV structural organization in which glycoproteins are included in the matrix formed by the protein p 17 and suggest that p24 is neither involved in the matrix nor closely bound to any viral proteins.

Proteolytic activation of influenza WSN virus in cultured cells is performed by homologous plasma enzymes.

The effect of chick embryo allantoic fluid, porcine plasma or canine plasma on virus progeny was studied in cultured chicken, porcine and canine cells infected with influenza WSN virus. Cells incubated either without plasma or with heterologous plasma produced virions which had uncleaved haemagglutinin and low infectivity. Cells incubated with homologous plasma produced highly infectious virions with cleaved haemagglutinin. Little increase of progeny virus infectivity was observed in canine cell-porcine plasma and porcine cell-canine plasma host systems. The addition of protease inhibitors to culture containing homologous plasma, in particular epsilon-amino-n-caproic acid (an inhibitor of plasminogen activation), suppressed cleavage of haemagglutinin, and virions which had uncleaved haemagglutinin and low infectivity were produced by the cells. It therefore follows that haemagglutinin cleavage and activation of influenza WSN virus infectivity in cultured cells is most efficiently performed by homologous plasma proteolytic enzyme(s). The mechanism of selective plasma-mediated influenza virus proteolytic activation in homologous cells is discussed.