Translation of Sindbis virus mRNA: effects of sequences downstream of the initiating codon

One incentive for developing the alphavirus Sindbis virus as a vector for the expression of heterologous proteins is the very high level of viral structural proteins that accumulates in infected cells. Although replacement of the structural protein genes by a heterologous gene should lead to an equivalent accumulation of the heterologous protein, the Sindbis virus capsid protein is produced at a level 10- to 20-fold higher than that of any foreign protein. Chimeric mRNAs which contain the first 275 nucleotides of the Sindbis virus 26S mRNA fused to the lacZ gene are also translated at the higher level. The enhancing sequences, located downstream of the AUG codon that initiates translation of the capsid protein, have a predicted hairpin-like structure; deletions in this region destroy the activity. These sequences enhance translation in infected cells but have the opposite effect in uninfected cells. Furthermore, translation of this RNA in infected cells is suppressed by a second viral RNA lacking the hairpin-like structure, but translation of the latter RNA is not affected. We propose that the hairpin-like structure presents a barrier to the movement of the ribosomes during translation of mRNA. In infected cells, under conditions in which this mRNA is essentially the only RNA being translated, a slowdown in the transit of the ribosomes gives factors present at low concentrations a chance to bind to the translation complex and permits a high level of functional complexes to be formed. In uninfected cells and in infected cells translating two different viral subgenomic mRNAs, a pause in the movement of the ribosomes along the RNA is no longer an advantage, because the required factors are now usurped by other translation complexes.     

Inhibition of Sindbis virus replication by cyclopentenone prostaglandins: a cell-mediated event associated with heat-shock protein synthesis

Cyclopentenone prostaglandins (PGs) have been shown to inhibit the replication of several DNA and RNA viruses. Here we report on the effect of prostaglandin A1 (PGA1) on the multiplication of a positive strand RNA virus, Sindbis virus, in Vero cells under one-step multiplication conditions. PGA1 was found to inhibit Sindbis virus production dose-dependently, and virus yield was reduced by more than 90% at the concentration of 8 micrograms/ml, which was non-toxic to the cells and did not inhibit DNA, RNA or protein synthesis in Vero cells. The cyclopentenone prostaglandin delta 12-PGJ2 was also shown to be a potent inhibitor of Sindbis virus replication. Virus-induced reduction of [3H]uridine uptake by cells was partially prevented by PGA1 treatment, which also caused a 1 h delay in the peak of virus RNA synthesis. SDS-PAGE analysis of [35S]methionine-labeled proteins showed that PGA1 moderately inhibited the synthesis of the viral structural proteins E1, E2 and C, and induced the synthesis of a 72 kDa M(r) protein, identified as a heat-shock protein related to the HSP70 group, in both virus-infected and uninfected cells. Actinomycin D treatment completely prevented PGA1-antiviral activity, indicating that a cellular product is responsible for this action. PGA1-induced HSP70 is a good candidate for this role.     

Microviscosity of togavirus membranes studied by fluorescence depolarization: influence of envelope proteins and the host cell

The microviscosities of the hydrophobic regions of the membranes of intact Semliki forest and Sindbis viruses grown on BHK-21 cells, of liposomes derived from the extracted viral lipids, and of protease-treated virions were measured by fluorescence depolorization using the fluorescence probe 1, 6-diphenyl-1,3,5-hexatriene. The intact virus membranes were found to have a higher microviscosity than did virus-derived liposomes, indicating the viral envelope proteins contribute to microviscosity. However, protease-treated virus, devoid of protruding spikes but with residual lipophilic peptide tails, was found to have a microviscosity more similar to that of the intact virus than to that of protein-free liposomes. Sindbis virus grown in BHK-21 cells at 37 C had a much higher microviscosity than did Sindbis virus grown on Aedes albopicuts cells at 22 C. Sindbis virus grwon in A. albopictus and BHK-21 cells also gave higher microviscosity values than did the intact host cells. These data indicate that both the virion proteins and the cellular lipids selected during viral growth and maturation contribute to the increased microviscosity of togavirus membranes.     

Effect of some synandrogens and antiandrogens on the conversion of testosterone to dihydrotestosterone in the cultured rat ventral prostate

Whereas defective interfering particles of Sindbis virus are readily produced in BHK-21 cells or chicken embryo fibroblasts by the techniques of serial undiluted passage, similar methods failed to generate such particles in Aedes albopictus cell cultures. In addition, Sindbis virus stocks produced in BHK-21 cells or chicken embryo fibroblasts and which contained defective interfering particles, when tested in A. albopictus cells, failed (i) to interfere with the replication of standard Sindbis virus and (ii) to change the pattern of intracellular viral RNA synthesis from that produced by infection with standard Sindbis virus alone. We conclude that defective interfering particles of Sindbis virus generated in chicken or hamster cells are silent or inert in mosquito cells.     

The von Hippel-Lindau tumor suppressor gene. A rare and intriguing disease opening new insight into basic mechanisms of carcinogenesis

Sindbis virus is a positive strand RNA virus that has provided a valuable model for studying virus structure and replication. It is also being developed as a vector for the expression of heterologous proteins. Many studies with this virus are carried out in cultured BHK cells where infection is usually highly cytopathic and within 1 or 2 days after infection all of the cells are dead. Weiss et al. had established a persistently infected culture of BHK cells by infecting the cells with a virus preparation highly enriched in defective interfering (DI) particles and had isolated an attenuated virus, SIN-1 virus, from the culture [Weiss et al. (1980) J. Virol. 33, 463-474]. SIN-1 virus, free of DI particles, was able to establish a persistent infection in BHK cells. We initiated studies to determine what changes in the genome of the virus were responsible for this phenotype. We describe here the cDNA cloning and sequencing of the 5' terminus and the four nonstructural protein genes from SIN-1 virus. A single coding mutation in the nsP2 gene (a predicted change of Pro-726 --> Ser) produced a virus that was able to establish persistent infection in BHK cells. Additional mutations in the other genes were required to decrease the synthesis of viral RNA to a level similar to that found in cells infected with SIN-1 virus. Incorporation of the nsP2 mutation into a Sindbis virus expression vector led to a higher level of synthesis of the reporter protein, beta-galactosidase, than that obtained with the original Sindbis virus replicon.     

Defective interfering passages of Sindbis virus: nature of the defective virion RNA

Defective interfering particles of Sindbis virus contain 20S RNA identical to that found in BHK cells co-infected with standard and defective virions. We have characterized these RNAs by their oligonucleotide fingerprints. Most of the oligonucleotides were identical to those found in the mRNA (26S RNA) that codes for the virion structural proteins. Three oligonucleotides found in 20S RNA were absent from the 26S RNA pattern and may represent sequences from the 5' end of the virion RNA. Previous difficulties in describing the nature of the defective virion RNA were due to the aggregated state of the RNA. Nucleocapsids obtained from standard and defective virions were essentially the same size and had about the same density, suggesting that defective particles contain more than a single molecule of 20S RNA.     

The formation of intramolecular disulfide bridges is required for induction of the Sindbis virus mutant ts23 phenotype

The Sindbis virus envelope protein spike is a hetero-oligomeric complex composed of a trimer of glycoprotein E1-E2 heterodimers. Spike assembly is a multistep process which occurs in the endoplasmic reticulum (ER) and is required for the export of E1 from the ER. PE2 (precursor to E2), however, can transit through the secretory pathway and be expressed at the cell surface in the absence of E1. Although oligomer formation does not appear to be required for the export of PE2, there is evidence that defects in E1 folding can affect PE2 transit from the ER. Temperature-sensitive mutant ts23 of Sindbis virus contains two amino acid substitutions in E1, while PE2 and capsid protein have the wild-type sequence; however, at the nonpermissive temperature, both E1 and PE2 are retained within the ER and can be isolated in protein aggregates with the molecular chaperone GRP78-BiP. We previously demonstrated that the temperature sensitivity for ts23 was lost as oligomer formation took place at the permissive temperature, suggesting that temperature sensitivity is initiated early in the process of viral spike assembly (M. Carleton and D. T. Brown, J. Virol. 70:952-959, 1996). Experiments described herein investigated the defects in envelope protein maturation that occur in ts23-infected cells and which result in retention of both envelope proteins in the ER. The data demonstrate that in ts23-infected cells incubated at the nonpermissive temperature, E1 folding is disrupted early after synthesis, resulting in the rapid incorporation of both E1 and PE2 into disulfide-stabilized aggregates. Furthermore, the aberrant E1 conformation which is responsible for induction of the ts phenotype requires the formation of intramolecular disulfide bridges formed prior to E1 association with PE2 and the completion of E1 folding.     

Segmented genome and nucleocapsid of La Crosse virus

La Crosse (LAC) virions purified by velocity and equilibrium gradient centrifugation contained three single-stranded RNA species. The three segments had sedimentation coefficients of 31S, 25S, and 12S by sodium dodecyl sulfate-sucrose gradient centrifugation. By comparison with other viral and cellular RNA species, the LAC viral RNAs had molecular weights of 2.9 x 10(6), 1.8 x 10(6), and 0.4 x 10(6). Phenol-sodium dodecyl sulfate-extracted LAC virion RNA was not infectious for BHK-21 cell cultures under conditions in which Sindbis viral RNA was infectious. Treatment of LAC virus with the nonionic detergent Triton X-100 and salt released three nucleocapsid structures, each containing one species of virion RNA. The nucleocapsids had sedimenation coefficients of 115S, 90S, and 65S. Negative-contrast electron microscopy of the nucleocapsids indicated that they were convoluted, supercoiled, and apparently circular. They had a mean diameter of 10 to 12 nm and modal lengths of 200, 510, and 700 nm (some were even longer). By chemical and enzymatic analysis of purified viral RNA, one type of 5' nucleotide (pppAp) present in the proportion of one per RNA segment was identified. After periodate oxidation, each virion RNA species was labeled by reduction with [3H]sodium borohydride. Taken together, these results suggest that although the nucleocapsids appear as closed loops, the viral RNA has free 5' and 3' ends and is, therefore, not circular.     

Production of avian leukosis virus particles in mammalian cells can be mediated by the interaction of the human immunodeficiency virus protein Rev and the Rev-responsive element

In human immunodeficiency virus type 1-infected cells, the efficient expression of viral proteins from unspliced and singly spliced RNAs is dependent on two factors: the presence in the cell of the viral protein Rev and the presence in the viral RNA of the Rev-responsive element (RRE). We show here that the HIV-1 Rev/RRE system can increase the expression of avian leukosis virus (ALV) structural proteins in mammalian cells (D-17 canine osteosarcoma) and promote the release of mature ALV virions from these cells. In this system, the Rev/RRE interaction appears to facilitate the export of full-length unspliced ALV RNA from the nucleus to the cytoplasm, allowing increased production of the ALV structural proteins. Gag protein is produced in the cytoplasm of the ALV-transfected cells even in the absence of a Rev/RRE interaction. However, a functional Rev/RRE interaction increases the amount of Gag present intracellularly and, more strikingly, results in the release of mature ALV particles into the supernatant. RCAS virus containing an RRE is replication-competent in chicken embryo fibroblasts; however, we have been unable to determine whether the particles produced in D-17 cells are as infectious as the particles produced in chicken embryo fibroblasts.     

An in-frame insertion into the Sindbis virus 6K gene leads to defective proteolytic processing of the virus glycoproteins, a trans-dominant negative inhibition of normal virus formation, and interference in virus shut off of host-cell protein synthesis

Encoded in the genomes of all alphaviruses is a hydrophobic polypeptide of 55 amino acids, which is post-translationally modified with 4 covalently bound palmitic acids. This protein, noted as 6K, associates with membranes and is transported along with the two virus transmembranal glycoproteins to the site of virus assembly at the infected cell's plasma membrane. Previous studies showed that mutations in the 6K protein led to the slow release of aberrant, multi-cored infectious virions. In this paper, we report that an in-frame insertion of 45 nucleotides into an internal site of the 6K gene of Sindbis virus produced single-cored infectious particles at about 5% the yield of wild-type virus when the mutant was grown on avian, mammalian, and insect cells. Although the 15 amino acids were inserted at position 29 of the 55-amino-acid 6K protein, the mutation interfered with the cotranslational proteolytic processing that cleaves the 6K at its amino terminus from the Sindbis virus p62 glycoprotein and at its carboxyl terminus from the E1 glycoprotein. As a result, the amounts of normal p62 and E1 proteins were only half that made in cells infected with wild-type virus. In addition, the post-translational proteolytic conversion of p62 to E2 occurred at 10% the rate of wild-type proteins and the extensive fatty acylation normally detected on wild-type 6K protein was not found on the altered 6K protein. None of the mutated 6K protein was detected in virions, which were morphologically indistinguishable from wild-type virus. The mutant 6K virions also were similar to wild type in their rate of attachment, uncoating, and formation of an early nonstructural virus protein in avian cells. When compared with the wild-type virus, 6K29-infected cells exhibited a decreased rate of host-cell protein synthesis shut off. However, the rates of virus capsid synthesis were the same, indicating that capsid protein, per se, is not involved in shut off of host-cell protein synthesis. In complementation studies, this mutant exhibited a trans-dominant phenotype. These data provide clues about the topology of 6K protein in the membrane and its function in virus maturation.     

Reduced infectivity of human immunodeficiency virus type 1 produced in the presence of a truncated Gag protein containing p7 gag and p6 gag

The C-terminal portion of human immunodeficiency virus type 1 p55gag protein, p15gag, contains two functional proteins; p6gag which is required for incorporation of Vpr into the virion, and p7gag which binds to viral RNA and is necessary for packaging of genomic RNA into virions. p7gag protein overexpressed in trans may compete with wild type p55gag for binding to genomic viral RNA, thereby inhibiting incorporation of RNA into the virions. To investigate if overexpression of the C-terminal portion of p55gag could interfere with generation of infectious virus, a plasmid producing a protein consisting of p2gag, p7gag and p6gag, termed p15gag*, was generated and cotransfected with an infectious proviral human immunodeficiency virus type 1 clone. Cells overexpressing p15gag* in trans produced approximately 40 fold less infectious virus than cells lacking exogenous p15gag*. These results demonstrated that expression of the C-terminal portion of p55gag efficiently reduced virus infectivity.     

Tmp21 and p24A, two type I proteins enriched in pancreatic microsomal membranes, are members of a protein family involved in vesicular trafficking

We report here on the isolation, cloning, and expression of two Mr 21,000 proteins from rat pancreatic acinar cells, the rat-Tmp21 (transmembrane protein, Mr 21,000) and the rat-p24A. Both proteins are transmembrane proteins with type I topology and share weak but significant homology to one another (23% identity). We further show the cloning and characterization of the human homologs, hum-Tmp21, which is expressed in two variants (Tmp21-I and Tmp21-II), and hum-p24A. Tmp21 proteins and p24A have highly conserved COOH-terminal tails, which contain motifs related to the endoplasmic reticulum retention and retrieval consensus sequence KKXX. The rat-p24 sequence is identical to the hamster CHOp24, a recently characterized component of coatomer-coated transport vesicles, which defines a family of proteins (called the p24 family) proposed to be involved in vesicular transport processes (Stamnes, M. A., Craighead, M. W., Hoe, M. H., Lampen, N., Geromanos, S., Tempst, P., and Rothman, J. E.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8011-8015). Sequence alignment and structural features identify the Tmp21 protein as a new member of this p24 family. Northern analysis of various tissues indicates that the Tmp21 proteins and the p24A protein are ubiquitously expressed. The integral membrane components Tmp21 and p24A are localized in microsomal membranes, zymogen granule membranes, and the plasma membrane and are absent from the cytosol. Both p24A and Tmp21 show weak homology to the yeast protein Emp24p, which recently has been shown to be involved in secretory protein transport from the endoplasmic reticulum to the Golgi apparatus. This leads us to conclude that the receptor-like Tmp21 and p24A are involved in vesicular targeting and protein transport.     

Evaluation of serological diagnosis of Borna disease virus infection using recombinant proteins in experimentally infected rats

We produced two recombinant Borna disease virus (BDV) proteins, p40 and p24, by using a baculovirus vector as a diagnostic antigen. Antigenicities of these recombinant proteins were evaluated by immune rabbit sera. Recombinant p40 was a more sensitive antigen than p24 for the detection of antibodies in infected rats. Rats inoculated with BDV within 24 hr after birth showed higher detection rates of viral RNA and viral proteins from the brain than rats inoculated at 4 weeks-old. Depending on the age of infection and the time postinfection, the detection of BDV RNA, protein, or anti-BDV antibody did not always correlate in individuals. We suggest both serological and molecular biological methods are needed in the diagnosis of BDV infection.     

Characterization of the ion channels formed by poliovirus in planar lipid membranes

The steps in poliovirus infection leading to viral entry and uncoating are not well understood. Current evidence suggests that the virus first binds to a plasma membrane-bound receptor present in viable cells, leading to a conformational rearrangement of the viral proteins such that the virus crosses the membrane and releases the genomic RNA. The studies described in this report were undertaken to determine if poliovirus (160S) as well as one of the subviral particles (135S) could interact with membranes lacking poliovirus receptors in an effort to begin to understand the process of uncoating of the virus. We report that both forms of viral particles, 160S and 135S, interact with lipid membranes and induce the formation of ion-permeable channels in a manner that does not require acid pH. The channels induced by the viral particles 160S have a voltage-dependent conductance which depends on the ionic composition of the medium. Our findings raise the possibility that viral entry into cells may be mediated by direct interaction of viral surface proteins with membrane lipids.     

Characterization of a temperature-sensitive, hexon transport mutant of type 5 adenovirus

Infection of KB cells at 39.5 degrees C with H5ts147, a temperature-sensitive (ts) mutant of type 5 adenovirus, resulted in the cytoplasmic accumulation of hexon antigen; all other virion proteins measured, however, were normally transported into the nucleus. Immunofluorescence techniques were used to study the intracellular location of viral proteins. Genetic studies revealed that H5ts147 was the single member of a nonoverlapping complementation group and occupied a unique locus on the adenovirus genetic map, distinct from mutants that failed to produce immunologically reactive hexons at 39.5 degrees C ("hexon-minus" mutants). Sedimentation studies of extracts of H5ts147-infected cells cultured and labeled at 39.5 degrees C revealed the production of 12S hexon capsomers (the native, trimeric structures), which were immunoprecipitable to the same extent as hexons synthesized in wild type (WT)-infected cells. In contrast, only 3.4S polypeptide chains were found in extracts of cells infected with the class of mutants unable to produce immunologically reactive hexon protein at 39.5 degrees C. Hexons synthesized in H5ts147-infected cells at 39.5 degrees C were capable of being assembled into virions, to the same extent as hexons synthesized in WT-infected cells, when the temperature was shifted down to the permissive temperature, 32 degrees C. Infectious virus production was initiated within 2 to 6 h after shift-down to 32 degrees C; de novo protein synthesis was required to allow this increase in viral titer. If ts147-infected cells were shifted up to 39.5 degrees C late in the viral multiplication cycle, viral production was arrested within 1 to 2 h. The kinetics of shutoff was similar to that of a WT-infected culture treated with cycloheximide at the time of shift-up. The P-VI nonvirion polypeptide, the precursor to virion protein VI, was unstable at 39.5 degrees C, whereas the hexon polypeptide was not degraded during the chase. It appears that there is a structural requirement for the transport of hexons into the nucleus more stringent than the acquisition of immunological reactivity and folding into the 12S form.     

Suppression of the metastatic phenotype of a mouse skin carcinoma cell line independent of E-cadherin expression and correlated with reduced Ha-ras oncogene products

The HaCa4 cell line, derived from a mouse skin carcinoma induced by Harvey murine sarcoma virus, is highly tumorigenic when injected into nude mice and produces multiple metastases in the lungs. HaCa4 cells express high levels of viral Ha-ras oncogene products, anomalously synthesize the embryonic/simple epithelial keratin K8, and have lost the expression of the cell-cell adhesion receptor E-cadherin (E-CD). E-CD(+) cell clones (E62 and E24), obtained by transfection of an exogenous E-CD cDNA into HaCa4 cells, had a decreased ability to migrate through type IV collagen matrices. However, the E-CD (+) E62 clone remained as metastatic as the parental cell line, whereas the E24 clone, which does not take up the exogenous cDNA but spontaneously switches on the endogenous E-CD gene, suppressed the metastatic phenotype although it maintained its tumorigenicity. E24 cells had fivefold to sixfold lower levels of viral Ha-ras mRNA and p21 protein than the other cell lines. In addition, they did not synthesize K8 but rather switched on keratin K19. The comparison of E-CD proteins synthesized by E62 and E24 cell lines revealed no structural or functional differences because both localized at cell-cell contacts and associated with alpha-catenin, beta-catenin, and plakoglobin. Furthermore, E-CD was still expressed in metastatic lung nodules produced by E62 cells. These results suggest that suppression of the metastatic phenotype in E24 cells occurs independently of E-CD expression and correlates with decreased levels of the oncogenic ras p21 protein.