The furin protease cleavage recognition sequence of Sindbis virus PE2 can mediate virion attachment to cell surface heparan sulfate.
(17/923)
Cell culture-adapted Sindbis virus strains attach to heparan sulfate (HS) receptors during infection of cultured cells (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). At least three E2 glycoprotein mutations (E2 Arg 1, E2 Lys 70, and E2 Arg 114) can independently confer HS attachment in the background of the consensus sequence Sindbis virus (TR339). In the studies reported here, we have investigated the mechanism by which the E2 Arg 1 mutation confers HS-dependent binding. Substitution of Arg for Ser at E2 1 resulted in a significant reduction in the efficiency of PE2 cleavage, yielding virus particles containing a mixture of PE2 and mature E2. Presence of PE2 was associated with an increase in HS-dependent attachment to cells and efficient attachment to heparin-agarose beads, presumably because the furin recognition site for PE2 cleavage also represents a candidate HS binding sequence. A comparison of mutants with partially or completely inhibited PE2 cleavage demonstrated that efficiency of cell binding was correlated with the amount of PE2 in virus particles. Viruses rendered cleavage defective due to deletions of portions or all of the furin cleavage sequence attached very poorly to cells, indicating that an intact furin cleavage sequence was specifically required for PE2-mediated attachment to cells. In contrast, a virus containing a partial deletion was capable of efficient binding to heparin-agarose beads, suggesting different requirements for heparin bead and cell surface HS binding. Furthermore, virus produced in C6/36 mosquito cells, which cleave PE2 more efficiently than BHK cells, exhibited a reduction in cell attachment efficiency correlated with reduced content of PE2 in particles. Taken together, these results strongly argue that the XBXBBX (B, basic; X, hydrophobic) furin protease recognition sequence of PE2 can mediate the binding of PE2-containing Sindbis viruses to HS. This sequence is very similar to an XBBXBX heparin-HS interaction consensus sequence. The attachment of furin protease cleavage sequences to HS may have relevance to other viruses whose attachment proteins are cleaved during maturation at positively charged recognition sequences. (+info)
Selective irreversible inactivation of replicating mengovirus by nucleoside analogues: a new form of viral interference.
(18/923)
We describe the selective irreversible inhibition of mengovirus growth in cultured cells by a combination of two pyrrolopyrimidine nucleoside analogues, 5-bromotubercidin (BrTu) and tubercidin (Tu). At a concentration of 5 microgram/ml, BrTu reversibly blocked the synthesis of cellular mRNA and rRNA but did not inhibit either mengovirus RNA synthesis or multiplication. BrTu is a potent inhibitor of adenosine kinase, and low concentrations of BrTu (e.g., 0.5 microgram/ml), which did not by themselves inhibit cell growth, blocked phosphorylation of Tu and thus protected uninfected cells against irreversible cytotoxicity resulting from Tu incorporation into nucleic acids. In contrast, in mengovirus-infected cells, BrTu did not completely inhibit Tu incorporation into mengovirus RNA, allowing the formation of Tu-containing functionally defective polynucleotides that aborted the virus development cycle. This increased incorporation of Tu coupled to mengovirus infection could be attributed either to a reduction in the inhibitory action of BrTu and/or its nucleotide derivatives at the level of nucleoside and nucleotide kinases and/or, perhaps, to an effect upon the nucleoside transport system. The virus life cycle in nucleoside-treated cells progressed to the point of synthesis of negative strands and probably to the production of a few defective new positive strands. Irreversible virus growth arrest was achieved if the nucleoside mixture of BrTu (0.5 to 10 microgram/ml) and Tu (1 to 20 microgram/ml) was added no later than 30 min after virus infection and maintained for periods of 2 to 8 h. The cultures thus "cured" of mengovirus infection could be maintained and transferred for several weeks, during which they neither produced detectable virus nor showed a visible cytopathic effect; however, the infected and cured cells themselves, while metabolically viable, were permanently impaired in RNA synthesis and unable to divide. Although completely resistant to superinfecting picornaviruses, they retained the ability to support the growth of several other viruses (vaccinia virus, reovirus, and vesicular stomatitis virus), showing that cured cells had, in general, retained the metabolic and structural machinery needed for virus production. The resistance of cured cells to superinfection with picornaviruses seemed attributable neither to interferon action nor to destruction or blockade of virus receptors but more likely to the consumption of some host factor(s) involved in the expression of early viral functions during the original infection. (+info)
Low-pH-dependent fusion of Sindbis virus with receptor-free cholesterol- and sphingolipid-containing liposomes.
(19/923)
There is controversy as to whether the cell entry mechanism of Sindbis virus (SIN) involves direct fusion of the viral envelope with the plasma membrane at neutral pH or uptake by receptor-mediated endocytosis and subsequent low-pH-induced fusion from within acidic endosomes. Here, we studied the membrane fusion activity of SIN in a liposomal model system. Fusion was followed fluorometrically by monitoring the dilution of pyrene-labeled lipids from biosynthetically labeled virus into unlabeled liposomes or from labeled liposomes into unlabeled virus. Fusion was also assessed on the basis of degradation of the viral core protein by trypsin encapsulated in the liposomes. SIN fused efficiently with receptor-free liposomes, consisting of phospholipids and cholesterol, indicating that receptor interaction is not a mechanistic requirement for fusion of the virus. Fusion was optimal at pH 5.0, with a threshold at pH 6.0, and undetectable at neutral pH, supporting a cell entry mechanism of SIN involving fusion from within acidic endosomes. Under optimal conditions, 60 to 85% of the virus fused, depending on the assay used, corresponding to all of the virus bound to the liposomes as assessed in a direct binding assay. Preincubation of the virus alone at pH 5.0 resulted in a rapid loss of fusion capacity. Fusion of SIN required the presence of both cholesterol and sphingolipid in the target liposomes, cholesterol being primarily involved in low-pH-induced virus-liposome binding and the sphingolipid catalyzing the fusion process itself. Under low-pH conditions, the E2/E1 heterodimeric envelope glycoprotein of the virus dissociated, with formation of a trypsin-resistant E1 homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the E1 trimer represents the fusion-active conformation of the viral spike. (+info)
Comparison of the protective efficacy of naked DNA, DNA-based Sindbis replicon, and packaged Sindbis replicon vectors expressing Hantavirus structural genes in hamsters.
(20/923)
Seoul virus (SEOV) is a member of the Hantavirus genus (family Bunyaviridae) and an etiological agent of hemorrhagic fever with renal syndrome. The medium (M) and small (S) gene segments of SEOV encode the viral envelope glycoproteins and nucleocapsid protein, respectively. We compared the immunogenicity and protective efficacy of naked DNA (pWRG7077), DNA-based Sindbis replicon (pSIN2.5), and packaged Sindbis replicon vectors (pSINrep5), containing either the M or S gene segment of SEOV in Syrian hamsters. All of the vectors elicited an anti-SEOV immune response to the expressed SEOV gene products. Vaccinated hamsters were challenged with SEOV and monitored for evidence of infection. Protection from infection was strongly associated with M-gene vaccination. A small number of S-gene-vaccinated animals also were protected. Hamsters vaccinated with the pWRG7077 vector expressing the M gene demonstrated the most consistent protection from SEOV infection and also were protected from heterologous hantavirus (Hantaan virus) infection. (+info)
TNFalpha, interferon, and stress response induction as a function of age-related susceptibility to fatal Sindbis virus infection of mice.
(21/923)
The age-related acquisition of resistance to fatal Sindbis virus infection was examined using a molecularly cloned laboratory strain of the AR339 isolate designated TRSB. TRSB caused 100% mortality in mice up to 5 days of age. Resistance to fatal infection developed abruptly between 5 and 9 days of age. Lethal Sindbis virus infection of mice inoculated at 4 days of age was characterized by high levels of virus replication, induction of high levels of interferon-alpha/beta and TNF-alpha and severe thymic involution indicative of a systemic stress response. These changes correlated with predominantly noninflammatory lesions. In contrast, TRSB infection of older mice was characterized by survival, more limited virus replication, reduced cytokine induction, and the development of inflammatory responses leading to encephalitis, myositis, and myocarditis. Previous studies utilized infections of neonatal mice with TRSB and an attenuated mutant of TRSB to compare fatal and nonfatal Sindbis infection (Trgovcich et al., 1996. Virology 224, 73-83). The experiments reported here utilize mouse age at the time of infection to create conditions for examination of fatal and nonfatal TRSB infections. Both experiments suggest that fatal infection is associated with a shock-like syndrome and little or no inflammatory pathology, while survival is correlated with greatly reduced cytokine levels and inflammatory lesions. (+info)
An amino acid change in the exodomain of the E2 protein of Sindbis virus, which impairs the release of virus from chicken cells but not from mosquito cells.
(22/923)
In order to obtain a mutant of Sindbis virus (SV) with a low methionine-resistant (LMR) phenotype, i.e., able to replicate in methionine-deprived Aedes albopictus mosquito cells, standard SV (SV(STD)) was passaged 17 times in mosquito cells maintained in a low methionine medium and then plaque-purified, also in mosquito cells. Although the virus obtained by this procedure, SV(LM17), did have the desired LMR phenotype, it also appeared to have acquired a host-range phenotype. We have now characterized the host-range phenotype of SV(LM17) in greater detail. In yield assays, the titer of SV(LM17) produced by chick embryo fibroblasts (CEF) was 100- to 1000-fold lower than that from mosquito cells. SV(STD), in contrast, produced a similar titer of virus from the two cell types. On the other hand, when SV(LM17) was assayed directly by plaque formation on CEF and on mosquito cell monolayers, no host restriction in CEF was observed. When CEF were infected with SV(LM17), viral proteins were synthesized normally, pE2 was processed to E2, and E2 was demonstrated by the fluorescent antibody method to reach the cell surface. However, electron microscopy of SV(LM17)-infected cells revealed an absence of extracellular virions and of budding particles; also, nucleocapsids were not aligned beneath the plasma membrane. By sequence determination and by site-directed mutagenesis, it was determined that the host restriction of SV(LM17) was due to a change from Ala to Val at position 251 of the E2 protein. Substitution of Gly or Leu at this position also resulted in the same host range phenotype. (+info)
Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA.
(23/923)
A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3'2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5' end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo. (+info)
Efficient trans-complementation of the flavivirus kunjin NS5 protein but not of the NS1 protein requires its coexpression with other components of the viral replicase.
(24/923)
Successful trans-complementation of the defective Kunjin virus (KUN) RNA FLdGDD with a deletion of the RNA polymerase motif GDD in the NS5 gene by using a BHK cell line, repBHK, that continuously produced a functionally active KUN replication complex (RC) from replicon RNA was recently reported (A. A. Khromykh, M. T. Kenney, and E. G. Westaway, J. Virol. 72:7270-7279, 1998). In order to identify whether this complementation of FLdGDD RNA was provided by the wild-type NS5 protein alone or with the help of other nonstructural (NS) proteins also expressed in repBHK cells, we generated BHK cell lines stably producing the individual NS5 protein (SRns5BHK) or the NS1-NS5 polyprotein (SRns1-5BHK) by using a heterologous expression vector based on a modified noncytopathic Sindbis replicon. Western blot analysis with anti-NS5 antibodies showed that the level of production of NS5 was significantly higher in SRns5BHK cells than in SRns1-5BHK cells. Despite the higher level of expressed NS5, trans-complementation of FLdGDD RNA was much less efficient in SRns5BHK cells than in SRns1-5BHK cells and produced at least 100-fold less of the secreted complemented virus. In contrast, efficient complementation of KUN RNA with lethal cysteine-to-alanine mutations in the NS1 gene was achieved both in BHK cells producing the individual KUN NS1 protein from the Sindbis replicon vector and in repBHK cells, with both cell lines expressing similar amounts of NS1 protein. These results clearly demonstrate that flavivirus NS5 coexpressed with other components of the viral replicase possesses much higher functional (trans-complementing) activity than individually expressed NS5 and that efficient trans-complementation of mutated flavivirus NS1 and NS5 proteins occurs by different mechanisms. The results are interpreted and discussed in relation to our proposed model of formation of the flavivirus RC largely based on previous ultrastructural and biochemical analyses of KUN replication. (+info)