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Mosquitoes are hematophagous insects that carry-on and transmit many human viruses. However, little information is available regarding the common mechanisms underlying the infection of mosquitoes by these viruses. In this study, we reveal that the hematophagous nature of mosquitoes contributes to arboviral infection after a blood meal, which suppresses antiviral innate immunity by activating the GABAergic pathway. dsRNA-mediated interruption of the GABA signaling and blockage of the GABA A receptor by the specific inhibitors both significantly impaired arbovirus replication. Consistently, inoculation of GABA enhanced arboviral infection, indicating that GABA signaling facilitates the arboviral infection of mosquitoes. The ingestion of blood by mosquitoes resulted in robust GABA production from glutamic acid derived from blood protein digestion. The oral introduction of glutamic acid increased virus acquisition by mosquitoes via activation of the GABAergic system. Our study reveals that blood meals enhance arbovirus replication in mosquitoes through activation of the GABAergic system.

Background: Semliki Forest virus (SFV) has a positive strand RNA genome and infects different cells of vertebrates and invertebrates. The 5' two-thirds of the genome encodes non-structural proteins that are required for virus replication and synthesis of subgenomic (SG) mRNA for structural proteins. SG-mRNA is generated by internal initiation at the SG-promoter that is located at the complementary minus-strand template. Different types of expression systems including replication-competent vectors, which represent alphavirus genomes with inserted expression units, have been developed. The replication-competent vectors represent useful tools for studying alphaviruses and have potential therapeutic applications. In both cases, the properties of the vector, such as its genetic stability and expression level of the protein of interest, are important., Results: We analysed 14 candidates of replication-competent vectors based on the genome of an SFV4 isolate that contained a duplicated SG promoter or an internal ribosomal entry site (IRES)-element controlled marker gene. It was found that the IRES elements and the minimal -21 to +5 SG promoter were non-functional in the context of these vectors. The efficient SG promoters contained at least 26 residues upstream of the start site of SG mRNA. The insertion site of the SG promoter and its length affected the genetic stability of the vectors, which was always higher when the SG promoter was inserted downstream of the coding region for structural proteins. The stability also depended on the conditions used for vector propagation. A procedure based on the in vitro transcription of ligation products was used for generation of replication-competent vector-based expression libraries that contained hundreds of thousands of different genomes, and maintained genetic diversity and the ability to express inserted genes over five passages in cell culture., Conclusion: The properties of replication-competent vectors of alphaviruses depend on the details of their construction. In the case of SFV4, such vectors should contain the SG promoter with structural characteristics for this isolate. The main factor for instability of SFV4-based replication-competent vectors was the deletion of genes of interest, since the resulting shorter genomes had a growth advantage over the original vector.

Semliki Forest virus RNA replication takes place in association with specific cytoplasmic vacuoles, derived from the endosomal apparatus. Of the four virus-encoded replicase proteins, nsP1 serves as the membrane anchor of the replication complex. An amphipathic peptide segment, G245STLYTESRKLLRSWHLPSV264, has been implicated in the membrane binding of nsP1. nsP1 variants with changes within the peptide were studied after protein expression and in the context of virus infection. Proteins with mutations R253E and W259A accumulated in the cytoplasm and were very poorly palmitoylated. The same mutations also drastically affected the localization of the precursor polyprotein P123, and they were lethal when introduced into the virus genome. Mutations R253A and L255A+L256A partially changed the localization of nsP1, and the respective viruses acquired compensatory changes. L255A+L256A only yielded virus encoding L255A+L256V, indicating the importance of a hydrophobic residue in the central 256 position. When fused to green fluorescent protein, the peptide was required in at least two tandem copies to effect a change in localization, but even then the fusion protein was associated with membranes in a nonspecific manner. Thus, the amphipathic peptide is a crucial element for the membrane association of nsP1 and the replication complex. It provides essential affinity for membranes, and other regions of nsP1 also appear to contribute to the localization of the protein.

Alphavirus nonstructural proteins are translated as a polyprotein that is ultimately cleaved into four mature proteins called nsP1, nsP2, nsP3, and nsP4 from their order in the polyprotein. The role of this nonstructural polyprotein, of cleavage intermediates, and of mature proteins in synthesis of Semliki Forest virus (SFV) RNA has been studied using mutants unable to cleave one or more of the sites in the nonstructural polyprotein or that had the arginine sense codon between nsP3 and nsP4 changed to an opal termination codon. The results were compared with those obtained for Sindbis virus (SINV), which has a naturally occurring opal codon between nsP2 and nsP3. We found that (1) an active nonstructural protease in nsP2 is required for RNA synthesis. This protease is responsible for all three cleavages in the nonstructural polyprotein. (2) Cleavage between nsP3 and nsP4 (the viral RNA polymerase) is required for RNA synthesis by SFV. (3) SFV mutants that are able to produce only polyprotein P123 and nsP4 synthesize minus-strand RNA early after infection as efficiently as SF wild type but are defective in the synthesis of plus-strand RNA. The presence of sense or opal following nsP3 did not affect this result. At 30 degrees C, they give rise to low yields of virus after a delay, but at 39 degrees C, they are nonviable. (4) SFV mutants that produce nsP1, P23, nsP4, as well as the precursor P123 are viable but produce an order of magnitude less virus than wild type at 30 degrees C and two orders of magnitude less virus at 39 degrees C. The ratio of subgenomic mRNA to genomic RNA is much reduced in these mutants relative to the parental viruses. (5) At 30 degrees C, the variants containing an opal codon grow as well as or slightly better than the corresponding virus with a sense codon. At 39 degrees C, however, the opal variants produce significantly more virus. These results support the conclusion that SFV and SINV, and by extension all alphaviruses, regulate their RNA synthesis in the same fashion after infection. P123 and nsP4 form a minus-strand replicase that synthesizes plus-strand RNA only inefficiently, especially at the higher temperatures found in mammals and birds. A replicase containing nsP1, P23, and nsP4 can make both plus and minus strands, but prefers the promoter for genomic plus sense RNA to that for subgenomic mRNA. The fully cleaved replicase can make only plus-strand RNA, and prefers the promoter for subgenomic mRNA to that for genomic RNA. Alphaviruses alternate between infection of hematophagous arthropods and higher vertebrates. Although the infection of higher vertebrates is acute and often accompanied by disease, continuing transmission of the virus in nature requires that infection of arthropods be persistent and relatively asymptomatic. We propose that this mechanism for control of RNA synthesis evolved to moderate the pathogenicity of the viruses in their arthropod hosts.

The Semliki Forest virus directs the synthesis of three virus-specific transmembrane proteins p62, 6K, and E1, which all are made in equimolar amounts from a polyprotein precursor molecule. The p62 and E1 spike proteins form heterodimeric complexes in the endoplasmic reticulum before being transported to the cell surface where virus budding occurs. In this study we show that the 6K protein becomes associated to the p62E1 complex in the endoplasmic reticulum and transported with the complex to the cell surface. During virus budding, E1 and p62 (which has matured into the E2 protein) are incorporated into new virions whereas the 6K is mostly excluded. Virus particles released from infected BHK cells contain only about 3% of 6K in their membrane as compared to the spike protein content. The relevance of these findings for the mechanism of SFV assembly is discussed.

Neurotropic RNA budding viruses such as Semliki Forest virus (SFV), influenza and measles were each grown in identical mouse brain cell cultures. Positive immunoelectron microscopical labelling with gold was seen in the envelope of these viruses using an anti-SFV derived glycolipid monoclonal antibody (MAb), 373 shown to be directed chiefly against galactocerebroside. The results indicate that each enveloped virus grown from the same cell type contains the same glycolipid in its envelope. The presence of common glycolipids derived from the host cell in the envelopes of various enveloped budding viruses may play a significant role in the pathogenesis of virus induced, immune mediated CNS autoimmunity and demyelination, particularly in multiple sclerosis (MS).

Several animal viruses are known to contain significant amounts of polyamines but so far the function of these viral components is poorly understood. In this study the role of polyamines in the replication of two different types of viruses, herpes simplex virus type 2 and Semliki Forest virus (SFV) has been investigated. Purified SFV was found to contain fairly small amounts of polyamines, sufficient to neutralize only about 3% of viral nucleic acid phosphate, i.e., 1/20 of that found in herpes simplex virus. The production of both viruses was, however, markedly inhibited in BHK21 cells depleted of polyamines by treatment with alpha-difluoromethylornithine, a specific inhibitor of polyamine synthesis. This inhibition was reversed by putrescine, spermidine and spermine, and at least partly reversed by several other diamines and polyamine homologs. The activity of viral RNA polymerase induced by SFV infection was markedly reduced in polyamine-depleted cells but increased rapidly after addition of spermidine to the culture medium. It appears that the inhibition of virus production in polyamine-depleted cells is due in part to malfunction of protein synthetic machinery of the host cell. The possibility that other steps in virus synthesis and assembly are affected by polyamine deficiency is currently being investigated.

A complex synthesizing Semliki Forest virus (SFV)-specific RNAs was purified from infected HeLa cells. During purification, the RNA-synthesizing complex was monitored by the presence of RNA chains synthesized during a 1 min pulse in vivo and the ability to synthesize 42S and 26S RNAs in vitro. Finally, the protein composition of the replication complex was analysed. Thirty to 40% of the pulse-labelled RNAs and 10 to 25% of the polymerase activity present in the postnuclear supernatant were recovered in smooth membranes. At this stage of purification single stranded 42S and 26S RNA were synthesized and released from the replication complex in vitro. After treatment of the smooth membrane fraction with Triton X-100 the replication complex was solubilized. When analysed by sucrose gradient centrifugation, the solubilized replication complex distributed heterogeneously. It had reduced RNA polymerase activity, but was still able to synthesize both 42S and 26S nascent RNA chains which were not released from RIs and RFs. The non-structural protein ns70 was the major virus-specified component associated with the replication complex.

Cordycepin (3'-deoxyadenosine), at a concentration of 20 microgram/ml, has a marked effect on Semliki Forest virus multiplication. The appearance of plaque forming units is delayed by about 2 hours and the yield greatly reduced. The incorporation of [3H] uridine into intracellular viral RNAs reaches less than 50 per cent of controls. However, no specific effect on poly (A) synthesis could be detected. The binding efficiency of viral RNAs on nitrocellulose membranes and poly (U) sepharose is not affected by cordycepin. The average poly (A) length of total intracellular viral RNA was calculated on the basis of the ratio of the radioactivity of adenosine-monophosphate: adenosine and found to be about 35 nucleotides in treated and untreated cells.

The route of transport of Semliki Forest virus (SFV) membrane glycoproteins to the plasma membrane was studied using immunoperoxidase electron microscopy. SFV glycoproteins were localized in cultured BHK-21 fibroblasts infected with a temperature-sensitive mutant ts-1 of SFV, which shows a temperature-dependent, reversible defect in the transport of membrane glycoproteins to the cell surface. At 39 degrees C (restrictive temperature) the viral proteins were retained in the endoplasmic reticulum and the nuclear membrane. After shift of the infected cultures to 28 degrees C (permissive temperature) the proteins were synchronously transported to the Golgi complex. In the Golgi complex the labeled proteins were first (at 2.5 min) detected in large Golgi-associated vacuoles (GAV). Subsequently, i.e., at 5-30 min, the viral glycoproteins appeared in the cisternal stack: at 5 min the label was found in one or two of the proximal cisternae whereas at 15 or 30 min also the more distal cisternae were partially or uniformly labeled. At all time points examined after the temperature-shift, peroxidase label was found in 50 nm vesicles which were frequently coated. At 30 min, in addition to the 50 nm vesicles, larger 80 nm vesicles, which often had a cytoplasmic coat were labeled in the Golgi region. These results identify two major size classes of both coated and smooth vesicles which appear to function in the transport of the viral membrane proteins from the endoplasmic reticulum via distinct GAV and the stacked Golgi cisternae to the plasma membrane.

Serial passage of Semliki Forest virus (SFV) in chicken embryo cells had little effect on SFV yield; however, high multiplicity infection of murine cells with one of the late passage pools (passage 9 SFV) resulted in a virus yield 10- to 20-fold lower than that obtained with earlier passage virus and 80-fold lower than the corresponding yield in chicken cells. This effect was accompanied by a striking decrease in the levels of 42S and 26S RNA and by increased proportions of a small single-stranded viral RNA (molecular weight, 9 x 10(5)) and of a low-molecular-weight replicative form. There was also a reduction in the number of specific membranous structures previously associated with the group A arbovirus replication complex. These results suggested that passage 9 SFV contained defective interfering particles which were detected more readily after one passage in a murine indicator host cell. Identical results were obtained with two different murine cell lines: one a leukemia virus-free clone of AKR cells and the other JLS-V9 cells chronically infected with Rauscher leukemia virus. Host production of RNA tumor virus particles apparently did not affect arbovirus replication.

INTRACYTOPLASMIC MEMBRANOUS STRUCTURES OF A UNIQUE TYPE WERE ASSOCIATED WITH THE REPLICATION OF THREE GROUP A ARBOVIRUSES: Semliki Forest virus (SFV), Sindbis virus, or Western equine encephalomyelitis virus. The structures, referred to as type 1 cytopathic vacuoles (CPV-1), were membrane-limited and characteristically lined by regular membranous spherules measuring 50 nm in diameter. The membranous spherules typically contained a fine central density, but were neither virus cores nor virions. Detection of CPV-1 by electron microscopy at 3 to 6 hr postinfection coincided with the time of rapid virus growth and preceded the accumulation of virus nucleocapsids. A range of 20 to 100 CPV-1 profiles were counted per 100 ultrathin cell sections at 6 to 9 hr postinfection when viruses were grown in chick embryo, baby hamster kidney, or mouse L cells. Maximum counts remained in the same range even when the multiplicity of infection was varied over 100-fold. Inhibition of cellular ribonucleic acid (RNA) and protein synthesis by actinomycin D during SFV infection did not decrease the counts of CPV-1; however, biogenesis of CPV-1 was decreased when viral replication was limited by inhibitors of viral RNA synthesis (guanidine) or of viral protein synthesis (cycloheximide). On the basis of present and earlier findings, we concluded that formation of CPV-1 must result from a virus-specified modification of pre-existing host cell macromolecules.