Temperature-sensitive Mutants of Sindbis Virus: Biochemical Correlates of Complementation

Temperature-sensitive mutants of Sindbis virus fail to grow at a temperature that permits growth of the wild type, but when certain pairs of these mutants, mixed together, infect cells at that temperature, viral growth (i.e., complementation) occurs. The yield from this complementation, however, is of the same order of magnitude as the infectivity in the inoculum. Since in animal virus infections the protein components of the virion probably enter the cell with the viral nucleic acid, it was necessary to demonstrate that the observed complementation required synthesis of new viral protein and nucleic acid rather than some sort of rearrangement of the structural components of the inoculum. To demonstrate that complementation does require new biosynthesis, three biochemical events of normal virus growth have been observed during complementation and correlated with the efficiency of viral growth seen in complementation. These events include: (i) entrance of parental viral ribonucleic acid (RNA) into a double-stranded form; (ii) subsequent synthesis of viral RNA; and (iii) synthesis and subsequent incorporation of viral protein(s) into cell membranes where they were detected by hemadsorption. Although the infecting single-stranded RNA genome of the wild type was converted to a ribonuclease-resistant form, the genome of a mutant (ts-11) incapable of RNA synthesis at a nonpermissive temperature was not so converted. However, during complementation with another mutant also defective in viral RNA synthesis, some of the RNA of mutant ts-11 was converted to a ribonuclease-resistant form, and total synthesis of virus-specific RNA was markedly enhanced. The virus-specific alteration of the cell surface, detected by hemadsorption, was also extensively increased during complementation. These observations support the view that complementation between temperature-sensitive mutants and replication of wild-type virus are similar processes.     

Agglutination of Sindbis Virus and of Cells Infected with Sindbis Virus by Plant Lectins

We have examined the agglutination of Sindbis virus and of chick and hamster cells infected with Sindbis virus by two of the plant lectins, concanavalin A and Ricinus communis agglutinin. Both lectins agglutinate the virus by binding to the polysaccharide chains of the envelope glycoproteins. Both chick and hamster cells exhibit increased agglutination by the lectins after infection by Sindbis virus. In the case of chick cells infected with Sindbis virus, this increase in agglutinability occurs between 3 and 5 h after infection. Infected and mock-infected cells bind the same amount of (3)H-labeled concanavalin A, which suggests that the increase in agglutination after infection is due to rearrangements at the cell surface rather than to insertion of new lectin binding sites per se.     

Genome of Sindbis Virus

³²P-labeled ribonucleic acid (RNA) from purified Sindbis virus was examined for the presence of hidden breaks. Viral RNA was treated with acid at pH 2.9 or with formaldehyde and was analyzed on sucrose gradients or by polyacrylamide gel electrophoresis. The sedimentation pattern and mobility on polyacrylamide gels of the 42S RNA was unaffected by heating and quick cooling and was not altered by denaturing agents such as dimethyl sulfoxide and urea. No evidence that Sindbis RNA is a polyaggregate of fragments was obtained. It is concluded that the genome consists of a continuous length of single-stranded polynucleotide.     

Phosphorylated Proteins of Sindbis Virus

The capsid and two membrane proteins of Sindbis virus, grown in chicken cells, contain 0.03 to 0.1 mol of phosphate per mol of protein.     

Temperature-Sensitive Virus from Aedes albopictus Cells Chronically Infected with Sindbis Virus

Cultures of Aedes albopictus cells persistently infected with wild-type Sindbis virus (SV-W) give rise to small plaque-forming mutants which are also temperature sensitive. These mutants, designated SV-C, are neutralized by antiserum produced against SV-W. Mutant ts clones were isolated from SV-C by plaque purification. After serial undiluted passage in BHK or mosquito cells, each of the clones gave rise to ts(+) revertants which, however, remained mutant with respect to plaque morphology. Nineteen of 20 clones derived from SV-C were RNA(+), and one was RNA(-) (SV-C-2). The RNA synthesizing activity, once induced in infected cells by SV-C-2, was stable at the nonpermissive temperature (39.5 C). All clones derived from SV-C were inactivated at 60 C much more quickly than was SV-W. It was not possible to demonstrate complementation between any of the SV-C clones.     

DNA Immunization against Herpes Simplex Virus: Enhanced Efficacy Using a Sindbis Virus-Based Vector

Previously we reported the development of a plasmid DNA expression vector system derived from Sindbis virus (T. W. Dubensky, Jr., et al., J. Virol. 70:508–519, 1996). In vitro, such vectors exhibit high-level heterologous gene expression via self-amplifying cytoplasmic RNA replication. In the present study, we demonstrated the in vivo efficacy of the Sindbis virus-based pSIN vectors as DNA vaccines. A single intramuscular immunization of BALB/c mice with pSIN vectors expressing the glycoprotein B of herpes simplex virus type 1 induced a broad spectrum of immune responses, including virus-specific antibodies, cytotoxic T cells, and protection from lethal virus challenge in two different murine models. In addition, dosing studies demonstrated that the pSIN vectors were superior to a conventional plasmid DNA vector in the induction of all immune parameters tested. In general, 100- to 1,000-fold-lower doses of pSIN were needed to induce the same level of responsiveness as that achieved with the conventional plasmid DNA vector. In some instances, significant immune responses were induced with a single dose of pSIN as low as 10 ng/mouse. These results indicate the potential usefulness of alphavirus-based vectors for DNA immunization in general and more specifically as a herpes simplex virus vaccine.     

Large-Molecular-Weight Precursors of Sindbis Virus Proteins

Infection of chicken embryo fibroblasts with a temperature-sensitive mutant of Sindbis virus at the nonpermissive temperature leads to the accumulation of a large-molecular-weight protein. We have shown that this protein contains (14)C-arginine tryptic peptides present in the three virion proteins. We have also found that a slightly smaller protein which is detected in Sindbis-infected BHK cells contains the (14)C-arginine tryptic peptides of the two envelope proteins but not those of the capsid protein. Pulse-chase experiments indicate that the Sindbis virus protein in BHK cells is cleaved to the envelope proteins.     

Enzymatic Iodination of Sindbis Virus Proteins

Sindbis virus was iodinated by using the enzyme lactoperoxidase, an iodination technique which labels only surface proteins. By this technique, the two viral glycoproteins are labeled, and the internal viral protein is not. The two glycoproteins are iodinated to strikingly different extents. This difference in susceptibility to iodination apparently is due to the position or conformation of the glycoproteins in the envelope spikes of the virion and not to differing contents of tyrosine, the amino acid substrate of lactoperoxidase. Both viral glycoproteins are iodinated by lactoperoxidase on the surface of Sindbis-infected chicken cells. Here, as in the virion, the glycoproteins are iodinated unequally, with the smaller glycoprotein again being preferentially iodinated. Another virus-specific protein found in large amounts in infected cells, and from which the preferentially iodinated virion glycoprotein is produced by a proteolytic cleavage, is not iodinated by lactoperoxidase. Thus it appears that the viral glycoproteins are present on the cell surface and that the precursor protein is not.     

Biosynthesis of the Sindbis Virus Carbohydrates

The sequence in which sugars are added to the Sindbis virus glycoproteins was studied. Infected cells contain three glycosylated virus-specific proteins: the two virion glycoproteins and the immediate precursor to the smaller virion glycoprotein. Larger Sindbis-specific proteins are not glycosylated. The cell-associated forms of both of the virion glycoproteins contain glucosamine, mannose, galactose, and fucose. The glycosylated precursor contains only glucosamine, mannose, and some galactose. The conversion of precursor to virion protein involves both the addition of galactose and fucose and the loss of mannose. The apparent extent of glycosylation of each virus-specific protein is not influenced by the host cell.     

Aspects of the Encapsidation of Simian Virus 40 Deoxyribonucleic Acid

Growing subcloned CV1-cells were infected with simian virus 40, and the time course of virus formation was determined. When infected cells were fractionated into cytoplasmic and nuclear fractions, most of the progeny virus particles were recovered in the cytoplasmic extract and not in the nuclei. This result was independent of the technique used for the preparation of nuclei and of the time after infection at which the extracts were prepared. Leakage of the virions from the nucleus occurred during the course of cell fractionation, suggesting that the nuclear membrane of the infected cells is damaged. Virions were found to accumulate in a nonlinear fashion, at the time when the number of viral deoxyribonucleic acid (DNA) molecules increases linearly with time after infection. This suggests that the size of the intracellular pool of capsid proteins increases constantly during the late phase of virus replication. Progeny viral DNA to become encapsidated is withdrawn at random from the pool of replicated DNA molecules.     

Baculovirus Per Os Infectivity Factors Form a Complex on the Surface of Occlusion-Derived Virus

Five highly conserved per os infectivity factors, PIF1, PIF2, PIF3, PIF4, and P74, have been reported to be essential for oral infectivity of baculovirus occlusion-derived virus (ODV) in insect larvae. Three of these proteins, P74, PIF1, and PIF2, were thought to function in virus binding to insect midgut cells. In this paper evidence is provided that PIF1, PIF2, and PIF3 form a stable complex on the surface of ODV particles of the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV). The complex could withstand 2% SDS-5% β-mercaptoethanol with heating at 50°C for 5 min. The complex was not formed when any of the genes for PIF1, PIF2, or PIF3 was deleted, while reinsertion of these genes into AcMNPV restored the complex. Coimmunoprecipitation analysis independently confirmed the interactions of the three PIF proteins and revealed in addition that P74 is also associated with this complex. However, deletion of the p74 gene did not affect formation of the PIF1-PIF2-PIF3 complex. Electron microscopy analysis showed that PIF1 and PIF2 are localized on the surface of the ODV with a scattered distribution. This distribution did not change for PIF1 or PIF2 when the gene for PIF2 or PIF1 protein was deleted. We propose that PIF1, PIF2, PIF3, and P74 form an evolutionarily conserved complex on the ODV surface, which has an essential function in the initial stages of baculovirus oral infection.The entry mechanism of enveloped viruses includes two major steps: virus binding to host receptors and subsequent fusion of the viral membrane with the cell membrane. For many viruses the processes of binding and fusion are mediated by a machinery composed of several membrane proteins working in concert with sequential events triggered by conformational changes upon interaction with host (co)receptors. Examples are herpes simplex virus (HSV) (4) and vaccinia virus (23), which have an entry machinery composed of four and eight proteins, respectively. The entry of the occlusion-derived virus (ODV) form of baculoviruses into insect midgut epithelial cells upon oral infection of insect larvae may involve a similar strategy, but little is known about the role of ODV membrane proteins.Baculovirus ODVs are orally infectious, enveloped virus particles embedded in a protein crystal called an occlusion body (OB) that infect midgut epithelial cells (24). After ingestion of OBs by the host, the proteinaceous OB crystal dissolves quickly due to the alkaline conditions (pH 10 to 11) in the midgut, and the ODV particles are released (reviewed in reference 24). After passage through the peritrophic membrane, ODVs bind and fuse with the microvilli of columnar epithelial cells, resulting in the release of nucleocapsids into the cytosol and subsequent initiation of infection (10, 12, 24). A second type of virus particle, the budded virus (BV), is produced in these cells and infects other cells and tissues in the insect, causing a systemic infection (reviewed in reference 22). While the entry mechanisms of BVs have been studied at least to a certain extent (16, 31, 32), the entry mechanism of ODVs is still rather enigmatic due to its complexity and the lack of proper cell lines supporting ODV entry.ODVs contain more than 10 different envelope proteins (3). Five of these, denoted PIF1, PIF2, PIF3, PIF4, and P74, have been identified to be essential for per os infection of insect larvae (6, 7, 14, 18, 20). These PIF proteins function in the early stage of virus infection, and deletion of any of these pif genes leads to a block in infection prior to viral gene expression in midgut epithelial cells (7, 10, 18). Until now, three of these proteins, PIF1, PIF2, and P74, have been reported to function in virus binding (10, 18). Deletion of any of these three proteins leads to a loss of oral infectivity, while only a 3-fold reduction in binding is measured, and no significant reduction in fusion efficiency is observed (10, 18). This suggests that the three PIF proteins, apart from binding to midgut epithelial cells, may have other unknown functions for which they may have to work together. The functions of PIF3 and PIF4 are rather enigmatic although there has been speculation that PIF3 functions in nucleocapsid translocation along the microvilli as it seemed to be dispensable for ODV binding and fusion (18, 24).All five proteins are highly conserved in Baculoviridae and are encoded by so-called core genes (3, 6, 11, 29). Recent work further revealed that these proteins have homologues in other large invertebrate DNA viruses which replicate in the nucleus, such as salivary gland hypertrophy viruses (SGHVs) (9), nudiviruses (30) and white spot syndrome virus (WSSV) (Nimaviridae) (J. A. Jehle, personal communication). pif genes are also found in polydnaviruses of braconid wasps (2). This high conservation of pif genes in a diverse range of large, circular, double-stranded DNA viruses suggests that the PIF proteins are associated with a conserved and evolutionarily ancient entry mechanism of viruses into invertebrate hosts.The aim of the present study is to follow the ODV entry process by investigating whether the PIF proteins form a complex on the ODV membrane. Based on immunogold labeling, cross-linking, differential temperature SDS-PAGE, and coimmunoprecipitation (CoIP) analysis with a panel of recombinant viruses, we provide strong evidence that PIF1, PIF2, PIF3, and P74 form a complex on the ODV surface. This complex is likely to play an essential role in virus entry into midgut epithelial cells of susceptible insect larvae.     

Preservation of Influenza Virus Infectivity by Lyophilization

A method of lyophilizing influenza virus in allantoic fluid with retention of high-titer of egg infectivity is described. Five strains of virus were lyophilized, and all were much more stable than fluid virus preparations, retaining 2 to 3 logs of infectivity after storage at 37 C for 60 to 95 days. Statistical analysis of an accelerated storage test by extrapolation of viral degradation indicates that the lyophilized viruses are stable indefinitely at or below room temperature.     

Low-pH-Dependent Fusion of Sindbis Virus with Receptor-Free Cholesterol- and Sphingolipid-Containing Liposomes

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.     

Morphology and Morphogenesis of Sindbis Virus as Seen with Freeze-Etching Techniques

Freeze-etch electron microscope studies of the morphogenesis and morphology of Sindbis virus confirmed results obtained by other workers employing thin-sectioning techniques. The 68-nm virion was found to have a nucleocapsid 36 nm in diameter surrounded by a double-layered, unit membrane. The membranous envelope is acquired as the capsid buds through the plasma membrane of the infected cell. The freeze-etch technique also provided the following new information. (i) At any one time, budding occurs in patches rather than evenly over the cell surface. (ii) The nucleocapsid is composed of capsomers 7 nm in diameter. (iii) The capsid interacts strongly with the membrane, both prior to budding and after maturation. (iv) The 7- to 10-nm particles characteristic of the internal faces of plasma membranes, which presumably represent host membrane proteins, are present in early stages of budding but disappear as morphogenesis progresses. (v) Fusion of the cell membrane at the base of the budding virion is a two-step process; the inner leaflet fuses into a sphere before the outer one. (vi) The outer surface of the viral envelope is covered with 4-nm subunits with a center-to-center spacing of 6 nm.     

Identification and Characterization of a Herpes Simplex Virus Gene Product Required for Encapsidation of Virus DNA

A mutant of herpes simplex virus type 1, 17tsVP1201, has a temperature-sensitive processing defect in a late virus polypeptide. Immunoprecipitation studies with monoclonal antibodies showed that the aberrant polypeptide in mutant virus-infected cells was the nucleocapsid polypeptide known as p40. Since a revertant, TS(+) for growth, processed the polypeptide normally under conditions restrictive for the mutant, the processing event must be essential for virus replication. Electron microscopic analysis of mutant virus-infected cells grown at the nonpermissive temperature revealed that the nuclei contained large aggregations of empty nucleocapsids possessing some internal structure. Therefore, although the mutant synthesized virus DNA at the nonpermissive temperature, the DNA was not packaged into nucleocapsids. When mutant virus-infected cells were shifted from 39 to 31 degrees C in the presence of cycloheximide, the polypeptide p40 was processed to lower-molecular-weight forms, and full nucleocapsids were detected in the cell nuclei. The aberrant polypeptide of the mutant, however, was not processed in cells mixedly infected with 17tsVP1201 and a revertant at the nonpermissive temperature, suggesting that the defect of the mutant was in the gene encoding p40 rather than in a gene of a processing enzyme.