The pathway by which semliki forest virus (SFV), a membrane-containing animal virus, enters BHK-21 cells was studied morphologically and biochemically. After attaching to the cell surface, the majority of viruses was rapidly trapped into coated pits, internalized by endocytosis in coated vesicles, and sequestered into intracellular vacuoles and lysosomes. Direct penetration of viruses through the plasma membrane was never observed. To assess the possible involvement of lysosomes in the release of the genome into the cytoplasm, the effect of five lysosomotropic agents, known to increase the lysosomal pH, was tested. All of these agents inhibited SFV infectivity and one, chloroquine (the agent studied in most detail), inhibited a very early step in the infection but had no effect on binding, endocytosis, or intracellular distribution of SFV. Thus, the inhibitory effect was concluded to be either on penetration of the nucelocapsid into the cytoplasm or on uncoating of the viral RNA. Possible mechanisms for the penetration of the genome into the cytoplasm were studied in vitro, using phospholipids-cholesterol liposomes and isolated SFV. When the pH was 6.0 or lower, efficient fusion of the viral membranes and the liposomal membranes occurred, resulting in the transfer of the nucleocapsid into the liposomes. Infection of cells could also be induced by brief low pH treatment of cells with bound SFV under conditions where the normal infection route was blocked. The results suggest that the penetration of the viral genome into the cytosol takes place intracellularly through fusion between the limiting membrane of intracellular vacuoles and the membrane of viruses contained within them. The low pH required for fusion together with the inhibitory effect of lysosomotropic agents implicate lysosomes, or other intracellular vacuoles with sufficiently low pH, as the main sites of penetration.

/pdf/on-the-entry-of-semliki-forest-virus-into-bhk-21-cells-2dy4tjqw3z.pdf

On the entry of Semliki Forest virus into BHK-21 cells.

Defective interfering virus (DI) particles represent a major controlling element of virus replication. They are constantly generated at low levels by infectious virus and only amplify to interfering levels when the parent helper vims is abundant. This autointerference phenomenon, as it was called when first discovered, is achieved by rearrangements and deletions of the standard virus genome such that the resulting “incomplete form” of the virus can preferentially replicate.

Origin and replication of defective interfering particles.

Defective interfering (DI) particles were discovered three decades ago by von Magnus (1947) using the influenza virus system. He called them “incomplete” or “immature” particles. Even though they could not be isolated or characterized biochemically, a great deal was learned about them from their physiological interactions with the host and from their interference with the multiplication of “infectious” standard virus (Gard and von Magnus, 1947; Bernkopf, 1950; von Magnus, 1951). Reviews by Henle (1950), by von Magnus (1954), and by Schlesinger (1959) on viral interference discuss the earlier work on this particular homologous interference caused by incomplete virus.

Defective Interfering Animal Viruses

This chapter will emphasize recently derived knowledge concerning the nature of defective interfering (DI) particles of RNA animal viruses, their biological origins and functions, and their involvement in long-term persistent infections. We will not attempt to review all of the DI literature, and we will confine ourselves to DI particles of RNA viruses. The previous review by Huang and Baltimore (1977) amply documents the occurrence and behavior of DI particles in a wide variety of DNA and RNA viruses and discusses their biological effects, and a very thorough recent review of rhabdovirus DI particles by Reichmann and Schnitzlein (1978) provides excellent in-depth coverage of many areas not covered by the present chapter as well as some alternate viewpoints of areas which are considered here. We will omit DNA virus DI particles from extensive consideration because of space limitations and because they are generally less well characterized at present.

/pdf/defective-interfering-rna-viruses-and-the-host-cell-response-4npo059tdp.pdf

Defective Interfering RNA Viruses and the Host-Cell Response

The nucleotide sequence coding for the nonstructural proteins of Semliki Forest virus has been determined from cDNA clones. The total length of this region is 7381 nucleotides, it contains an open reading frame starting at position 86 and ending at an UAA stop codon at position 7379-7381. This open reading frame codes for a 2431 amino acids long polyprotein, from which the individual nonstructural proteins are formed by proteolytic processing steps, so that nsPl is 537, nsP2 798, nsP3 482 and nsP4 614 amino acids. In the closely related Sindbis and Middelburg viruses there is an opal stop codon (UGA) between the genes for nsP3 and nsP4. Interestingly, no stop codon is found in frame in this region of the Semliki Forest virus 42S RNA. In other aspects the amino acid sequence homology between Sindbis, Middelburg and Semliki Forest virus nonstructural proteins is highly significant.

Complete nucleotide sequence of the nonstructural protein genes of Semliki Forest virus

Serial passaging of Semlike Forest virus in BHK cells at a constant input multiplicity of 50 p.f.u./cell resulted in a 4 log10 drop in yield of infectious virus by passage 9. An interference analysis showed that this drop was due to the presence of defective-interfering (DI) particles. Attempts were made to separate the DI particles from standard virus by equilibrium and velocity centrifugation. Only equilbrium centrifugation on CsCl resolved the DI particles (identified by interference analyses) from standard virus. The buoyant density of the DI particles (1.23 g/ml) was higher than that of standard virus (p = 1-20 g/ml). No difference was observed between the structural porteins of standard virus and DI particles. Analysis of the RNA of standard virus and DI particles showed that whereas standard virus contained only 42S RNA (mol. wt. approx. 4-2 X 10(6), DI particles contained two small pieces of RNA of mol. wt. 0.81 and 0-75 X 10(6) respectively. Infectivity assays showed that these low mol. wt. species were not only non-infectious but also interfered with the infectivity of 42S RNA from standard virus. Nucleocapsids derived from purified DI particles had a buoyant density 0-02 g/ml greater than the nucleocapsids from standard virus. Analysis of the RNA from DI nucleocapsids showed it to be entirely of the low mol. wt. class. To account therefore for the density difference not only between DI particles and standard virus but also between their respective nucleocapsids we propose that each SFV DI particle contains several molecules of the low mol. wt. RNA species.

Defective-interfering particles of Semliki Forest Virus: structural differences between standard virus and defective-interfering particles.

Summary
Three replicative forms of RNA (RF I, RF II, and RF III) have been isolated from BHK cells infected with Semliki Forest virus. Using analytical and rate-zonal sedimentation the mol. wt. of these replicative forms were estimated to be 8.5 × 106, 5.5 × 106 and 3.1 × 106 respectively. After continuous labelling from 1 to 6 h post-infection, RF I constituted more than 80% of the total replicative forms. Competition hybridization experiments showed that one strand of RF I was 42S RNA which had opposite (negative) polarity to that found in the virus particle. The positive strand of RF I was 42S RNA. The negative strand of replicative intermediate (RI) was also found to be 42S RNA. No evidence was found for an RI with a 26S negative strand. RF I was shown to contain non-hydrogen bounded poly A at or near the 3′ end of the component 42S positive strand. Isolation and analysis of the poly A tract from RF I on an acrylamide gel showed it to be of essentially the same average size as the poly A tract from virus particle RNA. About 30% of the RI molecules contained non-hydrogen bonded poly A. No poly U was detected in either RF I or RI. The kinetics of positive and negative strand synthesis were investigated during virus multiplication. These experiments showed that the rate of negative strand synthesis reaches a maximum 2½ h post-infection and thereafter rapidly falls. The rate of positive strand synthesis increases rapidly up to 3 h post-infection and then remains constant for a further 3 to 4 h.

Semliki Forest virus intracellular RNA: properties of the multi-stranded RNA species and kinetics of positive and negative strand synthesis.

Summary
Purified defective-interfering (DI) particles of Semliki Forest virus are unable to carry out any of the steps in virus multiplication except uncoating. Cells co-infected with DI particles and standard virus contain several virus-specified RNA species (DI particle-specific species) absent from cells infected with standard virus alone. Moreover, synthesis of all the virus-specified components distinctive of standard virus-infected cells is reduced. The DI particle-specific RNA species comprise two poly A-containing single-stranded RNAs (DIss1 and DIss2), identical to those found in purified DI particles, two double-stranded RNAs (RFs) and a new size class of replicative intermediate (RI).
Hybridization experiments showed that the nucleotide sequences of DIss1 and DIss2 (i) are present in the 42S genome of standard virus but absent from the 26S RNA — the RNA from standard virus-infected cells which encodes the structural proteins of the virion (Clegg & Kennedy, 1975a) and (ii) are complementary to the negative strands of the DI particle-specific RFs and RI. Oligonucleotide fingerprinting revealed extensive nucleotide sequence homology between DIss1 and DIss2. Analysis of the mRNA complement of standard virus-infected, co-infected and uninfected cells strongly indicated that neither DIss1 nor DIss2 can serve as a functional messenger RNA.
From these studies we propose a mechanism for the multiplication of and interference by DI particles of Semliki Forest virus.

Defective-interfering particles of Semliki Forest virus: intracellular events during interference.

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Defective-interfering particles of Semliki Forest Virus: structural differences between standard virus and defective-interfering particles.

Semliki Forest virus intracellular RNA: properties of the multi-stranded RNA species and kinetics of positive and negative strand synthesis.

Defective-interfering particles of Semliki Forest virus: intracellular events during interference.