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In an effort to enhance genetic interactions by eliminating spatial or physical barriers between variants of Sindbis virus, MgCl2 was used to aggregate infecting viral particles. Mixing viral samples in a 1:1 ratio with 0.5m MgCl2 produced maximal reduction in plaque forming units (PFU) with minimal cell damage due to MgCl2. Aggregate size was determined to be about 7 PFU. Samples taken at 3, 5 and 10 hours after infection with mixed aggregates composed of large and small plaque forming virus indicated that only one type of genome was represented among the progeny particles. In addition, aggregation enhanced complementation and the progeny were stable after several cycles of sonication and passage.

In chicken cells infected by Sindbis virus and exposed to a variety of membrane-active compounds, virus release was inhibited. In infected cells exposed to antiserum directed against the virion glycoproteins E1 or E2, retinol, cortisone, Pb++ or insulin, the processing of two Sindbis virus precursor polypeptides which lead to the formation of virion polypeptides was inhibited. The B-protein, which is the precursor to both envelope proteins, accumulated in cells treated by these compounds. This precursor is generally not detected in chicken cells, presumably because it is processed rapidly. The cleavage of the precursor PE2 to the envelope glycoprotein E2 was also inhibited. E2 was also absent in cells exposed to menadione or to antiserum prepared against uninfected chicken cells. Since each of the compounds tested interfered with Sindbis virus polypeptide cleavage, despite their diverse mechanisms of action, it suggests that perturbation of normal membrane fluidity can interfere with Sindbis virus budding.

The viral polypeptides synthesized in cells coinfected with group C and group D or E Sindbis virus mutants were studied. Cleavage of the ts2 protein occurs in cells coinfected with ts2 and ts20. Since the ts2 protein fails to chase in cells infected with ts2 alone, the activity effecting this cleavage must be, at least in part, virus specified.

We have demonstrated that the small plaque (SP) variant of Sindbis virus is more extensively neutralized by complete antibody and serum IgG fractions than is the large plaque (LP) variant. The Fc portion failed to demonstrate any neutralizing potential and did not block subsequent neutralization by IgG or Fab fragments. The LP and SP variants of Sindbis gave identical patterns upon isoelectric focusing. Treatment of the virus with intact serum IgG or isolated Fab fragments was found not to have an irreversible effect on the net charge of the virus population. The selective processes involved in the maturation of the nucleocapsid implicate the incorporation of charged host determinants which are then reflected in the charge of the virion. It is suggested that the virus core has a preferential affinity for those components of the plasma membranes which have an isoelectric point at pH 3.6.

The peptide composition of variants of Sindbis virus which differ in plaque-type, elution pattern from calcium phosphate columns and virulence in mice was analyzed. Differences in peptide number or composition were not detected in these variants. Since the Sindbis virion has an outer lipoprotein envelope which is derived at least in part from the plasma membrane of the host cell, peptide composition of purified chicken embryo membranes was analyzed. In contrast to the virions, the number of peptide units in the host cell membrane were too numerous to give resolution.

A method for concentrating and purifying enveloped viruses is described. Its salient feature is the use of an antihost immunoadsorbent column. Purified virus preparations were obtained in which viral-specific proteins per/mg total protein were enriched 800-fold, while host-specific complement fixing material became undetectable.

Sindbis virus, a group A arbovirus, is composed of a nucleocapsid and a glycolipoprotein membrane (Pfefferkorn & Hunter, 1963a; Strauss et al. 1968; Schelle & Pfefferkorn, 1969). The membrane protein is specified by the virus genome (Pfefferkorn & Clifford, 1964; Strauss et al. 1968; Strauss, Burge & Darnell, 1969) whereas the phospholipids and carbohydrate are derived from the host of origin (Pfefferkorn & Hunter, 1963b; Burge & Strauss, 1969; Strauss, Burge & Darnell, 1970) during maturation by budding through the membranes of cytopathic vacuoles (CPV) or the plasma membranes of the infected cell (Grimley, Berezesky & Friedman, 1968; Hackett et al. 1968; Holmes, Wark & Warburton, 1969; Nakai, Shand & Howatson, 1969). We have studied the maturation of Sindbis virus with the use of ferritin-conjugated antibody. After exhaustive adsorption with normal chick embryo cells, the globulin fraction precipitated from rabbit anti-Sindbis serum (Keckwick, 1940; Strauss et al. 1960) was conjugated with ferritin (Singer, 1959; Hsu, Rifkind & Zabriski, 1963; Rifkind, Hsu & Morgan, 1964).

Complement-fixing antigens were detected in the nucleocapsid and envelope of Sindbis virus. Neutralizing antibodies are directed against antigens in the viral envelope.

In cells infected with the Sindbis temperature-sensitive mutants ts-23 and ts-10 (complementation group D), which contain a defect in the envelope glycoprotein E1, the precursor polypeptide PE2 is not cleaved to the envelope glycoprotein E2 at the nonpermissive temperature. This defect is phenotypically identical to the defect observed in the complementation group E mutant, ts-20. The lesion in ts-23 is reversible upon shift to permissive temperature, whereas that of ts-10 is not. Antiserum against whole virus, E1, or E2 also prevents the cleavage of PE2 in cells infected with wild-type Sindbis virus. Because the cleavage of PE2 is inhibited by the lesion in mutants that are genotypically distinct and by anti-E1 or -E2 serum, it appears that PE2 and E1 exist as a complex in the membrane of the infected cell.

The development of resistance of outbred white mice to two plaque size variants of Sindbis virus was studied as a function of host age. A sudden drop in mortality occurred over a 3-day period. As little as 1 plaque-forming unit (PFU) per mouse was lethal in 1-day-old animals, whereas inocula of 10 9 PFU per mouse failed to produce high mortality in weanlings. A comparison of viral growth kinetics shows that early replication was similar in newborns and weanlings but that after 6 to 8 hr the production rate and, therefore, the maximal viral titer were suppressed in weanlings. The data demonstrate a lethal viral threshold. Titers in excess of this level lead to mortality, whereas those below it allow survival.

The mechanism of penetration and uncoating of enveloped animal viruses has recently been the subject of renewed controversy. Electron microscopic studies have led a number of investigators to conclude that penetration takes place by cellular engulfment (viropexis) of intact virus particles (Dales & Choppin, 1962; Mussgay & Weibel, 1962; Dales, 1963; Simpson, Hauser & Dales, 1969). Other investigators have concluded that enveloped animal viruses penetrate by fusion of the virus envelope with the host cell plasma membrane (Morgan & Howe, 1968; Morgan, Rose & Mednis, 1968a, b; Heine & Schnaitman, 1969). We now report on the role of the Sindbis virus envelope in the attachment of virus to host cells. Friedman & Berezesky (1967) and Sreevalsan & Allen (1968) noted that nucleocapsid components obtained from cytoplasmic extracts of arbovirus-infected cells or by detergent treatment of intact virus particles were non-infective for chicken embryo cells. However, the RNA released from arboviruses by detergent treatment is infective when assayed appropriately (Richter & Wecker, 1963; Nakamura & Eguchi, 1968).