Intracellular events in the replication of defective interfering particles of vesicular stomatitis virus.

During a synchronous infection with both wild-type vesicular stomatitis virus (VSV) and the MS-T defective interfering (DI) particle at levels of high interference, total RNA synthesis was both reduced by 75% and shut off early as compared to the VSV infection alone. In the interfered infection the production of both VSV intracellular nucleocapsids and progeny virus was suppressed by 90% while there was a dramatic increase in intracellular MS-T nucleocapsids. There was a linear accumulation of MS-T nucleocapsids for only 5 hr with the maximum rate of synthesis being observed at 3 hr postinfection. The MS-T nucleocapsids contained genome-length, single-stranded 19 S DI RNA and were 40% of the positive (+)- and 60% of the negative (?)-strand sense. Neither the (+)- nor (?)strand of MS-T nucleocapsid RNA contained poly(A) sequences since none of the RNA or its ribonuclease digestion products bound to oligo(dT)-cellulose. We can detect no methylation of the nucleocapsid RNA and the 5′-termini were not blocked since both the (+)- and (?)-strands initiated with pppA.     

Inhibition of SV40 DNA synthesis by vesicular stomatitis virus in doubly infected monkey kidney cells

Vesicular stomatitis virus (VSV) inhibited SV40 DNA synthesis in doubly infected synchronized Vero cells. Gel-electrophoretic profiles demonstrated that SV40 DNA monomers accumulated in all stages of supercoiling, regardless of whether cells were superinfected with VSV early or late in the S phase. These gel profiles were indistinguishable from ones obtained from SV40-infected, cycloheximide-treated cells in the absence of VSV infection. Radiolabel in the partial supercoils could be chased into supercoils, but only by restoring protein synthesis. The relative rates of SV40 DNA chain elongation were determined in VSV-superinfected and nonsuperinfected cells. The gradients of 3H incorporation as a function of distance from the origin of replication in pulse-labeled form I DNA were unaffected by VSV. It is concluded that VSV inhibition of SV40 DNA synthesis is an indirect result of inhibition of host cell protein synthesis and it is suggested that incompletely supercoiled SV40 chromatin is not a good template for DNA synthesis.     

Factors which influence inactivation of vesicular stomatitis virus by fresh human serum.

Vesicular stomatitis virus (VSV) was inactivated by fresh human serum, but was less susceptible when grown in human cells. The action of fresh serum differed from that of bromelain, thermolysin, or phospholipase C since inactivation by these enzymes did not depend on host origin of the virus, and since the mutant VSV-tl 17 was more susceptible to these enzymes than VSV, although more resistant to fresh human serum. Host cell antigens may be the target for virolysis by fresh human serum since this activity was specifically increased when VSV was treated with heated anticellular sera. Interaction of fresh human serum with VSV allowed access of ribonuclease to the viral RNA. VSV pseudotypes formed with the simian retraviruses, Mason-Pfizer virus (M-P V) and baboon endogenous virus (BeV), were neutralized by the corresponding antibodies to the retraviruses but also showed increased susceptibility to anticellular sera. Excess of host cell antigen may explain the extreme sensitivity to human serum of VSV (BeV) pseudotypes obtained in dog cells, as well as the resistance of VSV (M-P V) prepared in HeLa cells.     

A defective interfering vesicular stomatitis virus particle that directs the synthesis of functional proteins in the absence of helper virus.

A defective interfering (DI) particle derived from the HR strain of VSV, DI-LT, contains genetic information from the 3′ portion of the parental genome. In the absence of helper virus, this DI particle stimulates the synthesis of four proteins that comigrate with the VSV proteins G, NS, N, and M. The DI-LT-stimulated proteins are immunologically cross-reactive with VSV proteins and can be immune precipitated with antisera directed against VSV. These proteins are functional as demonstrated by the genetic complementation of VSV ts mutants (groups II–V) by DI-LT particles.     

Frequent generation of new 3'-defective interfering particles of vesicular stomatitis virus

We have isolated and partially characterized a number of different genome types of defective interfering (DI) particles newly generated by a highly heat-resistant strain of vesicular stomatitis virus in either Rat(B77) or Vero cells. Northern blot analyses revealed that many of these DI genomes contain N gene sequences and/or sequences of the NS, M, and G genes. One type contains NS sequences without any indication for the presence of either N, M, or G sequences. Another type of DI particle genomes did not contain any detectable sequences of N, NS, M, or G, but contain panhandle-type sequences and, thus, most likely resembles the 5'-panhandle-type DI particles. Unlike previously assumed, these data demonstrate that DI genomes which have the 3'-terminal N, NS, M, and G genes or portions of these genes conserved do frequently arise together with 5'-DI particle genomes after serial undiluted passages of the heat-resistant strain of vesicular stomatitis virus.     

Host range restriction of vesicular stomatitis virus on duck embryo cells.

Vesicular stomatitis virus (VSV) plaque formation and replication are restricted in duck embryo cells compared to chick embryo cells. Mutants of VSV which replicate normally on both chick and duck cells were isolated. The duck-adapted mutant, when passed through chick cells, retains its ability to grow normally on duck cells. This indicates that the ability of this virus to grow on duck cells is due to mutation rather than to host-controlled modification. VSV is restricted in duck cells but not in quail or pheasant embryo cells which follows the evolutionary relationship of these species. Newcastle disease virus and Rous sarcoma virus are restricted in duck cells also. Virus-specific protein synthesis of the wild-type VSV is greatly reduced in duck cells compared to chick cells. A structural protein of the duck cell-adapted mutant is altered.     

Persistent infection. I Interferon-inducing defective-interfering particles as mediators of cell sparing: possible role in persistent infection by vesicular stomatitis virus.

Defective-interfering (DI) particles have been implicated in modulating the lethal action of infectious virus during persistent infection. However, in the case of vesicular stomatitis (VS) virus, conventional [?]RNA DI particles (nonlethal themseleves) do not block the lethal action of infectious or cell-killing (CK) particles of VS virus, even though no new infectious virus is produced. The discovery of a class of interferon-inducing defective-interfering (DI → IF) particles with covalently linked, self-complementary [±]RNA, and the capacity of a single such particle to induce interferon, coupled with the demonstration that interferon action prevents cell killing by VS virus, led us to postulate that defective-interfering particles of the [±]RNA class present initially or arising during infection might activate the interferon system and prevent CK particle activity and hence contribute to the establishment and possible maintenance of persistent infection in some cell systems. We provide evidence to support this hypothesis: Single cell survival experiments established that GMK-BSC or mouse L cells retain the capacity to form colonies, i.e., are spared from killing, if they are pretreated with interferon-inducing [±]RNA DI-011 particles 24 hr prior to challenge with either homologous or heterologous viruses. Mouse interferon antiserum present during the pretreatment phase eliminated cell sparing. Pretreatment with DI-011 particles of GMK-Vero cells, which lack the capacity to produce interferon, did not spare these cells from the lethal action of VS virus. Activation of the interferon system by the [±]RNA class of defective-interfering particles provides a means whereby normally susceptible cells may survive in the presence of lethal virus — an aspect of persistent infection by VS virus not satisfactorily accounted for by current models.     

Molecular characterization of two strains of Shope fibroma virus

An isolate of Shope fibroma virus (SFV), designated Indiana (SFV-I), was previously described to be tumorigenic in vivo as SFV, cytocidal in vitro as the orthopoxviruses (vaccinia, rabbitpox, etc.), and to share antigenic determinants with SFV and vaccinia. The genetic relatedness of SFV-I to SFV and vaccinia was studied by means of Southern blotting and hybridization. The results indicated that SFV-I shares extensive DNA homology with vaccinia, but few common sequences with SFV. By contrast, SFV and vaccinia show no sequence homology. These findings suggest that SFV-I is an orthopoxvirus which carries some genetic information from the leporipoxviruses. Recombinants between two genera of poxviruses have not been reported before.     

Proteins of vesicular stomatitis virus IV. A comparison of tryptic peptides of the vesicular stomatitis group of rhabdoviruses

The methionme-containing tryptic peptides of the five constituent polypeptides of members of the vesicular stomatitis groups of rhabdoviruses were compared to the Indiana serotype by two-dimensional thin-layer chromatography-electrophoresis. While the corresponding protein of each virus serotype could be identified by its characteristic map, there were apparent common peptides shared between serologically related members. Three methionine-containing peptides of the N protein of VSV (Indiana) could not be separated from peptides of VSV (Cocal) and of these three, one peptide was also present in VSV (New Jersey). A comparison of the tryptic peptides of proteins from two members of the VSV (New Jersey) serotype revealed characteristic differences in the polypeptides from the closely related, but serologically distinguishable, New Jersey-Concan and New Jersey Missouri isolates.     

The 5' terminal nucleotide of RNA from vesicular stomatitis virus defective interfering particles.

The RNAs of three well-characterized and vastly dissimilar VSV defective interfering particles (DI_LT, DI_T, and DI₀₁₁) have the same 5′ terminus, pppA, as the infectious particle.     

Superinfection exclusion by vesicular stomatitis virus

The infection of baby hamster kidney (BHK21) cells by the Indiana strain of vesicular stomatitis virus (VSV) causes a rapid loss of the ability of the cells to be superinfected by VSV virions or defective-interfering particles. This exclusion phenomenon is at the level of virus penetration and requires viral gene expression and a functional VSV transmembrane glycoprotein G. Infection with the New Jersey serotype of VSV also inhibits the uptake of the Indiana serotype. However, infection of BHK21 cells with either encephalomyocarditis, Newcastle disease, or influenza A viruses does not inhibit superinfection by VSV.     

RNA synthesis in standard and autointerfered vesicular stomatitis virus infections.

The levels of intracellular viral RNA have been measured in BHK-21 cells undergoing standard and autointerfered infections with vesicular stomatitis virus (VSV). The total amount of intracellular nucleocapsid RNA is almost unaffected by autointerference. However, the composition of this class of RNA changes with increasing autointerference: Control cells undergoing standard infections contain only the 140 S, full genomic length nucleocapsid, while autointerfered cells contain, in addition, a smaller nucleocapsid which corresponds in size to that found in the defective interfering (DI) particle. There is a systematic decrease in viral mRNA with increasing autointerference. This decrease in mRNA closely parallels the reduced yield of total progeny particles eventually released from these cells. A causal relationship between the two is proposed. These results are compatible with explanations of autointerference that depend upon a competition between the DI and VSV genomes for the viral replicase.     

Diversity and generation of defective interfering influenza virus particles.

A number of individually isolated, plaque-purified clones of ts-52 WSN influenza virus were analyzed for their ability to generate defective interfering (DI) virus particles. Clonal stocks, when passaged four or five times serially in MDBK cells at high multiplicity produced DI viruses each with a characteristic set of DI RNA segments. Aliquots of different viral passages from a given clone (VP1 through VP3), when passaged independently produced the same DI virus suggesting that DI viruses are already present in VP1. Furthermore, additional experiments suggested that DI viruses characteristic of a given clone were even present in the clonal stock virus and became amplified during undiluted passages. When aliquots of a virus clone (directly isolated from a plaque) were passaged independently some clones produced identical DI viruses at VP4 suggesting that DI viruses were already present in the plaque whereas aliquots of another clone produced different DI viruses suggesting that DI viruses were absent in the clone and were generated subsequently during passages. Thus DI virus can be generated even in the plaque produced by a single infectious virus. Large quantities of a particular DI virus could be produced by coinfection with a helper infectious virus. However, continued amplification of DI viruses for a large number of passages by a helper infectious virus resulted in disappearance of some DI RNA segments with the appearance of possible new DI RNA segments. Mixed infections of two different DI viruses in the presence of helper virus resulted in the production of both types of major DI viruses. Furthermore, we have developed an assay for quantitating DI virus directly using an infectious center reduction method. Our data suggest that a single DI virus particle can inhibit infectious center formation by standard virus and that standard virus is replaced almost entirely by DI virus after four undiluted passages.