Defective interfering virus particles modulate virulence.

To determine whether defective interfering (DI) particles modulate virulence by initiating a cyclic pattern of virus growth in vivo, adult mice were infected with vesicular stomatitis virus (VSV), both with and without DI particles. A total of 184 mice divided into groups were inoculated intranasally. A majority of mice inoculated only with standard VSV developed paralysis, most of them between days 7 and 9. The addition of DI particles altered the development of paralysis in several ways. When there was significant protection, a few still became paralyzed on days 7 and 9. When overall mortality was unaffected or even slightly increased, the majority of mice became paralyzed between days 7 and 9 as well. Protection could not be predicted based on a single ratio of standard VSV to DI particles or on the absolute amount of DI particles inoculated. Infectious virus recovered from mouse brains at the time of paralysis and incipient death showed considerable variation, although the titer in a majority of the animals was between 10(5) and 10(7) PFU/ml. When the brains of these paralyzed mice were examined for hybridizable VSV RNA, the detection of standard VSV RNA correlated well with infectivity. The amount of DI RNA in the coinfected mice was more variable and independent of the amount of 40S RNA, although DI RNA was usually found when standard RNA was present. Survivors examined between days 14 and 21 did not contain infectious virus or any detectable viral RNA in their brains. Because these results were consistent with the hypothesis of viral cycling in vivo, rather than a gradual accumulation of total infectious virus, mice were coinfected with 10(8) PFU of standard VSV and 10(5) PFU equivalents of DI particles and sacrificed daily thereafter, irrespective of whether they developed paralysis. Infectivity measurements indicated a reproducible cycling pattern of VSV in the mouse brains with a periodicity of about 5 days. This cycling and the detection of DI RNA in brains several days after intranasal inoculation suggest that there is a dynamic continuous interaction between standard VSV and its DI particle beyond the initial site of replication as the virus population spreads into the host animal. Such cycling of virus production before the full development of specific immune responses from the host may have important implications for viral diagnostics and disease transmission.     

Replication-defective viruses modulate immune responses.

By immunizing inbred mice with purified replication-competent, defective virus particles, or an admixture of the two, differential effects on the cellular immune system have been uncovered. Defective virus, exemplified by the vesicular stomatitis virus (VSV) defective interfering particle (DI 0.33), induced in BALB/c mice low levels of proliferating, IL-2 secreting, and cytolytic Ag-specific T lymphocytes. This was not caused by a dominant suppressor cell response, or by a failure to stimulate lymphokine-secreting cells, but appeared to reflect a reduced efficiency of priming as compared with standard virus. Mice primed with a mixture of wt and DI virus showed reduced proliferation compared with mice primed with wt virus. When histocompatible target cells were sensitized by pure DI particles, they were neither recognized nor lysed by CD8+ CTL. Cells co-infected with wt and DI particles were not as readily lysed by CD8+ CTL as cells infected by VSV alone. The extent of this reduction was dependent on the concentration of DI particles. This suggests that DI particles may have prevented the proper presentation of endogenously synthesized Ag for recognition by CD8+ CTL. Metabolic labeling studies indicated that the presence of DI particles suppressed the synthesis of viral proteins in dually infected cells. However, CD4+ T lymphocyte clones recognized and efficiently lysed histocompatible Ia+ cells infected with DI particles alone or co-infected with replication-competent and defective virus.     

Vesicular stomatitis virus defective interfering particles can contain extensive genomic sequence rearrangements and base substitutions

We sequenced the 5' and 3' RNA termini of 16 defective interfering (DI) particles of vesicular stomatitis virus (VSV) isolated at intervals from persistent infections and from a series of undiluted lytic passages. All DI RNAs exhibited complementary termini, but sequences internal to these termini were extensively rearranged in a variety of ways. Despite extensive rearrangement, these internal sequences (in addition to the termini) apparently are important for DI particle interference properties. Some of these DI particles are derived from multiple intrastrand and interstrand recombination events, and the generation of each can be explained by current replicase error models. During viral evolution in persistent and acute infections, DI particles with specific termini base substitutions are selected. One DI particle exhibits a remarkable clustering of specific A----G (and complementary U----C) substitutions, apparently as a result of repetitive misincorporations by an error-prone viral polymerase complex.     

Utilization of homotypic and heterotypic proteins of vesicular stomatitis virus by defective interfering particle genomes for RNA replication and virion assembly: implications for the mechanism of homologous viral interference

Defective interfering (DI) particles of Indiana serotype of vesicular stomatitis virus (VSV(Ind)) are capable of interfering with the replication of both homotypic VSV(Ind) and heterotypic New Jersey serotype (VSV(NJ)) standard virus. In contrast, DI particles from VSV(NJ) do not interfere with the replication of VSV(Ind) standard virus but do interfere with VSV(NJ) replication. The differences in the interfering activities of VSV(Ind) DI particles and VSV(NJ) DI particles against heterotypic standard virus were investigated. We examined the utilization of homotypic and heterotypic VSV proteins by DI particle genomic RNAs for replication and maturation into infectious DI particles. Here we show that the RNA-nucleocapsid protein (N) complex of one serotype does not utilize the polymerase complex (P and L) of the other serotype for RNA synthesis, while DI particle genomic RNAs of both serotypes can utilize the N, P, and L proteins of either serotype without serotypic restriction but with differing efficiencies as long as all three proteins are derived from the same serotype. The genomic RNAs of VSV(Ind) DI particles assembled and matured into DI particles by using either homotypic or heterotypic viral proteins. In contrast, VSV(NJ) DI particles could assemble only with homotypic VSV(NJ) viral proteins, although the genomic RNAs of VSV(NJ) DI particles could be replicated by using heterotypic VSV(Ind) N, P, and L proteins. Thus, we concluded that both efficient RNA replication and assembly of DI particles are required for the heterotypic interference by VSV DI particles.     

Properties of DI particle resistant mutants of vesicular stomatitis virus isolated from persistent infections and from undiluted passages

We describe an assay procedure to quantitate relative DI resistance of a variety of DI particle resistant (Sdi^?) mutants of vesicular stomatitis virus (VSV). We show that numerous diverse Sdi^? mutants of VSV are selected continuously in a stepwise manner during persistent infections, and also during serial undiluted lytic passages initiated with cloned virus. Concurrently with the successive appearance and disappearance of different Sdi^? mutants of infectious VSV, new DI particle types with altered interference properites also appear and disappear, resulting in rapid “coevolution” of virus and DI particle populations. Complementation tests with Sdi^? mutants indicate that mutations in at least two different virus factors (presumably associated with replication-encapsidation) can give rise to Sdi^? mutants. Interference studies with chimeric DI particles indicate that DI particle template RNA rather than DI particle protein determines the interference properties of DI particles interacting with Sdi^? and Sdi⁺ mutants of helper virus.     

Inhibition of pseudorabies virus replication by vesicular stomatitis virus. II Activity of defective interfering particles.

Purified defective interfering (DI) particles of vesicular stomatitis virus (VSV) inhibit the replication of a heterologous virus, pseudorabies virus (PSR), in hamster (BHK-21) and rabbit (RC-60) cell lines. In contrast to infectious B particles of VSV, UV irradiation of DI particles does not reduce their ability to inhibit PSR replication. However, UV irradiation progressively reduces the ability of DI particles to cause homologous interference with B particle replication. Pretreatment with interferon does not affect the ability of DI particles to inhibit PSR replication in a rabbit cell line (RC-60) in which RNA, but not DNA, viruses are sensitive to the action of interferon. Under similar conditions of interferon pretreatment, the inhibition of PSR by B particles is blocked. These data suggest that de novo VSV RNA or protein synthesis is not required for the inhibition of PSR replication by DI particles. DI particles that inhibit PSR replication also inhibit host RNA and protein synthesis in BHK-21 and RC-60 cells. Based on the results described and data in the literature, it is proposed that the same component of VSV B and DI particles is responsible for most, if not all, of the inhibitory activities of VSV, except homologous interference.     

Comparison of vesicular stomatitis virus defective interfering particle synthesis in chick embryo and L cells.

A comparison of the ability of vesicular stomatitis virus (VSV) to generate and replicate defective interfering (DI) particles in primary chick embryo (CE) and mouse L cells was investigated as a means of analyzing host control over DI-particle synthesis and interfering capacity. Serial undiluted passage of VSV in CE and L cells indicate that VSV-DI particles are generated and (or) replicate with greater efficiency in CE than in L cells. When DI particles accumulate in L cells, they are able to interfere with infectious particle replication. The DI particles from CE cells interfered to the same extent with infectious particle replication in both CE and L cells. L cells, therefore, are not considered 'low-interference' hosts in which DI particles are produced and do not interfere with infectious virus replication, but rather hosts which restrict the production of DI particles.     

Synthesis and infectivity of vesicular stomatitis virus containing nonglycosylated G protein.

The replication of vesicular stomatitis virus (VSV) is inhibited by tunicamycin (TM), an antibiotic that blocks the formation of N-acetylglucosaminelipid intermediates. We had shown previously that the viral glycoprotein (G) synthesized in cells treated with TM is not glycosylated and is not found on the outer surface of the cell plasma membrane. In this report, we shown that cells exposed to TM produce a low yield of infectious particles. The yield is increased when the temperature during infection is lowered from 37 to 30 degrees C. At 30 degrees C in the presence of TM, both wild-type VSV and the temperature-sensitive mutant ts045 produce particles that do not bind to concanavalin A Sepharose and contain only the nonglycosylated form of G. These particles have a specific infectivity (pfu/cpm) comparable to that of VSV containing glycosylated G.     

Role of heterologous and homologous glycoproteins in phenotypic mixing between Sendai virus and vesicular stomatitis virus.

Phenotypic mixing between Sendai virus and vesicular stomatitis virus (VSV) or the mutant VSV ts045 was studied. Conditions were optimized for double infection, as shown by immunofluorescence microscopy. Virions from double-infected cells were separated by sequential velocity and isopycnic gradient centrifugations. Two types of particles with mixed protein compositions were found. One type was VSV particles with Sendai virus spikes, i.e., phenotypically mixed particles. A second type was Sendai virus-VSV associations, which in plaque assays also behaved as phenotypically mixed particles. The ratio of VSV G protein to Sendai virus glycoproteins on the cell surface was varied, using the VSV mutant ts045 in double infections. Thus, different amounts of the VSV G protein were allowed to reach the cell surface at 32, 38, and 39 degrees C in Sendai virus-infected cells. However, a fixed number of Sendai virus spikes was always found in the ts045 virions. This represented 12 to 16% of the number of G proteins present in normal VSV. Furthermore, the yield of ts045 virions was radically reduced during double infection when the temperature was raised to block G-protein transport to the cell surface, suggesting that the Sendai virus glycoproteins were not able to compensate for G protein in budding. These results emphasize the role of the G protein in VSV assembly.     

Homologous interference mediated by defective interfering influenza virus derived from a temperature-sensitive mutant of influenza virus.

A temperature-sensitive group II mutant of influenza virus, ts-52, with a presumed defect in viral RNA synthesis, readily produced von Magnus-type defective interfering virus (DI virus) when passed serially (four times) at high multiplicity in MDBK cells. The defective virus (ts-52 DI virus) had a high hemagglutinin and a low infectivity titer, and strongly interfered with the replication of standard infectious viruses (both ts-52 and wild-type ts+) in co-infected cells. Progeny virus particles produced by co-infection of DI virus and infectious virus were also defective and also had low infectivity, high hemagglutinating activity, and a strong interfering property. Infectious viruses ts+ and ts-52 were indistinguishable from ts-52 DI viruses by sucrose velocity or density gradient analysis. Additionally, these viruses all possessed similar morphology. However, when the RNA of DI viruses was analyzed by use of polyacrylamide gels containing 6 M urea, there was a reduction in the amount of large RNA species (V1 to V4), and a number of new smaller RNA species (D1 to D6) with molecular weights ranging from 2.9 X 10(5) to 1.05 X 10(5) appeared. Since these smaller RNA species (D1 to D6) were absent in some clones of infectious viruses, but were consistently associated with DI viruses and increased during undiluted passages and during co-infection of ts-52 with DI virus, they appeared to be a characteristic of DI viruses. Additionally, the UV target size of interfering activity and infectivity of DI virus indicated that interfering activity was 40 times more resistant to UV irradiation than was infectivity, further implicating small RNA molecules in interference. Our data suggest that the loss of infectivity observed among DI viruses may be due to nonspecific loss of a viral RNA segment(s), and the interfering property of DI viruses may be due to interfering RNA segments (DIRNA, D1 to D6). ts-52 DI virus interfered with the replication of standard virus (ts+) at both permissive (34 degrees C) and nonpermissive temperatures. The infectivity of the progeny virus was reduced to 0.2% for ts+ and 0.05% for ts-52 virus without a reduction in hemagglutinin titer. Interference was dependent on the concentration of DI virus. A particle ratio of 1 between DI virus (0.001 PFU/cell) and infectious virus (1.0 PFU/cell) produced a maximal amount of interference. Infectious virus yield was reduced 99.9% without any reduction of the yield of DI viruses Interference was also dependent on the time of addition of DI virus. Interference was most effective within the first 3 h of infection by infectious virus, indicating interference with an early function during viral replication.     

Virus protein changes and RNA termini alterations evolving during persistent infection

Cloned infectious vesicular stomatitis virus isolated following 5 years of persistent infection of BHK21 cells in vitro exhibits a number of peptide map changes in the G protein (spike glycoprotein), the M protein (membrane matrix protein) and the N protein (nucleocapsid structural protein). Only slight alterations have occurred in the peptide maps of the two VSV polymerase-associated proteins L and NS. Dideoxy sequencing of the 3′ ends of the cloned virus originally used to establish the persistent infection, and of the cloned virus recovered following 5 years of persistence, shows one base substitution in the three base junction between the 3′ leader sequence and the N protein-coding region. Repeated lytic passages of virus recovered from persistent infection led to no oligonucleotide map changes after 30 passages, but two map changes were present after 102 and remained after 133 lytic passages in BHK21 cells in vitro. Only one of these represented reversion to the original map position, and this “mutant” virus still exhibited a temperature-sensitive small plaque phenotype. Finally, the mutated virus recovered after more than $\text{5}\text{1}\text{2}$ years of persistent infection is now so slow-growing that it can establish persistent infection of BHK21 cells in the absence of DI particles (although DI particles are present constantly once the cells recover from the initial cytopathology).     

Interference among defective interfering particles of vesicular stomatitis virus

Three defective interfering (DI) particles of vesicular stomatitis virus (VSV), all derived from the same parental standard San Juan strain (Indiana serotype), were used in various combinations to infect cells together with the parental virus. The replication of their RNA genomes in the presence of other competing genomes was described by the hierarchical sequence: DI 0.52 particles greater than DI 0.45 particles less than or equal to DI-T particles greater than standard VSV. The advantage of one DI particle over another was not due simply to multiplicity effects nor to the irreversible occupation of limited cellular sites. Interference, however, did correlate with a change in the ratio of plus and minus RNA templates that accumulated intracellularly and with the presence of new sequences at the 3' end of the DI genomes. DI 0.52 particles contained significantly more nucleotides at the 3' end that were complementary to those at the 5' end of its RNA than did DI-T or DI 0.45 particles. The first 45 nucleotides at the 3' ends of all of the DI RNAs were identical. VSV and its DI particles can be separated into three classes, depending on their terminal RNA sequences. These sequences suggest two mechanisms, one based on the affinity of polymerase binding and the other on the affinity of N-protein binding, that may account for interference by DI particles against standard VSV and among DI particles themselves.     

Terminal sequences of vesicular stomatitis virus RNA are both complementary and conserved.

The nucleotide sequences at the 5' and 3' termini of RNA isolated from the New Jersey serotype of vesicular stomatitis virus [vsV(NJ)] and two of its defective interfering (DI) particles have been determined. The sequence differs from that previously demonstrated for the RNA from the Indiana serotype of VSV at only 1 of the first 17 positions from the 3' terminus and at only 2 of the first 17 positions from the 5' terminus. The 5'-terminal sequence of VSV(NJ) RNA is the complement of the 3'-terminal sequence, and duplexes which are 20 bases long and contain the 3' and 5' termini have been isolated from this RNA. The RNAs isolated from DI particles of VSV(NJ) have the same base sequences as do the RNAs from the parental virus. These results are in sharp contrast to those obtained with the Indiana serotype of VSV and its DI particles, in which the 3'-terminal sequences differ in 3 positions within the first 17. However, with both serotypes, the 3'-terminal sequence of the DI RNA is the complement of the 5'-terminal sequence of the RNA from the infectious virus. These findings suggest that the 3' and 5' RNA termini are highly conserved in both serotypes and that the 3' terminus of DI RNA is ultimately derived by copying the 5' end of the VSV genome, as recently proposed (D. Kolakofsky, M. Leppert, and L. Kort, in B. W. J. Mahy and R. D. Barry, ed., Negative-Strand Virus and the Host Cell, 1977; M. Leppert, L. Kort, and D. Kolakofsky, Cell 12:539-552, 1977; A. S. Huang, Bacteriol. Rev. 41:811-8218 1977).     

Establishment and maintenance of persistent infection by Sindbis virus in BHK cells.

We have established a persistent infection of BHK cells with a preparation of Sindbis virus heavily enriched in defective interfering (DI) particles. The small fraction of cells that survived the initial infection grew out to form a stable population of cells [BHK(Sin-1) cells], most of which synthesized viral RNA and viral antigens. The presence of DI particles in this virus stock was required to establish this persistent state. BHK(Sin-1) cells released a small-plaque, temperature-sensitive virus (Sin-1 virus) as well as DI particles containing DI RNAs larger than those present in the original stock used to establish the persistent state. A cloned stock of Sin-1 virus, free of detectable DI particles, was able to initiate a persistent infection more quickly and with greater cell survival than the original stock of Sindbis virus containing DI particles. About 2 weeks after the Sin-1 virus-infected cells were cultured, DI RNAs arose and soon became the dominant viral RNA species produced by these cells.     

Selective isolation of mutants of vesicular stomatitis virus defective in production of the viral glycoprotein.

We describe a procedure that enriches for temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV), Indiana serotype, which are conditionally defective in the biosynthesis of the viral glycoprotein. The selection procedure depends on the rescue of pseudotypes of known ts VSV mutants in complementation group V (corresponding to the viral G protein) by growth at 39.5 degrees C in cells preinfected with the avian retrovirus Rous-associated virus 1 (RAV-1). Seventeen nonleaky ts mutants were isolated from mutagenized stocks of VSV. Eight induced no synthesis of VSV proteins at the nonpermissive temperature and hence were not studied further. Four mutants belonged to complementation group V and resembled other ts (V) mutations in their thermolability, production at 39.5 degrees C of noninfectious particles specifically deficient in VSV G protein, synthesis at 39.5 degrees C of normal levels of viral RNA and protein, and ability to be rescued at 39.5 degrees C by preinfection of cells by avian retroviruses. Five new ts mutants were, unexpectedly, in complementation group IV, the putative structural gene for the viral nucleocapsid (N) protein. At 39.5 degrees C these mutants also induced formation of noninfectious particles relatively deficient in G protein, and production of infectious virus at 39.5 degrees C was also enhanced by preinfection with RAV-1, although not to the same extent as in the case of the group V mutants. We believe that the primary effect of the ts mutation is a reduced synthesis of the nucleocapsid and thus an inhibition of synthesis of all viral proteins; apparently, the accumulation of G protein at the surface is not sufficient to envelope all the viral nucleocapsids, or the mutation in the nucleocapsid prevents proper assembly of G into virions. The selection procedure, based on pseudotype formation with glycoproteins encoded by an unrelated virus, has potential use for the isolation of new glycoprotein mutants of diverse groups of enveloped viruses.     

Continuing coevolution of virus and defective interfering particles and of viral genome sequences during undiluted passages: virus mutants exhibiting nearly complete resistance to formerly dominant defective interfering particles

We quantitatively analyzed the interference interactions between defective interfering (DI) particles and mutants of cloned vesicular stomatitis virus passaged undiluted hundreds of times in BHK-21 cells. DI particles which predominated at different times in these serial passages always interfered most strongly (and very efficiently) with virus isolated a number of passages before the isolation of the DI particles. Virus isolated at the same passage level as the predominant DI particles usually exhibited severalfold resistance to these DI particles. Virus mutants (Sdi- mutants) isolated during subsequent passages always showed increasing resistance to these DI particles, followed by decreasing resistance as new DI particles arose to predominate and exert their own selective pressures on the virus mutant population. It appears that such coevolution of virus and DI particle populations proceeds indefinitely through multiple cycles of selection of virus mutants resistant to a certain DI particle (or DI particle class), followed by mutants resistant to a newly predominant DI particle, etc. At the peak of resistance, virus mutants were isolated which were essentially completely resistant to a particular DI particle; i.e., they were several hundred thousand-fold resistant, and they formed plaques of normal size and numbers in the presence of extremely high multiplicities of the DI particle. However, they were sensitive to interference by other DI particles. Recurring population interactions of this kind can promote rapid virus evolution. Complete sequencing of the N (nucleocapsid) and NS (polymerase associated) genes of numerous Sdi- mutants collected at passage intervals showed very few changes in the NS protein, but the N gene gradually accumulated a series of stable nucleotide and amino acid substitutions, some of which correlated with extensive changes in the Sdi- phenotype. Likewise, the 5' termini (and their complementary plus-strand 3' termini) continued to accumulate extensive base substitutions which were strikingly confined to the first 47 nucleotides. We also observed addition and deletion mutations in noncoding regions of the viral genome at a level suggesting that they probably occur at a high frequency throughout the genome, but usually with lethal or debilitating consequences when they occur in coding regions.