Studies of L cells persistently infected with VSV: factors involved in the regulation of persistent infection.

Infection of interferon-treated L cells with VSV led frequently to the establishment of L cells persistently infected with VSV (LVSV cells). These cells were characterized by the following properties; (I) no supplement of antiviral factors such as anti-VSV antiserum, interferon, was required for their maintenance; (2) virus antigens were detected in about 5 to 30% of the cells by immunofluorescence staining; (3) the cells were not only resistant to superinfection by homologous virus, but also resistant to challenge by heterologous viruses such as Mengo virus; (4) the cells were destroyed by co-cultivation with heterologous cells susceptible to VSV infection; (5) the cells could be cured by serial cultivation in medium containing antiviral antibody, and the cured cells were as susceptible to VSV as normal L cells. It was shown that at least three factors (interferon, defective interfering [DI] particles and a selection of small-plaque temperature-sensitive [ts] mutants) took part in the maintenance of LVSV cells although it was difficult to evaluate exactly the relative importance of these factors. The effect of antiviral antibody, interferon and incubation temperature upon the maintenance of LVSV cells are discussed further.     

Antigenic variants are not selected during persistent infection with Theiler's virus.

In this study, we demonstrated that non-neutralizable antigenic variants of Theiler's murine encephalomyelitis virus do not predominate during the course of chronic relapsing infection.     

Properties of temperature-sensitive mutants of parainfluenza virus type 3 selected during the course of persistent infection

Summary We investigated properties of thets mutants that were selected during the course of persistent infection of Vero cells by parainfluenza virus type 3. The mutants demonstrated leakiness when infecting cells at high MOI and interfered with the growth of wild type virus, apparently by inhibiting a step prior to RNA synthesis.With 1 Figure     

Polykaryocyte formation induced by VSV in mouse L cells.

Infection of mouse L cells with VSV leads to the formation of polykaryocytes about 4 to 12 h p.i. When anti-VSV immune serum was added during the course of infection, progression of cell fusion was soon suppressed. Cycloheximide completely suppressed the cell fusion when the drug was added within 1 h p.i., while the cell fusion was not suppressed at all when the drug was added at and after 3 h. Early polykaryocyte formation, 'fusion from without', was observed only at a low level in cells infected at very high multiplicities. The development of cell fusion induced by VSV was found to be different in several cell types, although all these cells produced a rather high yield of virus: L and C-243-3 mouse cell lines showed a high level of polykaryocytosis (80 to 100%), BHK and RK-13 cells responded at low level, and PS and Vero cells showed no cell fusion in response to VSV infection. In PS cells, however, cell fusion occurred when VSV-infected L cells were co-cultivated. From these observations, the mechanism of cell fusion induced by VSV was discussed.     

Selection of a mutant S1 gene during reovirus persistent infection of L cells: role in maintenance of the persistent state

LR-7 cells, variant L cells derived from a type 3 reovirus persistently infected (p.i.) carrier culture (R. Ahmed, W. M. Canning, R. S. Kauffman, A. H. Sharpe, J. V. Hallum, and B. N. Fields, Cell 25, 325-332, 1983) were used to define the viral genes critical for maintenance of the persistent state. A cloned viral isolate (L/C virus) derived from the p.i. culture replicated normally in LR-7 cells, while wild-type (wt) viruses of the three reovirus serotypes replicated less efficiently. To identify the viral gene(s) permitting enhanced replication of L/C virus in LR-7 cells, viral reassortants were prepared by mixed infection of L cells with L/C virus and type 1 wt. Study of the one-step growth curves and final yields of large numbers of reassortants in both L cells and LR-7 cells revealed that the presence of the S1 gene from L/C virus was critical for normal viral replication in LR-7 cells. However, this phenotype was suppressed by the simultaneous presence in reassortants of both the M2 and S4 genes from the type 1 wt parent. The critical change in the S1 gene occurred by passage 13 (63 days) after initiation of the carrier culture. Although multiple mutations are present in the viral population from p.i. cultures, certain specific mutations can be identified as critical for maintenance of the persistent state.     

Recombinant Sendai viruses with L1618V mutation in their L polymerase protein establish persistent infection, but not temperature sensitivity

The Sendai virus pi strain (SeVpi) isolated from cells persistently infected with SeV shows mainly two phenotypes: (1) temperature sensitivity and (2) an ability of establishing persistent infection (steady state). Three amino acid substitutions are found in the Lpi protein and are located at aa 1088, 1618, and 1664. Recombinant SeV(Lpi) (rSeV(Lpi)) having all these substitutions is temperature sensitive and is capable of establishing persistent infection (steady state). rSeVs carrying the fragment containing L1618V show both phenotypes. rSeV(L1618V), in which leucine at aa 1618 is replaced with valine, has the ability of establishing persistent infection, but is not a temperature-sensitive mutant, indicating that the ability of a virus to establish persistent infection can be separated from temperature sensitivity. The amino acid change at 1618(L-->V) coexisting with aa 1169 threonine is required for acquirement of a temperature-sensitive phenotype. Three amino acid substitutions are also found in the Ppi protein, but rSeV(Ppi) does not show these phenotypes.     

Quantitative electron microscopic analysis of the penetration of VSV into L cells

Vesicular stomatitis virus (VSV) has been reported to penetrate L cells both by viropexis and by fusing with the cellular plasma membrane. The procedures used to produce these apparently contradictory results were significantly different. Quantitative electron microscopic methods were used to analyze the effect of these different procedures and the presence or absence of serum on the adsorption and penetration of VSV into L-929 cells. When penetration was analyzed following adsorption in the cold, VSV entered cells almost exclusively by viropexis. There was a quantitative relationship between the disappearance of extracellular attached virus and the appearance of virus in invaginations, and, somewhat later, in small intracellular vacuoles. Fusion was only rarely observed. When the multiplicity was lowered to 10–15 particles per cell, the same findings were obtained. When adsorption was achieved by centrifuging virus-cell mixtures at 18,000 g for 15 min, viropexis was again the predominant mode of penetration, but fusion occurred at a significant level, indicating that centrifugation per se played a role in the incidence of fusion. In all cases, serum reduced the amount of virus adsorbed, but did not affect the mode of penetration.     

Polypeptide synthesis in alphavirus-infectedAedes albopictus cells during the establishment of persistent infection

Summary Polypeptide synthesis was examined in mosquito cells during the establishment of a persistent infection with two alphaviruses, Ross River virus (RRV) and Semliki Forest virus (SFV), and in vertebrate cells cytopathically-infected with the same viruses.InAedes albopictus cells, RRV reached peak titres at 34–48 hours p.i. At 12 hours 85 per cent of cells assayed as infected by infective centre assay; by 48 hours when persistence was established, virus production was reduced and <5 per cent of cells assayed as infected. There was no shut-down of host polypeptide synthesis during infection. Viral polypeptide synthesis was maximal between 10 and 24 hours p.i. The major viral polypeptides labelled were nucleocapsid protein and envelope protein(s). The precursor polypeptide p95 which was prominent in infected BHK cells was not detected in mosquito cells. Similar results were obtained on SFV infection.During the establishment of persistence there was a coordinate decline in the synthesis of RRV polypeptides, reaching undetectable levels by 72 hours p.i. Subculturing persistently-infected cells led to a small increase in viral polypeptide synthesis and virus titre.In contrast, during RRV growth in BHK cells host protein synthesis was severely inhibited and by 9–11 hours p.i. virus-specific polypeptide synthesis represented more than 90 per cent of total protein synthetic activity.With 6 Figures     

Effects of DEAE-dextran on infection and hemolysis by VSV. Evidence that nonspecific electrostatic interactions mediate effective binding of VSV to cells

The polycation DEAE-dextran increased the binding of VSV to BHK cells about fourfold over a wide range of VSV concentrations. The same proportion of bound virions was internalized by the cell in the presence or absence of DEAE-dextran. Viral primary RNA production was increased to the equivalent of a 4- to 4.5-fold increase in multiplicity of infection in the presence of DEAE-dextran, closely paralleling the increase in total VSV binding to the cell. Viral secondary RNA production was increased only to the equivalent of about twofold increase in multiplicity. The kinetics of both primary and secondary RNA production were indistinguishable in the presence or absence of DEAE-dextran. DEAE-dextran had to be present simultaneously with the input virions in order to enhance RNA production; addition even 30 min after infection was ineffective. Addition of the polycation DEAE-dextran was also required for appreciable VSV-induced hemolysis of human erythrocytes; erythrocytes of several other species were hemolyzed in the absence of DEAE-dextran, but hemolysis was enhanced by its presence. Maximal binding and hemolysis occurred at pH 5.0 and 37 degrees. Vesicles containing only G protein and viral lipid were 40% as hemolytic as intact virions at pH 5.0, but were inactive at pH 6.0; "spikeless" virions lacking G, or protein-free viral lipid vesicles were not hemolytic, showing that G protein is necessary for hemolysis. These results, together with other recent observations, suggest that multiple electrostatic interactions between VSV and the cell surface, rather than an affinity for specific surface molecules, mediated the productive (infection producing) binding of VSV to the cell surface.     

Tick-borne encephalitis virus NS1 glycoprotein during acute and persistent infection of cells.

Tick-borne encephalitis virus (TBEV) was propagated in porcine embryo kidney (PS) cells until 48 h whereas human kidney (RH) cells maintained the virus persistence during at least 2 months. One of possible reasons of flavivirus chronic infection might be abnormal NS1 gene expression. Immunoblotting with monoclonal antibodies (MAbs) revealed the similarity of the intracellular and secreted NS1 nonstructural glycoprotein size and linear antigenic determinants in both the infected cell lines. However, according to the competitive binding of MAbs with the TBEV NS1 extracellular glycoprotein, its contiguous epitopes differed for acute or persistent infection. To map the TBEV NS1 glycoprotein antigenic determinants its recombinant analogues were used. All the studied MAbs could bind with the full-length NS1 recombinant protein. Deletion of the TBEV NS1 gene internal region resulted in defective NS1d1 protein without the region between 269 and 333 a.a. Lack of NS1d1 binding with 20B4 MAb and diminished binding with 22H8 and 17C3 MAbs permitted to map their antigenic determinants within or nearby deleted region, respectively. Interaction of other MAbs with the NS1 and NS1d1 recombinant proteins did not differ, suggesting that their epitopes were located in the region of N-terminal 268 a.a. or C-terminal 19 a.a. of the TBEV NS1 protein. The second NS1d2 truncated protein contained the first N-terminal 33 a.a. of the TBEV NS1 protein and was able to bind with 29G9 MAb. Taken together the data stand for the differences in the N-terminal structure of the TBEV NS1 multimers secreted from the acute and persistent infected cells whereas the intracellular and secreted monomer processing was the same. The modified NS1 protein oligomers in the RH cellular line might slow virus replication and could result in the TBEV persistence.     

Genetic variation during persistent reovirus infection: isolation of cold-sensitive and temperature-sensitive mutants from persistently infected L cells

We have examined the evolution of reovirus in two independently established persistently infected (p.i.) cell lines. We found that reovirus undergoes extensive mutation during persistent infection in L cells. However, there was no consistent pattern of virus evolution; in one p.i. cell line temperature-sensitive (ts) mutants were selected, whereas cold-sensitive (cs) mutants were isolated from the second p.i. culture. Neither the cs nor the ts mutants isolated from the carrier cultures expressed their defect at 37 degrees, the temperature at which the p.i. cells were maintained, indicating that the cs and ts phenotypes were nonselected markers. These results emphasize the point that emergence of the ts or cs mutants during persistent infection only signifies that the virus has changed; it does not necessarily imply that the particular mutant is essential for the maintenance of the persistent infection. Given the high mutation rate of viruses, and the wide spectrum of viral mutants present in carrier cultures, it is essential to distinguish the relevant changes from those which may simply represent an epiphenomenon. In the accompanying paper (R. S. Kauffman, R. Ahmed, and B. N. Fields Virology, 130, 79-87, 1983), we show that by using a genetic approach, it is possible to identify the viral gene(s) which are critical for the maintenance of persistent reovirus infection.     

Bovine viral diarrhea virus quasispecies during persistent infection.

Analysis of viral genome sequences from two calves persistently infected with bovine viral diarrhea virus revealed a quasispecies distribution. The sequences encoding the glycoprotein E2 were variable, translating to a number of changes in predicted amino acid sequences. The NS3 region was found to be highly conserved in both animals. The number of E2 clones showing variant amino acids increased with the age of the animal and comparison of the consensus sequences at the different time points confirmed differences in the predicted E2 sequences over time. The immune tolerance that allows the lifelong persistence of this viral infection is highly specific. It is likely that some of the variant viruses generated within these animals will differ antigenically from the persisting virus and be recognized by the immune system. Evidence of an immune response to persisting virus infection was gathered from a larger sample of cattle. Serum neutralizing antibodies were found in 4 of 21 persistently infected animals. Accumulations of viral RNA in the lymph nodes of all animals examined, particularly in the germinal center light zone, may represent antigenic variants held in the form of immune complexes on the processes of follicular dendritic cells.     

Cyclic rickettsemia during persistent Anaplasma marginale infection of cattle.

Submicroscopic levels of Anaplasma marginale rickettsemia in persistently infected cattle were determined by using nucleic acid hybridization. Within individuals, the rickettsemia levels steadily increased from less than 10(4) infected erythrocytes per ml to a peak of more than 10(6) infected erythrocytes per ml and then rapidly declined. This logarithmic variation parallels the variation of the rickettsemia level seen in acute infection and suggests that cyclic emergence of antigenic variants is a mechanism of rickettsial persistence.     

Variable infection of Vero cells and homologous interference after co-cultivation with HeLa cells with persistent defective infection by Edmonston measles virus.

The HeLa subline K11A-HG-1 (line of HeLa cells persistently infected with Edomonston measles virus but containing little or no transmissible infectious virus) was co-cultivated with Vero cells. Focal syncytia were formed containing measles antigen and accumulations of nucleocapsid-like structures with no detectable production of transmissible infectious virus or positive hemadsorption. The infection aborted between 2 and 3 weeks after preparation of co-cultures. Upon subculture of co-cultures, occasionally complete infections (progressive syncytial degeneration, hemadsorption, and production of transmissible infectious virus) appeared. A linear dose response curve for nontransmissible infection was obtained along with evidence that measles antigen had to be present on the surface of K11A-HG-1 cells for their infectivity for Vero cells. The basis for initiation of Vero cell infection by living K11A-HG-1 cells, but not by nonviable intact K11A-HG-1 cells killed by a virus-preserving technique, nor by disrupted K11A-HG-1 cells, is, at present, a matter of speculation. However, several lines of evidence were obtained which suggested that subsequent development of delayed variable transmissible Vero cell infection occurred because of a type of viral interference, including the presence of an inhibitor in K11A-HG-1 cultures, the bulk of which was cell-associated.     

Characterization of persistent poliovirus mutants selected in human neuroblastoma cells

Six Sabin-derived persistent poliovirus mutants were selected in human neuroblastoma IMR-32 cells. The mutants had a titer 30 to 10⁵ times lower in nonneural HEp-2c cells than in IMR-32 cells. When the growth cycles of persistent viruses in the two cell lines were compared, the most striking feature was a delay of 2 to 4 hr in virus release from HEp-2c cells. In HEp-2c cells, type 1 mutants could spontaneously establish a persistent infection in the absence of any exogenous viral inhibitor. Mutations at a rate of 1 every 210 nucleotides had accumulated in the genome of the type 1 mutants selected in neuroblastoma cells, modifying cell specificity and conferring the ability to persist in some non-neural cells. These results indicate that mutants of poliovirus with highly modified biological properties can be selected in vitro in cells of neural origin.     

Inducible Vesicular Stomatitis Virus (VSV) L Cell Line for Packaging of Recombinant VSV

Recently, recombinant vesicular stomatitis viruses (VSV) have been developed as high-level expression vectors which serve as effective vaccine vectors in animals. An ideal approach for VSV vector production would be the development of stable packaging cell lines that can produce vector particles without transfection step. In this report, we describe generation of an inducible cell line that expresses the VSV polymerase gene (L) under the control of the reverse tetracycline-controlled transactivator (rtTA) system as a first step to make VSV-based packaging cell lines. Integrated polymerase (L) gene was controlled by an rtetR-dependent promoter in the rtTA-producing BHK cell line. When the cell lines were cultured in the presence of tet (tetracycline) or tetracycline derivative doxycycline, the recombinant VSV and wild type VSV were replicated, whereas in the absence of tet or tetracycline derivative doxycycline, the recombinant VSV was not replicated. Viral supernatants were harvested, diluted, and monitored by plaque assay for the presence of infectious VSV. Plaques of VSV containing an additional sequence encoding the EGFP protein allowed rapid detection of infection. Our results suggest wide applications of other surrogate viruses based on VSV. The availability of stable packaging cell lines represents a step toward the use of a VSV vector delivery system that can allow scale-up production of vector-stocks for gene therapy.     

Functional impairment of CD8(+) T cells by regulatory T cells during persistent retroviral infection

The establishment of viral persistence generally requires evasion of the host CD8(+) T cell response. Here we describe a form of evasion wherein the CD8(+) T cells are fully capable of recognizing their cognate antigen but their effector functions are suppressed by regulatory T cells. Virus-specific CD8(+) T cells adoptively transferred into mice persistently infected with Friend virus proliferated and appeared activated, but failed to produce IFNgamma or reduce virus loads. Cotransfer experiments revealed that a subpopulation of CD4(+) T cells from persistently infected mice suppressed IFNgamma production by the CD8(+) T cells. Treatment of persistently infected mice with anti-GITR antibody to ameliorate suppression by regulatory T cells significantly improved IFNgamma production by transferred CD8(+) T cells and allowed a significant reduction in viral loads. The results indicate that CD4(+) regulatory T cells contribute to viral persistence and demonstrate an immunotherapy for treating chronic retroviral infections.