Temperature-sensitive Mutants Of Vesicular Stomatitis Virus Research Articles

The hypervariable region 1 of the E2 glycoprotein of hepatitis C virus binds to glycosaminoglycans, but this binding does not lead to infection in a pseudotype system.

The hypervariable region 1 (HVR1) of hepatitis C virus (HCV) E2 envelope glycoprotein is a 27-amino-acid sequence located at its N terminus. In this study, we investigated the functional role of HVR1 for interaction with the mammalian cell surface. The C-terminal truncated E2 glycoprotein was appended to a transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein for generation of the chimeric E2-G gene construct. A deletion of the HVR1 sequence from E2 was created for the construction of E2DeltaHVR1-G. Pseudotype virus, generated separately by infection of a stable cell line expressing E2-G or E2DeltaHVR1-G with a temperature-sensitive mutant of VSV (VSVts045), displayed unique functional properties compared to VSVts045 as a negative control. Virus generated from E2DeltaHVR1-G had a reduced plaquing efficiency ( approximately 50%) in HepG2 cells compared to that for the E2-G virus. Cells prior treated with pronase (0.5 U/ml) displayed a complete inhibition of infectivity of the E2DeltaHVR1-G or E2-G pseudotypes, whereas heparinase I treatment (8 U/ml) of cells reduced 40% E2-G pseudotype virus titer only. E2DeltaHVR1-G pseudotypes were not sensitive to heparin (6 to 50 micro g/ml) as an inhibitor of plaque formation compared to the E2-G pseudotype virus. Although the HVR1 sequence itself does not match with the known heparin-binding domain, a synthetic peptide representing 27 amino acids of the E2 HVR1 displayed a strong affinity for heparin in an enzyme-linked immunosorbent assay. This binding was competitively inhibited by a peptide from the V3 loop of a human immunodeficiency virus glycoprotein subunit (gp120) known to bind with cell surface heparin. Taken together, our results suggest that the HVR1 of E2 glycoprotein binds to the cell surface proteoglycans and may facilitate virus-host interaction for replication cycle of HCV.

Functional role of hepatitis C virus chimeric glycoproteins in the infectivity of pseudotyped virus.

The putative envelope glycoproteins of hepatitis C virus (HCV) likely play an important role in the initiation of viral infection. Available information suggests that the genomic regions encoding the putative envelope glycoproteins, when expressed as recombinant proteins in mammalian cells, largely accumulate in the endoplasmic reticulum. In this study, genomic regions which include the putative ectodomain of the E1 (amino acids 174 to 359) and E2 (amino acids 371 to 742) glycoproteins were appended to the transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein. This provided a membrane anchor signal and the VSV incorporation signal at the carboxy termini of the E1 and E2 glycoproteins. The chimeric gene constructs exhibited expression of the recombinant proteins on the cell surface in a transient expression assay. When infected with a temperature-sensitive VSV mutant (ts045) and grown at the nonpermissive temperature (40.5 degrees C), cells transiently expressing the E1 or E2 chimeric glycoprotein generated VSV/HCV pseudotyped virus. The resulting pseudotyped virus generated from E1 or E2 surprisingly exhibited the ability to infect mammalian cells and sera derived from chimpanzees immunized with the homologous HCV envelope glycoproteins neutralized pseudotyped virus infectivity. Results from this study suggested a potential functional role for both the E1 and E2 glycoproteins in the infectivity of VSV/HCV pseudotyped virus in mammalian cells. These observations further suggest the importance of using both viral glycoproteins in a candidate subunit vaccine and the potential for using a VSV/HCV pseudotyped virus to determine HCV neutralizing antibodies.

Cytoplasmic domain requirement for incorporation of a foreign envelope protein into vesicular stomatitis virus

Incorporation of human immunodeficiency virus type 1 (HIV-1) envelope proteins into vesicular stomatitis virus (VSV) particles was studied in a system that allows expressed envelope proteins to rescue phenotypically a temperature-sensitive mutant of VSV (tsO45). This mutant exhibits defective transport of its own envelope glycoprotein (G) and can be rescued by simultaneous expression of wild-type G protein from cDNA. We report here that a hybrid HIV-1-VSV protein containing the extracellular and transmembrane domains of the HIV-1 envelope protein fused to the cytoplasmic domain of VSV G protein was able to rescue the tsO45 mutant lacking the G protein, while the wild-type HIV-1 envelope protein was not. The VSV(HIV) pseudotypes obtained infected only CD4+ cells and were neutralized specifically by anti-HIV-1 sera. Our results indicate that the cytoplasmic tail of the VSV glycoprotein contains an independent signal capable of directing a foreign protein into VSV particles. The VSV(HIV) pseudotypes generated here were prepared in the absence of HIV-1 and should be useful for identifying molecules that block HIV-1 entry.

Fatty acid acylation is not required for membrane fusion activity or glycoprotein assembly into VSV virions

We have investigated the role of fatty acid acylation on two properties of the glycoprotein (G protein) from the Indiana serotype of vesicular stomatitis virus (VSV). Using a mutated G protein described previously (CS-2) that is not palmitylated, we found that fatty acid acylation was not required for the low pH-induced membrane fusion activity of VSV G protein. Transient expression of CS in HeLa cells resulted in syncytia formation that was indistinguishable from that induced by wild-type G protein. In addition, we found that expression of CS complemented a temperature-sensitive mutant of VSV (tsO45) as well as the wild-type protein. These results indicate that the presence of palmitate on the cytoplasmic domain of VSV G protein is not required for any step in the life cycle of the virus.

Inhibition of Primary Transcription of Vesicular Stomatitis Virus by Prostaglandin A1

Prostaglandin A (PGA) exhibits antiviral activity against RNA and DNA viruses. The effect of PGA1 on vesicular stomatitis virus (VSV) was investigated. When VSV-infected L-1210 cells were kept in the presence of PGA1 the amount of all five viral proteins and their respective mRNAs was dose-dependently decreased. To determine whether the effect was on viral transcription or translation, the temperature-sensitive VSV mutant tsG 41 was employed. This is a good model system for the investigation of primary transcription; at the restrictive temperature of 39 degrees C, tsG 41 is unable to replicate but can transcribe viral mRNA. Mutant mRNA synthesis was strongly inhibited by PGA1 at this temperature, indicating that the major effect is on primary transcription. This conclusion is supported by data demonstrating that in vitro transcription of viral genomic RNA was also inhibited by PGA1.

The Nucleocapsid Protein of Vesicular Stomatitis Virus Isolated from the Brains of Nude Mice is Responsible for Abated Viral RNA Synthesis at the Normal Body Temperature of Mice

Six temperature-sensitive mutants of vesicular stomatitis virus (VSV) were isolated from the central nervous system (CNS) of athymic nude mice. The nude mice had been reconstituted with syngeneic T lymphocytes and then infected with a temperature-sensitive mutant of VSV, tsG31-KS5 VSV, for 20 days. In BHK-21 cells incubated at 38 degrees C, the normal body temperature of mice, all six CNS virus clones had diminished RNA synthesis, when compared to RNA production in BHK-21 cells incubated at 31 degrees C. In contrast, the original tsG31-KS5 VSV mutant synthesized more RNA at 38 degrees C than it did at 31 degrees C. In vitro transcription assays were exploited to discern which viral protein(s) was functionally accountable for the abated synthesis of RNA of the CNS VSV isolates. The ribonucleoprotein complexes from the CNS VSV isolates were disrupted and template (N protein and RNA) and enzyme (L and NS proteins) fractions were purified. In vitro transcription assays were performed with template fractions of the brain isolates, added to enzyme fractions either wild-type wt VSV or tsG31-KS5 VSV, or with template fractions of wt VSV or tsG31-KS5 VSV mixed with enzyme fractions of the CNS isolates. The template fraction was responsible for the decrease in RNA synthesis in all six of the brain-isolated clones. When the template fractions of wt or tsG31-KS5 VSV were mixed with enzyme fractions of all the CNS-derived VSV, except BP5A VSV, leader sequence RNA and large Mr transcripts were transcribed. One clone, BP5A VSV, did not synthesize RNA when mixed with either template or enzyme of wt VSV, and probably had more than one functional mutation that influenced viral RNA synthesis.

Viral transcription is necessary and sufficient for vesicular stomatitis virus to inhibit maturation of small nuclear ribonucleoproteins

Infection of baby hamster kidney cells with vesicular stomatitis virus (VSV) results in the accumulation of immature U1 and U2 small nuclear ribonucleoproteins (snRNPs) that contain precursor U RNAs and at least some of the proteins specific for U1 and U2 snRNAs but lack the Sm complex of proteins that is common to these U snRNAs. The VSV function required for this effect is not known, but direct inhibition of cellular transcription did not alter the maturation of U1 and U2 snRNPs. On the other hand, viral transcription but not viral translation was required to inhibit U1 and U2 snRNP maturation. Temperature shift experiments with the mutant G114 showed that ongoing viral transcription was necessary, but that viral mRNA was not required for this inhibition. Furthermore, the VSV function involved in the inhibition of maturation of U1 and U2 snRNPs had a small UV target size of approximately 10 to 20 nucleotides. We demonstrate that temperature-sensitive mutants of VSV can be used as a tool to initiate the assembly of snRNPs in infected cells. These results are compatible with the suggestion that perturbation of snRNP metabolism by VSV precedes and is distinct from the effect of VSV on cellular RNA synthesis, although VSV leader RNA may be involved in both these functions.

Structural effects of amino acid substitutions on the matrix protein of vesicular stomatitis virus

The structural effects of amino acid substitutions for Gly at position 21 in the amino-terminal segment (Lys 15-Pro 26) of the matrix (M) protein of vesicular stomatitis virus (VSV) have been investigated using conformational energy analysis. Monoclonal antibody-binding experiments and protein digestion studies of the M protein indicate that this segment is important to its ribonucleoprotein recognition and its transcription-inhibitory activity. Temperaturesensitive mutants of VSV that do not bind monoclonal antibody and that are devoid of transcription-inhibitory activity are known to have the substitution of Glu for Gly at position 21. The current findings demonstrate a significant conformational change at position 21 induced by the substitution of Glu for Gly, which could explain this alteration in antibody binding and transcription-inhibitory activity. Furthermore, the results indicate that the substitution of any noncyclic L-amino acid for Gly at position 21 may be expected to produce similar changes in M protein function.

Neuropeptide-induced hypothermia and the course of central nervous system disease mediated by temperature-sensitive mutants of vesicular stomatitis virus.

Mice inoculated with many temperature-sensitive (ts) vesicular stomatitis virus (VSV) mutants incur a less aggressive disease than mice infected with wild-type VSV. The normal body temperature of mice, 38 degrees C, is not a permissive temperature for replication of the temperature-sensitive VSV mutants in cell culture. To determine whether the body temperature of mice caused the alteration in disease states, a neuropeptide that induces hypothermia in rodents was injected into mice before their infection with a temperature-sensitive VSV mutant. Only 1.0 ng of the neuropeptide neurotensin, injected intracerebroventricularly, was required to lower the core temperatures of mice an average of 2.5 degrees C. A single injection of neurotensin before infection with tsG31 VSV (complementation group III) dramatically altered the course of disease. Without neurotensin only 3% of the mice infected with tsG31 VSV died, but when neurotensin was administered 24 h before the inoculation of the tsG31 VSV, 80% of the mice died. The course of disease in mice produced by infection with another temperature-sensitive VSV mutant, tsG11 VSV (complementation group I), also was altered when neurotensin was injected before inoculation of the virus. Instead of 3% of the mice dying as in a normal infection with tsG11 VSV, treatment with neurotensin before inoculation produced a rapidly fatal disease, killing 90% of the mice.

Direct visualization of protein transport and processing in the living cell by microinjection of specific antibodies

We have prepared polyclonal antibodies to the cytoplasmic portion of the envelope glycoprotein G of vesicular stomatitis virus (VSV) by using synthetic peptides corresponding to either the 22 or 11 ultimate carboxy-terminal residues of the G as immunogens. When antibodies to the 22 residue peptide are microinjected into monolayer baby hamster kidney cells before or shortly after infection with wildtype VSV, G protein accumulates in large intracellular patches and little G is observed in the Golgi complex or at the cell surface. In contrast, when antibodies to the 11 residue peptide are injected, no such patches are observed and G protein is seen colocalized with the injected antibody at the endoplasmic reticulum, in the Golgi complex, in transport vesicles, and at the plasma membrane. Microinjection of these antibodies does not disturb the pathway or kinetics of G-protein transport. In cells infected with a temperature-sensitive mutant of VSV, 045, the glycoprotein accumulates in the endoplasmic reticulum at 39.8°C, but rapidly moves through the Golgi apparatus and then to the cell surface after a temperature shift-down to 32°C. Using rhodamine-coupled antibodies to the 11 residue peptide, a microscope stage equipped for precise temperature control, and a silicon intensifier target video camera, we can visualize by video light microscopy the synchronized exocytotic transport of the G protein directly in the living cell.

Prolonged infection of L cells with vesicular stomatitis virus Defective interfering forms and temperature-sensitive mutants as factors in the infection

When L y cells were treated with 100 units per milliliter of mouse interferon and then infected with vesicular stomatitis virus (VSV) at a multiplicity of 10–60 PFU/cell, a prolonged infection of cultures ensued, lasting in one case longer than 60 days. After four passages in L y cells at high multiplicities of VSV from a prolonged infection, there was no inhibition of virus growth, and virus from the prolonged infection had the same distribution of intracellular and extracellular RNA forms as was found in wild-type virus passed four times in L y cells. Also, the intracellular and extracellular RNA forms of virus taken directly from a prolonged infection of L y cells were indistinguishable from those of the wild-type virus. A morphological examination of VSV from a prolonged infection did not show a significant number of small defective viral forms after four passages in L y cells at a high multiplicity of infection. There was, however, evidence for the emergence of temperature-sensitive mutants of VSV during the course of the prolonged infection. Changing the incubation temperature of the cultures from 37° to 32° resulted in an increase in virus production and virus-induced cytopathology. The virus produced during prolonged infections grew to higher titers at 32° than at 37° and its plaque size at 39° progressively decreased with the length of time that the infection persisted. Furthermore, interferon production also seemed to have a role in the persistence of infections such as the treatment of cultures with rabbit anti-mouse interferon globulin and resulted in a significant rise in virus production and viral cytopathology in the cell cultures.

Vesicular stomatitis virus glycoprotein is necessary for H-2-restricted lysis of infected cells by cytotoxic T lymphocytes.

Vesicular stomatitis virus (VSV) elicited cytotoxic thymus-derived lymphocytes (CTLs) in mice of the BALB/c and three congenic strains (BALB.b, BALB.k, BALB.HTG). CTL lysis of VSV-infected fibroblasts from the four strains was restricted by the target cells' major histocompatibility complex (H-2). Target cells were also infected with two temperature-sensitive mutants of VSV, tsM and tsG in which, respectively, the viral matrix protein and glycoprotein are not expressed at 39 degrees (restrictive temperature) on the infected cell's surface membrane. At the restrictive temperature, cells infected with wild-type VSV or tsM were lysed by CTLs, but cells infected with tsG were not. The requirement for the glycoprotein on the target cell was also evident from the ability of antisera to the glycoprotein to block completely CTL lysis of VSV-infected cells.

Inhibition of mouse L cell protein synthesis by ultraviolet-irradiated vesicular stomatitis virus requires viral transcription

Ultraviolet-irradiated vesicular stomatitis (VS) virus (Indiana serotype) was used to determine which viral gene functions are required for expression of cellular protein synthesis inhibition. To carry out this experiment a method, described in this report, was employed that allows the simultaneous quantitation of viral and host protein synthesis. We demonstrated earlier using ultraviolet irradiation that to express cell killing, only about one-fifth of the VS virus genome corresponding to proteins N and NS need be transcribed. Previous results using temperature-sensitive mutants of VS virus indicated that inhibition of protein synthesis and cell killing are conjointly expressed. In this report it is demonstrated that the inhibition of cellular protein synthesis by a high multiplicity ( m pfp = 100) of uv-irradiated VS virus, like cell killing, requires the transcription and translation of N (and possibly NS) protein.

Mechanism of formation of pseudotypes between vesicular stomatitis virus and murine leukemia virus

Pseudotypes of vesicular stomatitis virus (VSV) and Moloney murine leukemia virus (MuLV), defined by their resistance to neutralization by anti-VSV antiserum, are released preferentially at early times after infection of MuLV-producing cells with VSV. At later times, after synthesis of MuLV proteins has been inhibited by the VSV infection, neither MuLV virions nor the VSV(MuLV) pseudotypes are made. Infection of MuLV-producing cells with mutants of VSV having temperature-sensitive lesions in either G or M protein does not generate pseudotypes at nonpermissive temperature, indicating that both proteins are needed for pseudotypes to form. Although the pseudotypes resist neutralization by anti-VSV serum, they are inactivated by anti-VSV serum plus complement, and they can be precipitated by rabbit anti-VSV serum plus goat anti-rabbit IgG. These results, coupled with experiments using a temperature-sensitive mutant of VSV G protein grown at partly restrictive temperature, suggest that small numbers of VSV G protein are obligately incorporated into VSV(MuLV) pseudotypes. There appears to be a stringent requirement for recognition of the viral core by homologous envelope components as the nucleating step in the budding process. Only after such a nucleation can the envelope components of the second virus substitute into the membrane of the budding particle.