Inhibition of pseudorabies virus replication by vesicular stomicles virus I. Activity of infectious and inactivated B particles.

Infectious B particles of vesicular stomatitis virus (VSV) are capable of inhibiting the replication of pseudorabies virus (PSR) in a variety of cell lines. Even under conditions of an abortive infection in a continuous line of rabbit cornea cells (RC-6O), B particles interfere with the replication of PSR with high efficiency. Particle per cell dose-response analysis of B particle populations revealed that the number of VSV particles capable of inhibiting PSR replication exceeds the number of PFU by a factor of 32 to 64. When B particles are treated with UV irradiation, a drastic increase in the multiplicity of infection is required to inhibit PSR replication. Whereas one infective B particles per cell is sufficient to prevent replication of PSR, 800 to 1,000 VSV particles rendered noninfective by UV irradiation are required to compensate for the loss of VSV synthetic activity that results from irradiation. Temperature-sensitive mutants representing five complementation groups of VSV were tested at low multiplicities of infection for their effect on PSR replication at the nonpermissive temperature. Generally, the ability of the different complementation groups to amplify virion products at the nonpermissive temperature is associated with their ability to inhibit PSR replication. These results imply that at low multiplicities of infection, amplification of infecting VSV components is necessary for inhibition of PSR replication., but at high multiplicities of infection with VSV, a virion component can prevent PSR replication in the absence of de novo VSV RNA or protein synthesis.     

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.     

Formation of Viral Ribonucleic Acid and Virus in Cells that are Permissive or Nonpermissive for Murine Encephalomyelitis Virus (GDVII)

GDVII virus growth in BHK-21 cells, a permissive host for the virus, resembled productive infections with other picornaviruses. Virus yields ranged from 100 to 600 plaque-forming units (PFU)/cell. Virus replication in HeLa cells, a nonpermissive host for GDVII virus, was characterized by virus yields of only 0.1 to 5 PFU/cell. Similar low yields of virus have been obtained from HeLa cells at all multiplicities of input up to 6,000 per cell. The progeny particles from HeLa cells were, like the infecting particles, restricted in the HeLa cell host. Despite the great difference in final yields of virus from BHK-21 and HeLa cells, the times when maximal yields were reached were similar. GDVII virus stock grown in BHK-21 cells was designated HeLa(-). A variant of GDVII virus which is capable of extensive growth in HeLa cells was obtained. This variant, designated HeLa(+) GDVII virus, was passaged serially in HeLa cells. Virus yields of 50 to 150 infective virus particles per cell were obtained from infection of HeLa cells with HeLa(+) GDVII virus. The major species of HeLa(+) virus-specific ribonucleic acid (RNA) produced was single stranded and sedimented with an S value of 35S. The rate of accumulation of HeLa(+) virus-specific RNA in HeLa cell cultures was about four times that of HeLa(-) RNA. The amount of virus-specific HeLa(+) RNA formed in HeLa cells was several-fold greater than that of HeLa(-) RNA. With HeLa(-) parent GDVII virus undergoing productive replication in BHK-21 cells or abortive replication in HeLa cells, the major species of virus-specific RNA produced was single stranded and sedimented with an approximate S value of 35S. The amount of HeLa(-) virus-specific RNA extracted from BHK-21 cells was several-fold greater than the amount obtained from HeLa cells.     

Vesicular stomatitis virus in Drosophila melanogaster cells: lack of leader RNA transport into the nuclei and frequent abortion of the replication step.

In cultured Drosophila melanogaster cells, vesicular stomatitis virus (VSV) establishes a persistent, noncytopathic infection. No inhibition of host macromolecular synthesis occurs. We studied the synthesis of VSV plus-strand leader RNA, which may be directly involved in vertebrate host synthesis shut-off. Leader RNA accumulated in Drosophila cell cytoplasm, but in low amounts, it was either free or associated to structures larger than the leader RNA-N protein complexes found in vertebrate cells. Only a few leader RNA copies migrated into the cell nucleus; no increase of this transport was observed at any time during the virus cycle. Viral RNAs complementary to the 3' end of the genome and ranging in size from the leader to several hundred nucleotides were found to accumulate in Drosophila cell cytoplasm. Their synthesis was inhibited in the presence of cycloheximide, which blocks all protein synthesis and VSV replication. Correlation between the absence of VSV cytopathogenicity in Drosophila cells and the lack of leader RNA transport into their nuclei is discussed, as well as the possible relationship between the restriction of viral synthesis and the frequent initiation of an abortive replication step.     

Inhibition of vesicular stomatitis virus replication in dexamethasone-treated L929 cells

We previously demonstrated that dexamethasone treatment of L929 cells inhibited plaque formation by vesicular stomatitis virus (VSV), encephalomyocarditis virus, or vaccinia virus. We now have characterized the antiviral effects of glucocorticoids in L929 cells. Dexamethasone did not directly inactivate VSV nor did steroid treatment of L929 cells affect virion adsorption or penetration. The VSV yield in L929 cells treated with dexamethasone for a period of only 4 or 8 hr was decreased by 50% when cells were infected the day following steroid treatment. Treating L929 cells with dexamethasone for a longer period resulted in greater inhibitions of virus synthesis. Interferon activity (less than 5 units/ml) was not detected in L929 cell culture fluids and cell sonicates from steroid-treated cells and the addition of antiserum to murine alpha/beta-interferon had no effect on the ability of dexamethasone to inhibit VSV replication. Dexamethasone treatment of L929 cells did not induce the production of double-stranded RNA-dependent protein kinase but did result in a slight elevation of 2-5A oligoadenylate synthetase activity, two enzymatic activities associated with the antiviral state induced by interferon. However, the elevated 2-5A synthetase activity was not associated with an inhibition of VSV RNA accumulation in dexamethasone-treated L929 cells. By contrast, the synthesis of all five VSV proteins was reduced by 50-75% in dexamethasone-treated L929 cells as early as 4 hr after infection. Thus, the dexamethasone-mediated inhibition of VSV replication in L929 cells is associated with decreased production of VSV structural proteins.     

Sensitivity of Ribonucleic Acid and Deoxyribonucleic Acid Viruses to Different Species of Interferon in Cell Cultures

Although two deoxyribonucleic acid (DNA) viruses, pseudorabies (PsRV) and vaccinia, are as susceptible as a ribonucleic acid (RNA) virus, vesicular stomatitis (VSV), to interferon when tested in chicken or mouse cells, they are refractory to inhibition in interferon-treated primary rabbit kidney cells and in a continuous line (RK-13) of rabbit kidney cells. Superinfection with VSV of RK-13 cells first infected with PsRV completely blocks the replication of PsRV with no effect on VSV yield. When the same experiment is carried out in RK-13 cells pretreated with 1,000 units of interferon, VSV replication is inhibited, which permits PsRV to replicate normally. These findings demonstrate that in the same cell one virus (PsRV) can be refractory to interferon and a second virus (VSV) can be susceptible. These experiments show that rabbit kidney cell cultures are deficient in the synthesis of resistance factors active against the DNA viruses tested and raise the possibility that separate resistance factors may exist for RNA and DNA viruses. In the case of sequential infection of interferon-treated RK-13 cells with vaccinia and VSV, it was found that not only was vaccinia replication refractory to inhibition by interferon, but also that prior infection with vaccinia was able to partially reverse the effect of the inhibitor on the replication of the VSV used for superinfection. On the basis of these and other data it is postulated that a vaccinia virion component or a replication product of vaccinia virus, or both, enables VSV to escape the inhibiting action of interferoninduced resistance factors.     

The induction of interferon by vesicular stomatitis virus in mouse L cells.

Although no detectable interferon was produced when L cells were infected with wild-type VSV (VSV-o), considerable amounts of interferon were produced when cells were infected with UV-irradiated VSV-o at a multiplicity equivalent to 10 PFU/cell. Treatment of VSV-o with UV-light resulted in the marked reduction of the RNA synthesizing capacity and cytotoxity of the virus, and the UV-irradiated virus had neither infectivity nor interfering activity against homologous viruses. The amount of interferon induced by UV-VSV-o was markedly influenced by multiplicity of infection and incubation temperature. Less-virulent temperature-sensitive mutants (VSV-mp and VSV-sp) derived from L cells persistently infected with VSV induced interferon in L cells without treatment of the viruses with UV-light, but these viruses could not induce interferon if the infected cells were incubated at nonpermissive temperature, or if cells were infected at multiplicities of more than 10 PFU/cell. On the other hand, it was shown that treatment of cells with cycloheximide (100 μg/ml) delayed the expression of cell damage caused by non-irradiated VSV-o and resulted in the production of interferon when cycloheximide was removed from the cultures. These results indicate that VSV has intrinsically interferon-inducing capacity in L cells and can induce interferon if the induction is carried out under such condition that cell damage caused by VSV are suppressed or delayed. Furthermore, the effect of pretreatment of cells by interferon and undiluted passage of VSV-o on interferon induction was discussed in relation to persistent infection.     

Monensin-resistant mouse Balb/3T3 cell mutant with aberrant penetration of vesicular stomatitis virus

A mutant (MO-5) resistant to monensin (an ionophoric antibiotic) derived from the mouse Balb/3T3 cell line, was a poor host for vesicular stomatitis virus (VSV) or semliki forest virus (SFV) multiplication. The yield of VSV particles in MO-5 is one 100-fold reduced as is VSV-dependent RNA synthesis. In contrast to a pH-remedial mutant, the abortive production of infectious VSV particles in MO-5 cells was not restored by low pH treatment. The pH values in the endosome and the lysosome of MO-5 cells were 5.2 and 5.4, respectively, values that were comparable to the pH value in Balb/3T3 cells. Assays with [3H]uridine-labeled VSV indicated similar binding of VSV in MO-5: percoll gradient centrifugation analysis of [35S]methionine-labeled VSV-infected Balb/3T3 showed accumulation of VSV in the lysosome fraction 20 min after VSV infection, whereas VSV can be found mainly in endosome/Golgi fraction of MO-5 cells after 40 to 60 min on the percoll gradients. Degradation of [35S]methionine-labeled VSV was observed at a significant rate in Balb/3T3 cells, but not in MO-5 cells. The monensin-resistant somatic cell may thus provide a genetic route to study the mechanism of endocytosis or transport of enveloped viruses.     

Expression and characterization of bovine lactoperoxidase by recombinant vaccinia virus

Lactoperoxidase (LPO) is a 78 kDa heme-containing oxidation–reduction enzyme present in milk, found in physiological fluids of mammals. LPO has an antimicrobial activity, and presumably contribute to the protective functions of milk against infectious diseases. In this study, recombinant vaccinia virus expressing bovine LPO (vv/bLPO) was constructed. In rabbit kidney (RK13) cells infected with vv/bLPO, recombinant bLPO was detected in both cell extracts and culture supernatants. Tunicamycin treatment decreased the molecular weight of recombinant bLPO, indicating that recombinant bLPO contains a N-linked glycosylation site. The replication of recombinant vaccinia viruses expressing bovine lactoferrin (vv/bLF) at a multiplicity of infection (moi) of 5 plaque-forming units (PFU)/cell was inhibited by antiviral activity of recombinant bLF, suggesting that vv/bLF has an antiviral effect against vaccinia virus. On the other hand, the replication of vv/bLPO at a moi of 5 PFU/cell was not inhibited by antiviral activity of recombinant bLPO, indicating that this recombinant virus could be used as a suitable viral vector. These results indicate that a combination of bLPO and vaccinia virus vector may be useful for medical and veterinary applications in vivo.     

Double-stranded RNA deaminase ADAR1 increases host susceptibility to virus infection

The RNA-editing enzyme ADAR1 is a double-stranded RNA (dsRNA) binding protein that modifies cellular and viral RNA sequences by adenosine deamination. ADAR1 has been demonstrated to play important roles in embryonic erythropoiesis, viral response, and RNA interference. In human hepatitis virus infection, ADAR1 has been shown to target viral RNA and to suppress viral replication through dsRNA editing. It is not clear whether this antiviral effect of ADAR1 is a common mechanism in response to viral infection. Here, we report a proviral effect of ADAR1 that enhances replication of vesicular stomatitis virus (VSV) through a mechanism independent of dsRNA editing. We demonstrate that ADAR1 interacts with dsRNA-activated protein kinase PKR, inhibits its kinase activity, and suppresses the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) phosphorylation. Consistent with the inhibitory effect on PKR activation, ADAR1 increases VSV infection in PKR+/+ mouse embryonic fibroblasts; however, no significant effect was found in PKR-/- cells. This proviral effect of ADAR1 requires the N-terminal domains but does not require the deaminase domain. These findings reveal a novel mechanism of ADAR1 that increases host susceptibility to viral infection by inhibiting PKR activation.     

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.     

Influenza A virus interactions with macrophages: Lessons from epithelial cells

Influenza viruses are an important cause of respiratory infection worldwide. In humans, infection with seasonal influenza A virus (IAV) is generally restricted to the respiratory tract where productive infection of airway epithelial cells promotes viral amplification, dissemination, and disease. Alveolar macrophages (MΦ) are also among the first cells to detect and respond to IAV, where they play a pivotal role in mounting effective innate immune responses. In contrast to epithelial cells, IAV infection of MΦ is a “dead end” for most seasonal strains, where replication is abortive and newly synthesised virions are not released. Although the key replicative stages leading to productive IAV infection in epithelial cells are defined, there is limited knowledge about the abortive IAV life cycle in MΦ. In this review, we will explore host factors and viral elements that support the early stages (entry) through to the late stages (viral egress) of IAV replication in epithelial cells. Similarities, differences, and unknowns for each key stage of the IAV replicative cycle in MΦ will then be highlighted. Herein, we provide mechanistic insights into MΦ‐specific control of seasonal IAV replication through abortive infection, which may in turn, contribute to effective host defence.     

Intracellular chelation of iron by bipyridyl inhibits DNA virus replication: ribonucleotide reductase maturation as a probe of intracellular iron pools

The efficient replication of large DNA viruses requires dNTPs supplied by a viral ribonucleotide reductase. Viral ribonucleotide reductase is an early gene product of both vaccinia and herpes simplex virus. For productive infection, the apoprotein must scavenge iron from the endogenous, labile iron pool(s). The membrane-permeant, intracellular Fe(2+) chelator, 2,2'-bipyridine (bipyridyl, BIP), is known to sequester iron from this pool. We show here that BIP strongly inhibits the replication of both vaccinia and herpes simplex virus, type 1. In a standard plaque assay, 50 microm BIP caused a 50% reduction in plaque-forming units with either virus. Strong inhibition was observed only when BIP was added within 3 h post-infection. This time dependence was observed also in regards to inhibition of viral late protein and DNA synthesis by BIP. BIP did not inhibit the activity of vaccinia ribonucleotide reductase (RR), its synthesis, nor its stability indicating that BIP blocked the activation of the apoprotein. In parallel with its inhibition of vaccinia RR activation, BIP treatment increased the RNA binding activity of the endogenous iron-response protein, IRP1, by 1.9-fold. The data indicate that the diiron prosthetic group in vaccinia RR is assembled from iron taken from the BIP-accessible, labile iron pool that is sampled also by ferritin and the iron-regulated protein found in the cytosol of mammalian cells.     

Human immunodeficiency virus infection of monoblastoid cells: cellular differentiation determines the pattern of virus replication.

Stringent control of human immunodeficiency virus (HIV) replication was observed in the human monoblastoid cell line U937. A low-multiplicity infection of these cells by the LAV1 strain of HIV was productive for 2.5 days; then virus replication became restricted and no further evidence of virion production was observed. The dramatic decrease in HIV production was due in part of reduced accumulation of cytoplasmic viral RNA and occurred in the absence of evident cytopathic effects. In contrast, infected cells induced to differentiate by phorbol ester, vitamin D3, or lymphokine supernatant did not release markers of HIV despite the accumulation of significant levels of cytoplasmic viral RNA. HIV infection altered the pattern of c-myc RNA accumulation in U937 cells. Expression of this gene changes normally in response to the state of cellular differentiation; in infected cells the level of c-myc expression was correlated to the levels of viral RNA accumulation and not to cellular differentiation. These results suggest that restricted replication of HIV in monocytes might be an important mechanism of virus persistence and demonstrate a relationship between HIV replication and monocyte differentiation.     

Vesicular stomatitis virus oncolysis of T lymphocytes requires cell cycle entry and translation initiation

Vesicular stomatitis virus (VSV) is a candidate oncolytic virus that replicates and induces cell death in cancer cells while sparing normal cells. Although defects in the interferon antiviral response facilitate VSV oncolysis, other host factors, including translational and growth regulatory mechanisms, also appear to influence oncolytic virus activity. We previously demonstrated that VSV infection induces apoptosis in proliferating CD4(+) T lymphocytes from adult T-cell leukemia samples but not in resting T lymphocytes or primary chronic lymphocytic leukemia cells that remain arrested in G(0). Activation of primary CD4(+) T lymphocytes with anti-CD3/CD28 is sufficient to induce VSV replication and cell death in a manner dependent on activation of the MEK1/2, c-Jun NH(2)-terminal kinase, or phosphatidylinositol 3-kinase pathway but not p38. VSV replication is specifically impaired by the cell cycle inhibitor olomoucine or rapamycin, which induces early G(1) arrest, but not by aphidicolin or Taxol, which blocks at the G(1)1S or G(2)1M phase, respectively; this result suggests a requirement for cell cycle entry for efficient VSV replication. The relationship between increased protein translation following G(0)/G(1) transition and VSV permissiveness is highlighted by the absence of mTOR and/or eIF4E phosphorylation whenever VSV replication is impaired. Furthermore, VSV protein production in activated T cells is diminished by small interfering RNA-mediated eIF4E knockdown. These results demonstrate that VSV replication in primary T lymphocytes relies on cell cycle transition from the G(0) phase to the G(1) phase, which is characterized by a sharp increase in ribogenesis and protein synthesis.     

IFN mimetic as a therapeutic for lethal vaccinia virus infection: possible effects on innate and adaptive immune responses

We have developed small peptide mimetics of IFN-gamma that can bypass the poxvirus virulence factor B8R protein, which binds to intact IFN-gamma and prevents its interaction with receptor extracellular domain. Thus, these peptides inhibit vaccinia virus replication in cell culture where intact IFN-gamma is ineffective. We demonstrate here that the mouse IFN-gamma-mimetic peptide, IFN-gamma(95-132), protects C57BL/6 mice against overwhelming lethal vaccinia virus infection. The mimetic peptide was synthesized with an attached lipophilic group for penetration of cell plasma membrane. Injection of mimetic i.p. before and at the time of intranasal (10(6) PFU) or i.p. (10(7) PFU) challenge with virus resulted in complete protection at 200 microg of mimetic and 40-60% protection at 5 microg of mimetic. Initiation of treatment of mice with IFN-gamma mimetic up to 2 days postinfection resulted in complete protection against death, whereas initiation of treatment at 6 days postinfection resulted in 40% protection. Administration of mimetic by the oral route also completely protected mice against the intranasal route of a lethal dose of vaccinia virus challenge. In addition to its direct antiviral effect, the mimetic also possessed adjuvant effects in boosting humoral and cellular immunity to vaccinia virus. The combination of antiviral and adjuvant effects by the IFN mimetic probably plays a role in its potent anti-vaccinia virus properties. These results suggest an effective therapeutic against ongoing, lethal poxvirus infections that taps into innate and adaptive host defenses.