Virucidal Activity of a GT-Rich Oligonucleotide against Herpes Simplex Virus Mediated by Glycoprotein B

ABSTRACT We have investigated the antiviral mechanism of a phosphorothioate oligonucleotide, ISIS 5652, which has activity against herpes simplex virus (HSV) in the low micromolar range in plaque reduction assays. We isolated a mutant that is resistant to this compound. Marker rescue and sequencing experiments showed that resistance was due to at least one of three mutations in the UL27 gene which result in amino acid changes in glycoprotein B (gB). Because gB has a role in attachment and entry of HSV, we tested the effects of ISIS 5652 at these stages of infection. The oligonucleotide potently inhibited attachment of virus to cells at 4°C; however, the resistant mutant did not exhibit resistance at this stage. Moreover, a different oligonucleotide with little activity in plaque reduction assays was as potent as ISIS 5652 in inhibiting attachment. Similarly, ISIS 5652 was able to inhibit entry of preattached virions into cells at 37°C, but the mutant did not exhibit resistance in this assay. The mutant did not attach to or enter cells more quickly than did wild-type virus. Strikingly, incubation of wild-type virus with 1 to 2 μM ISIS 5652 at 37°C led to a time-dependent, irreversible loss of infectivity (virucidal activity). No virucidal activity was detected at 4°C or with an unrelated oligonucleotide at 37°C. The resistant mutant and a marker-rescued derivative containing its gB mutations exhibited substantial resistance to this virucidal activity of ISIS 5652. We hypothesize that the GT-rich oligonucleotide induces a conformational change in gB that results in inactivation of infectivity.

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Herpes Simplex Virus 1 Induces Cytoplasmic Accumulation of TIA-1/TIAR and both Synthesis and Cytoplasmic Accumulation of Tristetraprolin, Two Cellular Proteins That Bind and Destabilize AU-Rich RNAs

Journal of Virology ◽

10.1128/jvi.78.16.8582-8592.2004 ◽

2004 ◽

Vol 78 (16) ◽

pp. 8582-8592 ◽

Cited By ~ 60

Author(s):

Audrey Esclatine ◽

Brunella Taddeo ◽

Bernard Roizman

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Mutant Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Herpes Simplex Virus 1 ◽

Infected Cells ◽

Cytoplasmic Accumulation ◽

Simplex Virus

ABSTRACT Herpes simplex virus 1 causes a shutoff of cellular protein synthesis through the degradation of RNA that is mediated by the virion host shutoff (Vhs) protein encoded by the UL41 gene. We reported elsewhere that the Vhs-dependent degradation of RNA is selective, and we identified RNAs containing AU-rich elements (AREs) that were upregulated after infection but degraded by deadenylation and progressive 3′-to-5′ degradation. We also identified upregulated RNAs that were not subject to Vhs-dependent degradation (A. Esclatine, B. Taddeo, L. Evans, and B. Roizman, Proc. Natl. Acad. Sci. USA 101:3603-3608, 2004). Among the latter was the RNA encoding tristetraprolin, a protein that binds AREs and is known to be associated with the degradation of RNAs containing AREs. Prompted by this observation, we examined the status of the ARE binding proteins tristetraprolin and TIA-1/TIAR in infected cells. We report that tristetraprolin was made and accumulated in the cytoplasm of wild-type virus-infected human foreskin fibroblasts as early as 2 h and in HEp-2 cells as early as 6 h after infection. The amounts of tristetraprolin that accumulated in the cytoplasm of cells infected with a mutant virus lacking UL41 were significantly lower than those in wild-type virus-infected cells. The localization of tristetraprolin was not modified in cells infected with a mutant lacking the gene encoding infected cell protein 4 (ICP4). TIA-1 and TIAR are two other proteins that are associated with the regulation of ARE-containing RNAs and that normally reside in nuclei. In infected cells, they started to accumulate in the cytoplasm after 6 h of infection. In cells infected with the mutant virus lacking UL41, TIA-1/TIAR accumulated in the cytoplasm in granular structures reminiscent of stress granules in a significant percentage of the cells. In addition, an antibody to tristetraprolin coprecipitated the Vhs protein from lysates of cells late in infection. The results indicate that the Vhs-dependent degradation of ARE-containing RNAs correlates with the transactivation, cytoplasmic accumulation, and persistence of tristetraprolin in infected cells.

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Posttranslational Processing of Infected Cell Proteins 0 and 4 of Herpes Simplex Virus 1 Is Sequential and Reflects the Subcellular Compartment in Which the Proteins Localize

Journal of Virology ◽

10.1128/jvi.75.17.7904-7912.2001 ◽

2001 ◽

Vol 75 (17) ◽

pp. 7904-7912 ◽

Cited By ~ 27

Author(s):

Sunil J. Advani ◽

Ryan Hagglund ◽

Ralph R. Weichselbaum ◽

Bernard Roizman

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Infected Cell ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Herpes Simplex Virus 1 ◽

Simplex Virus ◽

Made In ◽

In Cells

ABSTRACT The herpes simplex virus 1 (HSV-1) infected cell proteins 0 and 4 (ICP0 and ICP4) are multifunctional proteins extensively posttranscriptionally processed by both cellular and viral enzymes. We examined by two-dimensional separations the posttranslational forms of ICP0 and ICP4 in HEp-2 cells and in human embryonic lung (HEL) fibroblasts infected with wild-type virus, mutant R325, lacking the sequences encoding the US1.5 protein and the overlapping carboxyl-terminal domain of ICP22, or R7914, in which the aspartic acid 199 of ICP0 was replaced by alanine. We report the following (i) Both ICP0 and ICP4 were sequentially posttranslationally modified at least until 12 h after infection. In HEL fibroblasts, the processing of ICP0 shifted from A+B forms at 4 h to D+G forms at 8 h and finally to G, E, and F forms at 12 h. The ICP4 progression was from the A′ form noted at 2 h to B′ and C′ forms noted at 4 h to the additional D′ and E′ forms noted at 12 h. The progression tended to be toward more highly charged forms of the proteins. (ii) Although the overall patterns were similar, the mobility of proteins made in HEp-2 cells differed from those made in HEL fibroblasts. (iii) The processing of ICP0 forms E and F was blocked in HEL fibroblasts infected with R325 or with wild-type virus and treated with roscovitine, a specific inhibitor of cell cycle-dependent kinases cdc2, cdk2, and cdk5. R325-infected HEp-2 cells lacked the D′ form of ICP4, and roscovitine blocked the appearance of the most highly charged E′ form of ICP4. (iv) A characteristic of ICP0 is that it is translocated into the cytoplasm of HEL fibroblasts between 5 and 9 h after infection. Addition of MG132 to the cultures late in infection resulted in rapid relocation of cytoplasmic ICP0 back into the nucleus. Exposure of HEL fibroblasts to MG132 late in infection resulted in the disappearance of the highly charged ICP0 G isoform. The G form of ICP0 was also absent in cells infected with R7914 mutant. In cells infected with this mutant, ICP0 is not translocated to the cytoplasm. (v) Last, cdc2 was active in infected cells, and this activity was inhibited by roscovitine. In contrast, the activity of cdk2 exhibited by immunoprecipitated protein was reduced and resistant to roscovitine and may represent a contaminating kinase activity. We conclude from these results that the ICP0 G isoform is the cytoplasmic form, that it may be phosphorylated by cdc2, consistent with evidence published earlier (S. J., Advani, R. R. Weichselbaum, and B. Roizman, Proc. Natl. Acad. Sci. USA 96:10996–11001, 2000), and that the processing is reversed upon relocation of the G isoform from the cytoplasm into the nucleus. The processing of ICP4 is also affected by R325 and roscovitine. The latter result suggests that ICP4 may also be a substrate of cdc2 late in infection. Last, additional modifications are superimposed by cell-type-specific enzymes.

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Requirements for the Nuclear-Cytoplasmic Translocation of Infected-Cell Protein 0 of Herpes Simplex Virus 1

Journal of Virology ◽

10.1128/jvi.75.8.3832-3840.2001 ◽

2001 ◽

Vol 75 (8) ◽

pp. 3832-3840 ◽

Cited By ~ 69

Author(s):

Pascal Lopez ◽

Charles Van Sant ◽

Bernard Roizman

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Regulatory Protein ◽

Wild Type Virus ◽

Cell Protein ◽

Type Virus ◽

Wild Type ◽

Infected Cells ◽

Infected Cell Protein ◽

Simplex Virus

ABSTRACT Earlier studies have shown that wild-type infected-cell protein 0 (ICP0), a key herpes simplex virus regulatory protein, translocates from the nucleus to the cytoplasm of human embryonic lung (HEL) fibroblasts within several hours after infection (Y. Kawaguchi, R. Bruni, and B. Roizman, J. Virol. 71:1019–1024, 1997). Translocation of ICP0 was also observed in cells infected with thed120 mutant, in which both copies of the gene encoding ICP4, the major regulatory protein, had been deleted (V. Galvan, R. Brandimarti, J. Munger, and B. Roizman, J. Virol. 74:1931–1938, 2000). Furthermore, a mutant (R7914) carrying the D199A substitution in ICP0 does not bind or stabilize cyclin D3 and is retained in the nucleus (C. Van Sant, P. Lopez, S. J. Advani, and B. Roizman, J. Virol. 75:1888–1898, 2001). Studies designed to elucidate the requirements for the translocation of ICP0 between cellular compartments revealed the following. (i) Translocation of ICP0 to the cytoplasm in productive infection maps to the D199 amino acid, inasmuch as wild-type ICP0 delivered in trans to cells infected with an ICP0 null mutant was translocated to the cytoplasm whereas the D199A-substituted mutant ICP0 was not. (ii) Translocation of wild-type ICP0 requires a function expressed late in infection, inasmuch as phosphonoacetate blocked the translocation of ICP0 in wild-type virus-infected cells but not in d120 mutant-infected cells. Moreover, whereas in d120 mutant-infected cells ICP0 was translocated rapidly from the cytoplasm to the nucleus at approximately 5 h after infection, the translocation of ICP0 in wild-type virus-infected cells extended from 5 to at least 9 h after infection. (iii) In wild-type virus-infected cells, the MG132 proteasomal inhibitor blocked the translocation of ICP0 to the cytoplasm early in infection, but when added late in infection, it caused ICP0 to be relocated back to the nucleus from the cytoplasm. (iv) MG132 blocked the translocation of ICP0 in d120 mutant-infected cells early in infection but had no effect on the ICP0 aggregated in vesicle-like structures late in infection. However, ind120 mutant-infected cells treated with MG132 at late times, proteasomes formed a shell-like structure around the aggregated ICP0. These structures were not seen in wild-type virus or R7914 mutant-infected cells. The results indicate the following. (i) In the absence of β or γ protein synthesis, ICP0 dynamically associates with proteasomes and is translocated to the cytoplasm. (ii) In cells productively infected beyond α gene expression, ICP0 is retained in the nucleus until after the onset of viral DNA synthesis and the synthesis of γ2 proteins. (iii) Late in infection, ICP0 is actively sequestered in the cytoplasm by a process mediated by proteasomes, inasmuch as interference with proteasomal function causes rapid relocation of ICP0 to the nucleus.

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Recombinant Herpes Simplex Virus Type 1 Expressing Murine Interleukin-4 Is Less Virulent than Wild-Type Virus in Mice

Journal of Virology ◽

10.1128/jvi.75.19.9029-9036.2001 ◽

2001 ◽

Vol 75 (19) ◽

pp. 9029-9036 ◽

Cited By ~ 22

Author(s):

Homayon Ghiasi ◽

Yanira Osorio ◽

Guey-Chuen Perng ◽

Anthony B. Nesburn ◽

Steven L. Wechsler

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Herpes Simplex Virus Type ◽

Interleukin 4 ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Simplex Virus ◽

Hsv 1

ABSTRACT The effect of interleukin-4 (IL-4) on herpes simplex virus type 1 (HSV-1) infection in mice was evaluated by construction of a recombinant HSV-1 expressing the gene for murine IL-4 in place of the latency-associated transcript (LAT). The mutant virus (HSV-IL-4) expressed high levels of IL-4 in cultured cells. The replication of HSV-IL-4 in tissue culture and in trigeminal ganglia was similar to that of wild-type virus. In contrast, HSV-IL-4 appeared to replicate less well in mouse eyes and brains. Although BALB/c mice are highly susceptible to HSV-1 infection, ocular infection with HSV-IL-4 resulted in 100% survival. Furthermore, 57% of the mice survived coinfection with a mixture of HSV-IL-4 and a lethal dose of wild-type McKrae, compared with only 10% survival following infection with McKrae alone. Similar to wild-type BALB/c mice, 100% of IL-4−/− mice also survived HSV-IL-4 infection. T-cell depletion studies suggested that protection against HSV-IL-4 infection was mediated by a CD4+-T-cell response.

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Translocation and Colocalization of ICP4 and ICP0 in Cells Infected with Herpes Simplex Virus 1 Mutants Lacking Glycoprotein E, Glycoprotein I, or the Virion Host Shutoff Product of the UL41 Gene

Journal of Virology ◽

10.1128/jvi.02157-07 ◽

2007 ◽

Vol 82 (4) ◽

pp. 1701-1713 ◽

Cited By ~ 16

Author(s):

Maria Kalamvoki ◽

Jianguo Qu ◽

Bernard Roizman

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Herpes Simplex Virus 1 ◽

Glycoprotein E ◽

Glycoprotein I ◽

Infected Cells ◽

Simplex Virus

ABSTRACT In wild-type herpes simplex virus 1-infected cells, the major regulatory protein ICP4 resides in the nucleus whereas ICP0 becomes dynamically associated with proteasomes and late in infection is translocated and dispersed in the cytoplasm. Inhibition of proteasomal function results in retention or transport of ICP0 to the nucleus. We report that in cells infected with mutants lacking glycoprotein E (gE), glycoprotein I (gI), or the product of the UL41 gene, both ICP4 and ICP0 are translocated to the cytoplasm and coaggregate in small dense structures that, in the presence of proteasomal inhibitor MG132, also contain proteasomal components. Gold particle-conjugated antibody to ICP0 reacted in thin sections with dense protein aggregates in the cytoplasm of mutant virus-infected cells. Similar aggregates were present in the nuclei but not in the cytoplasm of wild-type virus-infected cells. Exposure of cells early in infection to MG132 does not result in retention of ICP0 as in wild-type virus-infected cells. The results suggest that the retention of ICP4 and ICP0 in the nucleus is a dynamic process that involves the function of other viral proteins that may include the Fc receptor formed by the gE/gI complex and is not merely the consequence of expression of a nuclear localization signal. It is noteworthy that in ΔUL41-infected cells gE is retained in the trans-Golgi network and is not widely dispersed in cellular membranes.

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A Double Mutation in Glycoprotein gB Compensates for Ineffective gD-Dependent Initiation of Herpes Simplex Virus Type 1 Infection

Journal of Virology ◽

10.1128/jvi.01633-10 ◽

2010 ◽

Vol 84 (23) ◽

pp. 12200-12209 ◽

Cited By ~ 32

Author(s):

Hiroaki Uchida ◽

Janet Chan ◽

William F. Goins ◽

Paola Grandi ◽

Izumi Kumagai ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Entry ◽

Growth Factor Receptor ◽

Mutant Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Double Mutation ◽

Simplex Virus

ABSTRACT Herpes simplex virus (HSV) entry into cells is triggered by the binding of envelope glycoprotein D (gD) to a specific receptor, such as nectin-1 or herpesvirus entry mediator (HVEM), resulting in activation of the fusion effectors gB and gH and virus penetration. Here we report the identification of a hyperactive gB allele, D285N/A549T, selected by repeat passage of a gD mutant virus defective for nectin-1 binding through cells that express a gD-binding-impaired mutant nectin-1. The gB allele in a wild-type virus background enabled the use of other nectins as virus entry receptors. In addition, combination of the mutant allele with an epidermal growth factor receptor (EGFR)-retargeted gD gene yielded dramatically increased EGFR-specific virus entry compared to retargeted virus carrying wild-type gB. Entry of the gB mutant virus into nectin-1-bearing cells was markedly accelerated compared to that of wild-type virus, suggesting that the gB mutations affect a rate-limiting step in entry. Our observations indicate that ineffective gD activation can be complemented by hypersensitization of a downstream component of the entry cascade to gD signaling.

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Enhanced Pathogenesis of an Attenuated Herpes Simplex Virus for Mice Lacking Stat1

Journal of Virology ◽

10.1128/jvi.00297-08 ◽

2008 ◽

Vol 82 (12) ◽

pp. 6052-6055 ◽

Cited By ~ 26

Author(s):

Tracy Jo Pasieka ◽

Betty Lu ◽

David A. Leib

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Wild Type Virus ◽

Growth Defect ◽

Type Virus ◽

Interferon Signaling ◽

Wild Type ◽

Virion Host Shutoff ◽

Simplex Virus

ABSTRACT Mice lacking the Stat1 interferon signaling gene were infected with herpes simplex virus type 1 (HSV-1) or an attenuated recombinant lacking virion host shutoff (Δvhs). Δvhs virus-infected Stat1−/− mice showed levels of replication equivalent to that of the wild-type virus-infected control mice but reduced relative to wild-type virus-infected Stat1−/− mice. Stat1 deficiency relieves the immunomodulatory deficiency of Δvhs virus, but not its inherent growth defect. Also Vhs is dispensable for reactivation.

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Replication of Herpes Simplex Virus 1 Depends on the γ134.5 Functions That Facilitate Virus Response to Interferon and Egress in the Different Stages of Productive Infection

Journal of Virology ◽

10.1128/jvi.78.14.7653-7666.2004 ◽

2004 ◽

Vol 78 (14) ◽

pp. 7653-7666 ◽

Cited By ~ 27

Author(s):

Xianghong Jing ◽

Melissa Cerveny ◽

Kui Yang ◽

Bin He

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Productive Infection ◽

Wild Type Virus ◽

Compensatory Mutation ◽

Type Virus ◽

Ns1 Protein ◽

Wild Type ◽

Amino Terminal ◽

Simplex Virus

ABSTRACT The ability of the γ134.5 protein to suppress the PKR response plays a crucial role in herpes simplex virus pathogenesis. In this process, the γ134.5 protein associates with protein phosphatase 1 to form a large complex that dephosphorylates eIF-2α and thereby prevents translation shutoff mediated by PKR. Accordingly, γ134.5 null mutants are virulent in PKR-knockout mice but not in wild-type mice. However, γ134.5 deletion mutants, with an extragenic compensatory mutation, inhibit PKR activity but remain avirulent, suggesting that the γ134.5 protein has additional functions. Here, we show that a substitution of the γ134.5 gene with the NS1 gene from influenza A virus renders viral resistance to interferon involving PKR. The virus replicates as efficiently as wild-type virus in SK-N-SH and CV-1 cells. However, in mouse 3T6 cells, the virus expressing the NS1 protein grows at an intermediate level between the wild-type virus and the γ134.5 deletion mutant. This decrease in growth, compared to that of the wild-type virus, is due not to an inhibition of viral protein synthesis but rather to a block in virus release or egress. Virus particles are predominantly present in the nucleus and cytoplasm. Notably, deletions in the amino terminus of the γ134.5 protein lead to a significant decrease in virus growth in mouse 3T6 cells, which is independent of eIF-2α dephosphorylation. In correlation, a series of deletions in the amino-terminal domain impair nuclear as well as cytoplasmic egress. These results indicate that efficient viral replication depends on the γ134.5 functions required to prevent the PKR response and to facilitate virus egress in the different stages during virus infection.

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In Vivo Immune Evasion Mediated by the Herpes Simplex Virus Type 1 Immunoglobulin G Fc Receptor

Journal of Virology ◽

10.1128/jvi.72.7.5351-5359.1998 ◽

1998 ◽

Vol 72 (7) ◽

pp. 5351-5359 ◽

Cited By ~ 86

Author(s):

Thandavarayan Nagashunmugam ◽

John Lubinski ◽

Liyang Wang ◽

Lester T. Goldstein ◽

Benjamin S. Weeks ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Immunoglobulin G ◽

Mutant Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcγR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcγR activity without affecting other gE functions, we constructed a mutant virus, NS-gE339, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcγRs. NS-gE339 expresses gE and gI, is FcγR−, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcγR does not bind murine IgG; therefore, the absence of an FcγR should not affect virulence in mice. NS-gE339 causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcγR− mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcγR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE339-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcγR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.

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Diverse Herpes Simplex Virus Type 1 Thymidine Kinase Mutants in Individual Human Neurons and Ganglia

Journal of Virology ◽

10.1128/jvi.00166-07 ◽

2007 ◽

Vol 81 (13) ◽

pp. 6817-6826 ◽

Cited By ~ 28

Author(s):

Kening Wang ◽

Gowtham Mahalingam ◽

Susan E. Hoover ◽

Erik K. Mont ◽

Steven M. Holland ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Herpes Simplex Virus Type ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Mixed Populations ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Mutations in the thymidine kinase gene (tk) of herpes simplex virus type 1 (HSV-1) explain most cases of virus resistance to acyclovir (ACV) treatment. Mucocutaneous lesions of patients with ACV resistance contain mixed populations of tk mutant and wild-type virus. However, it is unknown whether human ganglia also contain mixed populations since the replication of HSV tk mutants in animal neurons is impaired. Here we report the detection of mutated HSV tk sequences in human ganglia. Trigeminal and dorsal root ganglia were obtained at autopsy from an immunocompromised woman with chronic mucocutaneous infection with ACV-resistant HSV-1. The HSV-1 tk open reading frames from ganglia were amplified by PCR, cloned, and sequenced. tk mutations were detected in a seven-G homopolymer region in 11 of 12 ganglia tested, with clonal frequencies ranging from 4.2 to 76% HSV-1 tk mutants per ganglion. In 8 of 11 ganglia, the mutations were heterogeneous, varying from a deletion of one G to an insertion of one to three G residues, with the two-G insertion being the most common. Each ganglion had its own pattern of mutant populations. When individual neurons from one ganglion were analyzed by laser capture microdissection and PCR, 6 of 14 HSV-1-positive neurons were coinfected with HSV tk mutants and wild-type virus, 4 of 14 were infected with wild-type virus alone, and 4 of 14 were infected with tk mutant virus alone. These data suggest that diverse tk mutants arise independently under drug selection and establish latency in human sensory ganglia alone or together with wild-type virus.

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