A Mutant Standard Virus Isolated from Vesicular Stomatitis Virus Persistent Infection Interferes Specifically with Wild-type Virus Replication

ABSTRACT We report here the generation of recombinant vesicular stomatitis virus (VSV) able to produce the suicide gene product thymidine kinase (TK) or cytokine interleukin 4 (IL-4). In vitro cells infected with the engineered viruses expressed remarkably high levels of biologically active TK or IL-4 and showed no defects in replication compared to the wild-type virus. Recombinant viruses retained their ability to induce potent apoptosis in a variety of cancer cells, while normal cells were evidently more resistant to infection and were completely protected by interferon. Significantly, following direct intratumoral inoculation, VSV expressing either TK or IL-4 exhibited considerably more oncolytic activity against syngeneic breast or melanoma tumors in murine models than did the wild-type virus or control recombinant viruses expressing green fluorescent protein (GFP). Complete regression of a number of tumors was achieved, and increased granulocyte-infiltrating activity with concomitant, antitumor cytotoxic T-cell responses was observed. Aside from discovering greater oncolytic activity following direct intratumoral inoculation, however, we also established that VSV expressing IL-4 or TK, but not GFP, was able to exert enhanced antitumor activity against metastatic disease. Following intravenous administration of the recombinant viruses, immunocompetent BALB/c mice inoculated with mammary adenocarcinoma exhibited prolonged survival against lethal lung metastasis. Our data demonstrate the validity of developing novel types of engineered VSV for recombinant protein production and as a gene therapy vector for the treatment of malignant and other disease.

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Temperature-sensitive mutants of vesicular stomatitis virus are conditionally defective particles that interfere with and are rescued by wild-type virus.

Journal of Virology ◽

10.1128/jvi.19.1.102-107.1976 ◽

1976 ◽

Vol 19 (1) ◽

pp. 102-107 ◽

Cited By ~ 26

Author(s):

J S Youngner ◽

D O Quagliana

Keyword(s):

Vesicular Stomatitis Virus ◽

Wild Type Virus ◽

Temperature Sensitive ◽

Type Virus ◽

Wild Type ◽

Temperature Sensitive Mutants ◽

Vesicular Stomatitis

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Moving the Glycoprotein Gene of Vesicular Stomatitis Virus to Promoter-Proximal Positions Accelerates and Enhances the Protective Immune Response

Journal of Virology ◽

10.1128/jvi.74.17.7895-7902.2000 ◽

2000 ◽

Vol 74 (17) ◽

pp. 7895-7902 ◽

Cited By ~ 49

Author(s):

E. Brian Flanagan ◽

L. Andrew Ball ◽

Gail W. Wertz

Keyword(s):

Immune Response ◽

Protein Expression ◽

Vesicular Stomatitis Virus ◽

Wild Type Virus ◽

Wild Type ◽

Gene Encoding ◽

N Protein ◽

Glycoprotein G ◽

Vesicular Stomatitis ◽

Negative Sense

ABSTRACT Vesicular stomatitis virus (VSV) is the prototype of the Rhabdoviridae and contains nonsegmented negative-sense RNA as its genome. The 11-kb genome encodes five genes in the order 3′-N-P-M-G-L-5′, and transcription is obligatorily sequential from the single 3′ promoter. As a result, genes at promoter-proximal positions are transcribed at higher levels than those at promoter-distal positions. Previous work demonstrated that moving the gene encoding the nucleocapsid protein N to successively more promoter-distal positions resulted in stepwise attenuation of replication and lethality for mice. In the present study we investigated whether moving the gene for the attachment glycoprotein G, which encodes the major neutralizing epitopes, from its fourth position up to first in the gene order would increase G protein expression in cells and alter the immune response in inoculated animals. In addition to moving the G gene alone, we also constructed viruses having both the G and N genes rearranged. This produced three variant viruses having the orders 3′-G-N-P-M-L-5′ (G1N2), 3′-P-M-G-N-L-5′ (G3N4), and 3′-G-P-M-N-L-5′ (G1N4), respectively. These viruses differed from one another and from wild-type virus in their levels of gene expression and replication in cell culture. The viruses also differed in their pathogenesis, immunogenicity, and level of protection of mice against challenge with wild-type VSV. Translocation of the G gene altered the kinetics and level of the antibody response in mice, and simultaneous reduction of N protein expression reduced replication and lethality for animals. These studies demonstrate that gene rearrangement can be exploited to design nonsegmented negative-sense RNA viruses that have characteristics desirable in candidates for live attenuated vaccines.

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Interference of wild type virus replication by an RNA negative temperature-sensitive mutant of Semliki Forest virus

Virology ◽

10.1016/0042-6822(77)90376-2 ◽

1977 ◽

Vol 80 (1) ◽

pp. 1-11 ◽

Cited By ~ 11

Author(s):

Sirkka Keränen

Keyword(s):

Virus Replication ◽

Semliki Forest Virus ◽

Negative Temperature ◽

Wild Type Virus ◽

Temperature Sensitive ◽

Type Virus ◽

Wild Type ◽

Sensitive Mutant ◽

Temperature Sensitive Mutant

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Ubiquitination of the Cytoplasmic Domain of Influenza A Virus M2 Protein is Crucial for Production of Infectious Virus Particles

Journal of Virology ◽

10.1128/jvi.01972-17 ◽

2017 ◽

pp. JVI.01972-17 ◽

Cited By ~ 8

Author(s):

Wen-Chi Su ◽

Wen-Ya Yu ◽

Shih-Han Huang ◽

Michael M.C. Lai

Keyword(s):

Virus Replication ◽

Influenza A ◽

Functional Role ◽

Infectious Virus ◽

Virus Production ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Virus Particles

Virus replication is mediated by interactions between virus and host. Here, we demonstrate that influenza A virus membrane protein 2 (M2) can be ubiquitinated. The lysine residue at position 78, which is located in the cytoplasmic domain of M2, is essential for M2 ubiquitination. An M2-K78R (Lys78→Arg78) mutant, which produces ubiquitination-deficient M2, showed a severe defect in production of infectious virus particles. M2-K78R mutant progeny contained more HA proteins, less viral RNAs and less internal viral proteins, including M1 and NP, than the wild-type virus. Furthermore, most of the M2-K78R mutant viral particles lacked viral ribonucleoproteins upon examination under electron microscopy and exhibited slightly lower densities. We also found that mutant M2 colocalized with M1 protein to a lesser extent than for wild-type virus. These findings may account for the reduced incorporation of viral ribonucleoprotein into virions. By blocking the second round of virus infection, we showed that the M2 ubiquitination-defective mutant exhibited normal level of virus replication during the first round of infection, thereby proving that M2 ubiquitination is involved in the virus production step. Finally, we found that M2-K78R mutant virus induced autophagy and apoptosis earlier than wild-type virus. Collectively, these results suggest that M2 ubiquitination plays an important role in infectious virus production by coordinating efficient packaging of the viral genome into virus particles and timing of viral-induced cell death.IMPORTANCEAnnual epidemics and recurring pandemics of influenza viruses represent a very high global health and economic burden. Influenza virus M2 protein has been extensively studied for its important roles in virus replication, particularly in viral entry and release. Rimantadine, one of the most commonly used antiviral drugs, binds to the channel lumen near the N-terminus of M2 proteins. However, viruses resistant to Rimantadine have emerged. M2 undergoes several posttranslational modifications, such as phosphorylation and palmitoylation. Here, we reveal that ubiquitination mediates the functional role of M2. A ubiquitination-deficient M2 mutant predominately produced virus particles either lacking viral ribonucleoproteins or containing smaller amounts of internal viral components, resulting in lower infectivity. Our findings offer insights into the mechanism of influenza virus morphogenesis, particularly the functional role of M1-M2 interactions in viral particle assembly, and can be applied to the development of new influenza therapies.

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A Gammaherpesvirus 68 Gene 50 Null Mutant Establishes Long-Term Latency in the Lung but Fails To Vaccinate against a Wild-Type Virus Challenge

Journal of Virology ◽

10.1128/jvi.80.3.1592-1598.2006 ◽

2006 ◽

Vol 80 (3) ◽

pp. 1592-1598 ◽

Cited By ~ 36

Author(s):

Janice M. Moser ◽

Michael L. Farrell ◽

Laurie T. Krug ◽

Jason W. Upton ◽

Samuel H. Speck

Keyword(s):

B Cells ◽

Virus Replication ◽

Antiviral Drug ◽

Lytic Replication ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Murine Gammaherpesvirus ◽

Gammaherpesvirus 68

ABSTRACT The gammaherpesvirus immediate-early genes are critical regulators of virus replication and reactivation from latency. Rta, encoded by gene 50, serves as the major transactivator of the lytic program and is highly conserved among all the gammaherpesviruses, including Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and murine gammaherpesvirus 68 (γHV68). Introduction of a translation stop codon in γHV68 gene 50 (gene 50.stop γHV68) demonstrated that Rta is essential for virus replication in vitro. To investigate the role that virus replication plays in the establishment and maintenance of latency, we infected mice with gene 50.stop γHV68. Notably, the gene 50.stop virus established a long-term infection in lung B cells following intranasal infection of mice but was unable to establish latency in the spleen. This complete block in the establishment of latency in the spleen was also seen when lytic virus production was inhibited by treating mice infected with wild-type virus with the antiviral drug cidofovir, implicating virus replication and not an independent function of Rta in the establishment of splenic latency. Furthermore, we showed that gene 50.stop γHV68 was unable to prime the immune system and was unable to protect against a challenge with wild-type γHV68, despite its ability to chronically infect lung B cells. These data indicate gammaherpesviruses that are unable to undergo lytic replication in vivo may not be viable vaccine candidates despite the detection of cells harboring viral genome at late times postinfection.

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A double deletion prevents replication of the pestivirus bovine viral diarrhea virus in the placenta of pregnant heifers

PLoS Pathogens ◽

10.1371/journal.ppat.1010107 ◽

2021 ◽

Vol 17 (12) ◽

pp. e1010107

Author(s):

Jolene Carlson ◽

Robert Kammerer ◽

Jens Peter Teifke ◽

Julia Sehl-Ewert ◽

Christiane Pfarrer ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Virus Replication ◽

Post Infection ◽

Deletion Mutant ◽

Mutant Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Double Deletion

In contrast to wild type bovine viral diarhea virus (BVDV) specific double deletion mutants are not able to establish persistent infection upon infection of a pregnant heifer. Our data shows that this finding results from a defect in transfer of the virus from the mother animal to the fetus. Pregnant heifers were inoculated with such a double deletion mutant or the parental wild type virus and slaughtered pairwise on days 6, 9, 10 and 13 post infection. Viral RNA was detected via qRT-PCR and RNAscope analyses in maternal tissues for both viruses from day 6 p.i. on. However, the double deletion mutant was not detected in placenta and was only found in samples from animals infected with the wild type virus. Similarly, high levels of wild type viral RNA were present in fetal tissues whereas the genome of the double deletion mutant was not detected supporting the hypothesis of a specific inhibition of mutant virus replication in the placenta. We compared the induction of gene expression upon infection of placenta derived cell lines with wild type and mutant virus via gene array analysis. Genes important for the innate immune response were strongly upregulated by the mutant virus compared to the wild type in caruncle epithelial cells that establish the cell layer on the maternal side at the maternal–fetal interface in the placenta. Also, trophoblasts which can be found on the fetal side of the interface showed significant induction of gene expression upon infection with the mutant virus although with lower complexity. Growth curves recorded in both cell lines revealed a general reduction of virus replication in caruncular epithelial cells compared to the trophoblasts. Compared to the wild type virus this effect was dramtic for the mutant virus that reached only a TCID50 of 1.0 at 72 hours post infection.

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The NB Protein of Influenza B Virus Is Not Necessary for Virus Replication In Vitro

Journal of Virology ◽

10.1128/jvi.77.10.6050-6054.2003 ◽

2003 ◽

Vol 77 (10) ◽

pp. 6050-6054 ◽

Cited By ~ 46

Author(s):

Masato Hatta ◽

Yoshihiro Kawaoka

Keyword(s):

Cell Culture ◽

Virus Replication ◽

Wild Type Virus ◽

Influenza B Virus ◽

Influenza B ◽

Type Virus ◽

Wild Type ◽

B Virus

ABSTRACT The NB protein of influenza B virus is thought to function as an ion channel and therefore would be expected to have an essential function in viral replication. Because direct evidence for its absolute requirement in the viral life cycle is lacking, we generated NB knockout viruses by reverse genetics and tested their growth properties both in vitro and in vivo. Mutants not expressing NB replicated as efficiently as the wild-type virus in cell culture, whereas in mice they showed restricted growth compared with findings for the wild-type virus. Thus, the NB protein is not essential for influenza B virus replication in cell culture but promotes efficient growth in mice.

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