scholarly journals A recombinant rotavirus harboring a spike protein with a heterologous peptide reveals a novel role of VP4 in viroplasm stability

2021 ◽  
Author(s):  
Guido Papa ◽  
Janine Vetter ◽  
Michael Seyffert ◽  
Kapila Gunasekera ◽  
Giuditta De Lorenzo ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Similar Distribution ◽  
Cell Tropism ◽  
Wild Type ◽  
Lectin Domain ◽  
Infected Cells ◽  
Sirna Silencing ◽  
Genetically Manipulated ◽  
Replication Fitness

The rotavirus (RV) VP4 spike protrudes as a trimeric structure from the five-fold axes of the virion triple-layer. Infectious RV particles need to be proteolytically cleaved in VP4 into two subunits, VP8* and VP5*, constituting both the distal part and central body of the virus spike. Modification of VP4 has been challenging as it is involved in biological processes such as the interaction with sialic acid and integrins, cell tropism and hemagglutinin activity. Using RV reverse genetics, four loops in the lectin domain of the VP8* subunit were engineered independently to harbor a small biotin acceptor peptide (BAP) tag and then tested for their ability to rescue virus. Only a single recombinant virus, rRV/VP4-BAP, harboring VP4 with a modified loop at position K145-G150 was rescued. This rRV/VP4-BAP internalizes, replicates, and generates virus progeny, demonstrating that the VP4 spike of RV particles can be genetically manipulated by the incorporation of at least 15 exogenous amino acids. VP4-BAP had a similar distribution as VP4 in infected cells by localizing in the cytoskeleton and surrounding viroplasms. However, compared to wild-type RV, rRV/VP4-BAP featured a reduced replication fitness and impaired viroplasm stability. Upon treatment with 1,6-hexanediol, a drug disrupting liquid-liquid phase-separated condensates, the kinetic of rRV/VP4-BAP viroplasm recovery was delayed, and their size and numbers reduced when compared to viroplasms of wild type RV. Moreover, siRNA silencing of VP4 expression in RV strain SA11 showed similar recovery patterns as rRV/VP4-BAP, revealing a novel function of VP4 in viroplasm stability.

scholarly journals The Human Cytomegalovirus UL116 Glycoprotein Is a Chaperone to Control gH-Based Complexes Levels on Virions

2021 ◽  
Vol 12 ◽  
Author(s):  
Giacomo Vezzani ◽  
Diego Amendola ◽  
Dong Yu ◽  
Sumana Chandramouli ◽  
Elisabetta Frigimelica ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Wild Type Virus ◽  
Cell Tropism ◽  
Wild Type ◽  
Viral Membrane ◽  
Viral Glycoproteins ◽  
Fusion Glycoprotein ◽  
Infected Cells ◽  
Viral Membrane Fusion

Human cytomegalovirus (HCMV) relies in large part upon the viral membrane fusion glycoprotein B and two alternative gH/gL complexes, gH/gL/gO (Trimer) and gH/gL/UL128/UL130/UL131A (Pentamer) to enter into cells. The relative amounts of Trimer and Pentamer vary among HCMV strains and contribute to differences in cell tropism. Although the viral ER resident protein UL148 has been shown to interact with gH to facilitate gO incorporation, the mechanisms that favor the assembly and maturation of one complex over another remain poorly understood. HCMV virions also contain an alternative non-disulfide bound heterodimer comprised of gH and UL116 whose function remains unknown. Here, we show that disruption of HCMV gene UL116 causes infectivity defects of ∼10-fold relative to wild-type virus and leads to reduced expression of both gH/gL complexes in virions. Furthermore, gH that is not covalently bound to other viral glycoproteins, which are readily detected in wild-type HCMV virions, become undetectable in the absence of UL116 suggesting that the gH/UL116 complex is abundant in virions. We find evidence that UL116 and UL148 interact during infection indicating that the two proteins might cooperate to regulate the abundance of HCMV gH complexes. Altogether, these results are consistent with a role of UL116 as a chaperone for gH during the assembly and maturation of gH complexes in infected cells.

scholarly journals The human cytomegalovirus UL116 glycoprotein is a chaperone to control gH-based complexes levels on virions

2020 ◽  
Author(s):  
Giacomo Vezzani ◽  
Diego Amendola ◽  
Dong Yu ◽  
Sumana Chandramuli ◽  
Elisabetta Frigimelica ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Wild Type Virus ◽  
Cell Tropism ◽  
Wild Type ◽  
Viral Membrane ◽  
Viral Glycoproteins ◽  
Fusion Glycoprotein ◽  
Infected Cells ◽  
Viral Membrane Fusion

ABSTRACTHuman cytomegalovirus (HCMV) relies in large part upon the viral membrane fusion glycoprotein B (gB) and two alternative gH/gL complexes, gH/gL/gO (Trimer) and the gH/gL/UL128/UL130/UL131A (Pentamer) to enter into cells. The relative amounts of the Trimer and Pentamer vary among HCMV strains and contribute to differences in cell tropism. Although the viral ER resident protein UL148 has been shown to interact with gH to facilitate gO incorporation, the mechanisms that favor the assembly and maturation of one complex over another remain poorly understood. HCMV virions also contain an alternative non-disulfide bound heterodimer comprised of gH and UL116 whose function remains unknown. Here, we show that disruption of HCMV gene UL116 causes infectivity defects of ~10-fold relative to wild-type virus and leads to reduced expression of both gH/gL complexes in virions. Furthermore, gH that is not covalently bound to other viral glycoproteins, which are readily detected in wild-type HCMV virions, become undetectable in the absence of UL116 suggesting that the gH/UL116 complex is abundant in virions. We find evidence that UL116 and UL148 interact during infection indicating that the two proteins might cooperate to regulate the abundance of HCMV gH complexes. Altogether, these results are consistent with a role of UL116 as a chaperone for gH during the assembly and maturation of gH complexes in infected cells.

scholarly journals An MDM2 inhibitor achieves synergistic cytotoxic effects with adenoviruses lacking E1B55kDa gene on mesothelioma with the wild-type p53 through augmenting NFI expression

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Thao Thi Thanh Nguyen ◽  
Masato Shingyoji ◽  
Michiko Hanazono ◽  
Boya Zhong ◽  
Takao Morinaga ◽  
...  
Keyword(s):  
Wild Type ◽  
Inhibitory Effects ◽  
Cytotoxic Effects ◽  
P53 Expression ◽  
Nuclear Factor I ◽  
Infected Cells ◽  
Wild Type P53 ◽  
Mdm2 Inhibitor ◽  
P16 Expression

AbstractA majority of mesothelioma specimens were defective of p14 and p16 expression due to deletion of the INK4A/ARF region, and the p53 pathway was consequently inactivated by elevated MDM2 functions which facilitated p53 degradaton. We investigated a role of p53 elevation by MDM2 inhibitors, nutlin-3a and RG7112, in cytotoxicity of replication-competent adenoviruses (Ad) lacking the p53-binding E1B55kDa gene (Ad-delE1B). We found that a growth inhibition by p53-activating Ad-delE1B was irrelevant to p53 expression in the infected cells, but combination of Ad-delE1B and the MDM2 inhibitor produced synergistic inhibitory effects on mesothelioma with the wild-type but not mutated p53 genotype. The combination augmented p53 phosphorylation, activated apoptotic but not autophagic pathway, and enhanced DNA damage signals through ATM-Chk2 phosphorylation. The MDM2 inhibitors facilitated production of the Ad progenies through augmented expression of nuclear factor I (NFI), one of the transcriptional factors involved in Ad replications. Knocking down of p53 with siRNA did not increase the progeny production or the NFI expression. We also demonstrated anti-tumor effects by the combination of Ad-delE1B and the MDM2 inhibitors in an orthotopic animal model. These data collectively indicated that upregulation of wild-type p53 expression contributed to cytotoxicity by E1B55kDa-defective replicative Ad through NFI induction and suggested that replication-competent Ad together with augmented p53 levels was a therapeutic strategy for p53 wild-type mesothelioma.

scholarly journals Role of Herpes Simplex Virus ICP27 in the Degradation of mRNA by Virion Host Shutoff RNase

2010 ◽  
Vol 84 (19) ◽  
pp. 10182-10190 ◽  
Author(s):  
Brunella Taddeo ◽  
Weiran Zhang ◽  
Bernard Roizman
Keyword(s):  
Mutant Virus ◽  
Structural Proteins ◽  
Wild Type Virus ◽  
Tegument Protein ◽  
Wild Type ◽  
Virion Host Shutoff ◽  
Infected Cells ◽  
Host Shutoff ◽  
Simplex Virus

ABSTRACT The virion host shutoff (VHS) RNase tegument protein released into cells by infecting virus has two effects. Preexisting stable mRNAs (e.g., GAPDH [glyceraldehyde-3-phosphate dehydrogenase]) are rapidly degraded. Stress response RNAs containing AU-rich elements (AREs) in the 3′ untranslated region (3′UTR) are deadenylated and cleaved, but the cleavage products persist for hours, in contrast to the short half-lives of ARE-containing mRNAs in uninfected cells. At late times, the VHS RNase is neutralized by the viral structural proteins VP16 and VP22. A recent study (J. A. Corcoran, W. L. Hsu, and J. R. Smiley, J. Virol. 80:9720-9729, 2006) reported that, at relatively late times after infection, ARE RNAs are rapidly degraded in cells infected with ΔICP27 mutant virus and concluded that ICP27 “stabilizes” ARE mRNAs. We report the following. (i) The rates of degradation of ARE mRNA at early times (3 h) after infection with the wild type or the ΔICP27 mutant virus are virtually identical, and hence ICP27 plays no role in this process. (ii) In noncomplementing cells, VHS RNase or VP22 is not synthesized. Therefore, the only VHS that is active is brought into cells by the ΔICP27 mutant. (ii) The VHS RNase brought into the cells by the ΔICP27 virus is reduced in potency relative to that of wild-type virus. Hence the rapid degradation of ARE mRNAs noted in ΔICP27 mutant-infected cells at late times is similar to that taking place in mock-infected or in ΔVHS RNase mutant-virus-infected cells and does not by itself support the hypothesis that ICP27 stabilizes ARE mRNAs. (iii) Concurrently, we present the first evidence that VHS RNase interacts with ICP27 most likely when bound to cap- and poly(A)-binding proteins, respectively.

scholarly journals Role of Tachykinins in the Host Response to Murine Gammaherpesvirus Infection

2001 ◽  
Vol 75 (21) ◽  
pp. 10467-10471 ◽  
Author(s):  
Catherine M. Payne ◽  
Caroline J. Heggie ◽  
David G. Brownstein ◽  
James P. Stewart ◽  
John P. Quinn
Keyword(s):  
Virus Infection ◽  
Host Response ◽  
Infectious Virus ◽  
Inflammatory Cells ◽  
Direct Influence ◽  
Wild Type ◽  
Murine Gammaherpesvirus ◽  
Latently Infected ◽  
Infected Cells

ABSTRACT Tachykinins function not only as neurotransmitters but also as immunological mediators. We used infection of tachykinin-deficient (PPT-A −/−) mice and wild-type controls with murine gammaherpesvirus to assess the role of tachykinins in the host response to a virus infection. Although infection was ultimately controlled in PPT-A −/− mice, there were higher titers of infectious virus in the lungs, accompanied by a more rapid influx of inflammatory cells. Clearance of latently infected cells from the spleen was also delayed. This is the first report of the direct influence of tachykinins in the host response to a virus infection.

scholarly journals A large panel of chicken cells are invaded in vivo by Salmonella Typhimurium even when depleted of all known invasion factors

Open Biology ◽  
2021 ◽  
Vol 11 (11) ◽  
Author(s):  
S. M. Roche ◽  
S. Holbert ◽  
Y. Le Vern ◽  
C. Rossignol ◽  
A. Rossignol ◽  
...  
Keyword(s):  
Gall Bladder ◽  
Cell Types ◽  
Human Infection ◽  
Cell Tropism ◽  
Wild Type ◽  
Phagocytic Cells ◽  
Bladder Cells ◽  
First Time

Poultry are the main source of human infection by Salmonella . As infected poultry are asymptomatic, identifying infected poultry farms is difficult, thus controlling animal infections is of primary importance. As cell tropism is known to govern disease, our aim was therefore to identify infected host–cell types in the organs of chicks known to be involved in Salmonella infection and investigate the role of the three known invasion factors in this process (T3SS-1, Rck and PagN). Chicks were inoculated with wild-type or isogenic fluorescent Salmonella Typhimurium mutants via the intracoelomic route. Our results show that liver, spleen, gall bladder and aortic vessels could be foci of infection, and that phagocytic and non-phagocytic cells, including immune, epithelial and endothelial cells, are invaded in vivo in each organ. Moreover, a mutant defective for the T3SS-1, Rck and PagN remained able to colonize organs like the wild-type strain and invaded non-phagocytic cells in each organ studied. As the infection of the gall bladder had not previously been described in chicks, invasion of gall bladder cells was confirmed by immunohistochemistry and infection was shown to last several weeks after inoculation. Altogether, for the first time these findings provide insights into cell tropism of Salmonella in relevant organs involved in Salmonella infection in chicks and also demonstrate that the known invasion factors are not required for entry into these cell types.

scholarly journals Unraveling the role of B cells in the pathogenesis of an oncogenic avian herpesvirus

2018 ◽  
Vol 115 (45) ◽  
pp. 11603-11607 ◽  
Author(s):  
Luca D. Bertzbach ◽  
Maria Laparidou ◽  
Sonja Härtle ◽  
Robert J. Etches ◽  
Bernd Kaspers ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Reverse Genetics ◽  
Gene Segment ◽  
T Cell Subsets ◽  
Bursa Of Fabricius ◽  
Wild Type ◽  
Ig Heavy Chain ◽  
Peripheral B Cells

Marek’s disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes immunosuppression, paralysis, and deadly lymphomas in chickens. In infected animals, B cells are efficiently infected and are thought to amplify the virus and transfer it to T cells. MDV subsequently establishes latency in T cells and transforms CD4+T cells, resulting in fatal lymphomas. Despite many years of research, the exact role of the different B and T cell subsets in MDV pathogenesis remains poorly understood, mostly due to the lack of reverse genetics in chickens. Recently, Ig heavy chain J gene segment knockout (JH-KO) chickens lacking mature and peripheral B cells have been generated. To determine the role of these B cells in MDV pathogenesis, we infected JH-KO chickens with the very virulent MDV RB1B strain. Surprisingly, viral load in the blood of infected animals was not altered in the absence of B cells. More importantly, disease and tumor incidence in JH-KO chickens was comparable to wild-type animals, suggesting that both mature and peripheral B cells are dispensable for MDV pathogenesis. Intriguingly, MDV efficiently replicated in the bursa of Fabricius in JH-KO animals, while spread of the virus to the spleen and thymus was delayed. In the absence of B cells, MDV readily infected CD4+and CD8+T cells, allowing efficient virus replication in the lymphoid organs and transformation of T cells. Taken together, our data change the dogma of the central role of B cells, and thereby provide important insights into MDV pathogenesis.

scholarly journals Effects of intrabodies specific for rotavirus NSP5 during the virus replicative cycle

2004 ◽  
Vol 85 (11) ◽  
pp. 3285-3290 ◽  
Author(s):  
Fulvia Vascotto ◽  
Michela Campagna ◽  
Michela Visintin ◽  
Antonino Cattaneo ◽  
Oscar R. Burrone
Keyword(s):  
Structural Protein ◽  
Wild Type ◽  
Single Chain ◽  
Single Chain Fv ◽  
Infected Cells ◽  
Antibody Capture ◽  
Virus Replication Cycle ◽  
First Time ◽  
Replicative Cycle

Intracellular antibodies or intrabodies (ICAbs) have great potential in protein knockout strategies for intracellular antigens. In this study, they have been used to investigate the role of the rotavirus non-structural protein NSP5 in the virus replication cycle. Intracellular antibody-capture technology was used to select single-chain Fv format (scFv) ICAbs against an NSP5 mutant. Five different specific ICAbs were selected and expressed in MA104 cells, in the scFv format, as cytoplasmic- and nuclear-tagged forms. By confocal microscopy, it was found that three of these ICAbs recognized the full-length wild-type NSP5 specifically, forming antigen-specific aggresomes in the cytoplasm of cotransfected cells. Expression of the ICAbs in rotavirus-infected cells largely reduced the assembly of viroplasms and cellular cytopathic effect. Replication of dsRNA was partially inhibited, despite there being no reduction in virus titre. These results demonstrate for the first time a key role for NSP5 during the virus replicative cycle.

scholarly journals Human Cytomegalovirus Latency-Associated Protein pORF94 Is Dispensable for Productive and Latent Infection

2000 ◽  
Vol 74 (19) ◽  
pp. 9333-9337 ◽  
Author(s):  
Kirsten Lofgren White ◽  
Barry Slobedman ◽  
Edward S. Mocarski
Keyword(s):  
Human Cytomegalovirus ◽  
Latent Infection ◽  
Cultured Cells ◽  
Wild Type Virus ◽  
Wild Type ◽  
Reading Frame ◽  
Latently Infected ◽  
Infected Cells ◽  
Latent Phase

ABSTRACT Human cytomegalovirus latency in bone marrow-derived myeloid progenitors is characterized by the presence of latency-associated transcripts encoded in the ie1/ie2 region of the viral genome. To assess the role of ORF94 (UL126a), a conserved open reading frame on these transcripts, a recombinant virus (RC2710) unable to express this gene was constructed. This virus replicated at wild-type levels and expressed productive as well as latency-associatedie1/ie2 region transcripts. During latency in granulocyte-macrophage progenitors, RC2710 DNA was detected at levels indistinguishable from wild-type virus, latent-phase transcription was present, and RC2710 reactivated when latently infected cells were cocultured with permissive fibroblasts. These data suggest pORF94 is not required for either productive or latent infection as assayed in cultured cells despite being the only known nuclear latency-associated protein.

scholarly journals Baculovirus lef-12 Is Not Required for Viral Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 12032-12043 ◽  
Author(s):  
Linda A. Guarino ◽  
Toni-Ann Mistretta ◽  
Wen Dong
Keyword(s):  
Trichoplusia Ni ◽  
Late Phase ◽  
Growth Curves ◽  
Single Step ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Infected Cells ◽  
Step Growth

ABSTRACT The baculovirus lef-12 (orf41) gene is required for transient expression of baculovirus late genes. To analyze the role of LEF-12 in the context of infected cells, two mutant viruses were constructed. Both mutants were viable in Trichoplusia ni High 5 and Spodoptera frugiperda Sf9 cells. Single-step growth curves, however, indicated that virus yields were reduced approximately fivefold in the absence of LEF-12. Pulse-labeling of infected cells revealed that LEF-12 mutant viruses entered the late phase and synthesized late proteins at levels equivalent to or only twofold lower than those of wild-type virus-infected cells. Western blot analyses confirmed that LEF-12 was not synthesized in cells infected with mutant virus. In wild-type virus-infected cells, LEF-12 was not detected until 18 h postinfection, and accumulation of LEF-12 peaked at 24 to 36 h postinfection. Primer extension mapping revealed that lef-12 mRNA was synthesized by 12 h postinfection and peaked between 18 and 24 h postinfection. Furthermore, synthesis of lef-12 mRNA and LEF-12 protein were inhibited by the addition of aphidicolin, indicating that lef-12 is expressed after DNA replication.

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