Frequent Coinfection of Cells Explains Functional In Vivo Complementation between Cytomegalovirus Variants in the Multiply Infected Host

ABSTRACT In contrast to many other virus infections, primary cytomegalovirus (CMV) infection does not fully protect against reinfection. Accordingly, clinical data have revealed a coexistence of multiple human CMV variants/strains in individual patients. Notably, the phenomenon of multiple infection was found to correlate with increased virus load and severity of CMV disease. Although of obvious medical relevance, the mechanism underlying this correlation is unknown. A weak immune response in an individual could be responsible for a more severe disease and for multiple infections. Alternatively, synergistic contributions of variants that differ in their biological properties can lead to qualitative changes in viral fitness by direct interactions such as genetic recombination or functional complementation within coinfected host cells. We have addressed this important question paradigmatically with the murine model by differently designed combinations of two viruses employed for experimental coinfection of mice. Specifically, a murine cytomegalovirus (MCMV) mutant expressing Cre recombinase was combined for coinfection with a mutant carrying Cre-inducible green fluorescent protein gene, and attenuated mutants were combined for coinfection with wild-type virus followed by two-color in situ hybridization studies visualizing the replication of the two viruses in infected host organs. These different approaches concurred in the conclusion that coinfection of host cells is more frequent than statistically predicted and that this coinfection alters virus fitness by functional trans-complementation rather than by genetic recombination. The reported findings make a major contribution to our molecular understanding of enhanced CMV pathogenicity in the multiply infected host.

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Reverse Genetic System for the Analysis of Parvovirus Telomeres Reveals Interactions between Transcription Factor Binding Sites in the Hairpin Stem

Journal of Virology ◽

10.1128/jvi.77.16.8650-8660.2003 ◽

2003 ◽

Vol 77 (16) ◽

pp. 8650-8660 ◽

Cited By ~ 9

Author(s):

Erik Burnett ◽

Peter Tattersall

Keyword(s):

Binding Site ◽

Simian Virus 40 ◽

Viral Fitness ◽

Host Cells ◽

Initiation Factor ◽

Wild Type Virus ◽

Reverse Genetic System ◽

Wild Type ◽

Left Hand ◽

Factor Binding

ABSTRACT The left-hand or 3′-terminal hairpin of minute virus of mice (MVM) contains sequence elements essential for both viral DNA replication at the left-hand origin (oriL) and for the modulation of the P4 promoter, from which the viral nonstructural gene cassette is transcribed. This hairpin sequence has proven difficult to manipulate in the context of the viral genome. Here we describe a system for generating mutant viruses using synthetic hairpin oligonucleotides and a truncated form of the infectious clone. This allows manipulation of the sequence of the left-hand hairpin and examination of the effects in the context of the viral life cycle. We have confirmed the requirement for a functional parvovirus initiation factor (PIF) binding site and determined that an optimized PIF binding site, with 6 bases between the half-sites, was actually detrimental to viral growth. The distal PIF half-site overlaps a cyclic AMP-responsive element (CRE), which was shown to play an important role in initiating infection, particularly in 324K simian virus 40-transformed human fibroblasts. Interestingly, reducing the spacing of the PIF half-sites, and thus the affinity of the binding site for PIF, increased viral fitness relative to wild type in 324K cells, but not in murine A9 cells. These results indicate that the relative importance of factor binding to the CRE and PIF sites during the establishment of an infection differs markedly between these two host cells and suggest that the suboptimal spacing of PIF half-sites found in wild-type virus represents a necessary reduction in the affinity of the PIF interaction in favor of CRE function.

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Bacteria facilitate viral co-infection of mammalian cells and promote genetic recombination

10.1101/154021 ◽

2017 ◽

Cited By ~ 2

Author(s):

A.K. Erickson ◽

P.R. Jesudhasan ◽

M.J. Mayer ◽

A. Narbad ◽

S.E. Winter ◽

...

Keyword(s):

Mammalian Cells ◽

Genetic Recombination ◽

Enteric Viruses ◽

Intestinal Bacteria ◽

Enteric Virus ◽

Viral Fitness ◽

Host Cells ◽

Bacterial Strains ◽

Viral Recombination ◽

Viral Genomes

SUMMARYIntestinal bacteria promote infection of several mammalian enteric viruses, but the mechanisms and consequences are unclear. We screened a panel of 41 bacterial strains as a platform to determine how different bacteria impact enteric viruses. We found that most bacterial strains bound poliovirus, a model enteric virus. Given that each bacterium bound multiple virions, we hypothesized that bacteria may deliver multiple viral genomes to a mammalian cell even when very few virions are present, such as during the first replication cycle after inter-host transmission. We found that exposure to certain bacterial strains increased viral co-infection even when the ratio of virus to host cells was low. Bacteria-mediated viral co-infection correlated with bacterial adherence to cells. Importantly, bacterial strains that induced viral co-infection facilitated viral fitness restoration through genetic recombination. Thus, bacteria-virus interactions may increase viral fitness through viral recombination at initial sites of infection, potentially limiting abortive infections.

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Genetic Recombination between Human Immunodeficiency Virus Type 1 (HIV-1) and HIV-2, Two Distinct Human Lentiviruses

Journal of Virology ◽

10.1128/jvi.01937-07 ◽

2007 ◽

Vol 82 (4) ◽

pp. 1923-1933 ◽

Cited By ~ 25

Author(s):

Kazushi Motomura ◽

Jianbo Chen ◽

Wei-Shau Hu

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Fluorescent Protein ◽

Genetic Recombination ◽

Green Fluorescent Protein Gene ◽

Low Frequencies ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) and HIV-2 are genetically distinct viruses that each can cause AIDS. Approximately 1 million people are infected with both HIV-1 and HIV-2. Additionally, these two viruses use the same receptor and coreceptors and can therefore infect the same target cell populations. To explore potential genetic interactions, we first examined whether RNAs from HIV-1 and HIV-2 can be copackaged into the same virion. We used modified near-full-length viruses that each contained a green fluorescent protein gene (gfp) with a different inactivating mutation. Thus, a functional gfp could be reconstituted via recombination, which was used to detect the copackaging of HIV-1 and HIV-2 RNAs. The GFP-positive (GFP+) phenotype was detected in approximately 0.2% of the infection events, which was 35-fold lower than the intrasubtype HIV-1 rates. We isolated and characterized 54 GFP+ single-cell clones and determined that all of them contained proviruses with reconstituted gfp. We then mapped the general structures of the recombinant viruses and characterized the recombination junctions by DNA sequencing. We observed several different recombination patterns, including those that had crossovers only in gfp. The most common hybrid genomes had heterologous long terminal repeats. Although infrequent, crossovers in the viral sequences were also identified. Taken together, our study demonstrates that HIV-1 and HIV-2 can recombine, albeit at low frequencies. These observations indicate that multiple factors are likely to restrict the generation of viable hybrid HIV-1 and HIV-2 viruses. However, considering the large coinfected human population and the high viral load in patients, these rare events could provide the basis for the generation of novel human immunodeficiency viruses.

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Chromosomalgfplabelling ofPseudomonas aeruginosausing a mini-Tn7transposon: application for studies of bacteria–host interactions

Canadian Journal of Microbiology ◽

10.1139/w07-118 ◽

2008 ◽

Vol 54 (1) ◽

pp. 48-57 ◽

Cited By ~ 7

Author(s):

Rebecca J. Barnes ◽

Kam Tin Leung ◽

Heidi Schraft ◽

Marina Ulanova

Keyword(s):

Matrix Protein ◽

Fluorescent Protein ◽

Cell Interactions ◽

Lung Epithelial Cells ◽

Host Cells ◽

Extracellular Matrix Protein ◽

Green Fluorescent Protein Gene ◽

Host Interactions

Analysis of bacterial interactions with host cells using multiple techniques is essential for studies on microbial pathogenesis and for the development of new antimicrobial therapies. Pseudomonas aeruginosa is an important opportunistic pathogen that can cause severe, often life-threatening pulmonary infections in individuals with impaired host defense mechanisms. Using a mini-Tn7 transposon delivery system, we have chromosomally labelled the strain P. aeruginosa PAK with a green fluorescent protein gene (gfp) and tested PAKgfp as a research tool for studies of bacteria–host interactions. We were able to reliably and rapidly measure the interactions of PAKgfp with A549 human lung epithelial cells by using flow cytometry, a fluorometric microplate reader-based assay, and fluorescence microscopy. With these analytical tools, we have demonstrated the adhesion of PAKgfp to the extracellular matrix protein fibronectin and the involvement of fibronectin in PAKgfp–A549 cell interactions. PAKgfp can be successfully used to explore the effects of various pharmacological compounds on P. aeruginosa – host cell interactions in both in vitro and in vivo systems, with potentially important medical applications.

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Identification of the Lymantria disparNucleopolyhedrovirus Envelope Fusion Protein Provides Evidence for a Phylogenetic Division of the Baculoviridae

Journal of Virology ◽

10.1128/jvi.74.13.6126-6131.2000 ◽

2000 ◽

Vol 74 (13) ◽

pp. 6126-6131 ◽

Cited By ~ 116

Author(s):

Margot N. Pearson ◽

Christoph Groten ◽

George F. Rohrmann

Keyword(s):

Fusion Protein ◽

Plasma Membranes ◽

Fluorescent Protein ◽

Protein Characterization ◽

Wild Type Virus ◽

Green Fluorescent Protein Gene ◽

Computer Assisted ◽

Reading Frame ◽

Infected Cells ◽

Close Relatives

ABSTRACT The complete genome sequences of a number of diverse members of theBaculoviridae including both nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) revealed that they lack a homolog of GP64, the envelope fusion protein of the budded form of Autographa californica multinucleocapsid NPV (AcMNPV) and its close relatives. Computer-assisted analyses of the genome of one of these viruses, Lymantria dispar MNPV (LdMNPV), revealed a single open reading frame (ld130) whose product had the predicted properties of a membrane protein. Characterization of the localization of the products of the full-length ld130gene and of an ld130-enhanced green fluorescent protein gene (egfp) fusion using both immunofluorescence and fluorescence microscopy revealed that LD130 accumulates at the plasma membranes of cells infected with LdMNPV or transfected withld130-egfp. In addition, cells transfected with eitherld130 or ld130-egfp or infected with wild-type virus undergo membrane fusion at pH 5. Western blot analyses indicate that LD130 is present in infected cells as an 83-kDa protein and is also present in budded virions as a protein doublet containing bands of 81 and 83 kDa. Tunicamycin treatment of infected cells resulted in an immunoreactive band of about 72 kDa, indicating that LD130 is N-glycosylated. Whereas the distribution ofgp64 appears to be confined to a relatively closely related group of NPVs, homologs of ld130 are present in a diverse number of both NPVs and GVs. This suggests that LD130 may be the primordial baculovirus envelope fusion protein.

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Faculty Opinions recommendation of Chlamydia trachomatis-infected host cells resist dsRNA-induced apoptosis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.5397956.5431066 ◽

2010 ◽

Author(s):

John Brumell ◽

Danielle Brabant

Keyword(s):

Chlamydia Trachomatis ◽

Host Cells ◽

Infected Host ◽

Induced Apoptosis

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Cytokine-dependent and–independent gene expression changes and cell cycle block revealed in Trypanosoma cruzi-infected host cells by comparative mRNA profiling

BMC Genomics ◽

10.1186/1471-2164-10-252 ◽

2009 ◽

Vol 10 (1) ◽

pp. 252 ◽

Cited By ~ 31

Author(s):

Jaime A Costales ◽

Johanna P Daily ◽

Barbara A Burleigh

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Trypanosoma Cruzi ◽

Host Cells ◽

Infected Host ◽

Mrna Profiling ◽

Independent Gene ◽

Cell Cycle Block

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Resveratrol Inhibits Venezuelan Equine Encephalitis Virus Infection by Interfering with the AKT/GSK Pathway

Plants ◽

10.3390/plants10020346 ◽

2021 ◽

Vol 10 (2) ◽

pp. 346

Author(s):

Caitlin W. Lehman ◽

Kylene Kehn-Hall ◽

Megha Aggarwal ◽

Nicole R. Bracci ◽

Han-Chi Pan ◽

...

Keyword(s):

Antiviral Activity ◽

Fluorescent Protein ◽

Glycogen Synthase ◽

Venezuelan Equine Encephalitis Virus ◽

Encephalitis Virus ◽

Host Cells ◽

Dynamics Simulation ◽

Luciferase Reporter ◽

Venezuelan Equine Encephalitis ◽

Equine Encephalitis Virus

The host proteins Protein Kinase B (AKT) and glycogen synthase kinase-3 (GSK-3) are associated with multiple neurodegenerative disorders. They are also important for the replication of Venezuelan equine encephalitis virus (VEEV), thereby making the AKT/GSK-3 pathway an attractive target for developing anti-VEEV therapeutics. Resveratrol, a natural phytochemical, has been shown to substantially inhibit the AKT pathway. Therefore, we attempted to explore whether it exerts any antiviral activity against VEEV. In this study, we utilized green fluorescent protein (GFP)- and luciferase-encoding recombinant VEEV to determine the cytotoxicity and antiviral efficacy via luciferase reporter assays, flow cytometry, and immunofluorescent assays. Our results indicate that resveratrol treatment is capable of inhibiting VEEV replication, resulting in increased viability of Vero and U87MG cells as well as reduced virion production and viral RNA contents within host cells for at least 48 h with a single treatment. Furthermore, the suppression of apoptotic signaling adaptors, caspase-3, caspase-7, and annexin V may also be implicated in resveratrol-mediated antiviral activity. We found that decreased phosphorylation of the AKT/GSK-3 pathway, mediated by resveratrol, can be triggered during the early stages of VEEV infection, suggesting that resveratrol disrupts the viral replication cycle and consequently promotes cell survival. Finally, molecular docking and dynamics simulation studies revealed that resveratrol can directly bind to VEEV glycoproteins, which may interfere with virus attachment and entry. In conclusion, our results suggest that resveratrol exerts inhibitory activity against VEEV infection and upon further modification could be a useful compound to study in neuroprotective research and veterinary sciences.

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Intercellular Transmission of Naked Viruses through Extracellular Vesicles: Focus on Polyomaviruses

Viruses ◽

10.3390/v12101086 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1086

Author(s):

Francois Helle ◽

Lynda Handala ◽

Marine Bentz ◽

Gilles Duverlie ◽

Etienne Brochot

Keyword(s):

Immune System ◽

Extracellular Vesicles ◽

Rna Viruses ◽

Viral Transmission ◽

Viral Fitness ◽

Host Cells ◽

Dna Viruses ◽

Recent Advances ◽

Viral Particles ◽

Similar Strategy

Extracellular vesicles have recently emerged as a novel mode of viral transmission exploited by naked viruses to exit host cells through a nonlytic pathway. Extracellular vesicles can allow multiple viral particles to collectively traffic in and out of cells, thus enhancing the viral fitness and diversifying the transmission routes while evading the immune system. This has been shown for several RNA viruses that belong to the Picornaviridae, Hepeviridae, Reoviridae, and Caliciviridae families; however, recent studies also demonstrated that the BK and JC viruses, two DNA viruses that belong to the Polyomaviridae family, use a similar strategy. In this review, we provide an update on recent advances in understanding the mechanisms used by naked viruses to hijack extracellular vesicles, and we discuss the implications for the biology of polyomaviruses.

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