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

2003 ◽  
Vol 77 (16) ◽  
pp. 8650-8660 ◽  
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.

scholarly journals The Influenza A Virus M2 Cytoplasmic Tail Is Required for Infectious Virus Production and Efficient Genome Packaging

2005 ◽  
Vol 79 (6) ◽  
pp. 3595-3605 ◽  
Author(s):  
Matthew F. McCown ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Infectious Virus ◽  
Virus Production ◽  
Cytoplasmic Tail ◽  
Host Cells ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Particles

ABSTRACT The M2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2 proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2 protein in trans restored the replication of the M2 truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.

scholarly journals Clathrin- and caveolin-1–independent endocytosis

2005 ◽  
Vol 168 (3) ◽  
pp. 477-488 ◽  
Author(s):  
Eva-Maria Damm ◽  
Lucas Pelkmans ◽  
Jürgen Kartenbeck ◽  
Anna Mezzacasa ◽  
Teymuras Kurzchalia ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Knockout Mouse ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Endocytic Pathway ◽  
Host Cells ◽  
Human Hepatoma ◽  
Wild Type ◽  
Embryonic Fibroblasts ◽  
Caveolin 1

Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)–deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1–independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.

scholarly journals Changes in Human Immunodeficiency Virus Type 1 Gag at Positions L449 and P453 Are Linked to I50V Protease Mutants In Vivo and Cause Reduction of Sensitivity to Amprenavir and Improved Viral Fitness In Vitro

2002 ◽  
Vol 76 (15) ◽  
pp. 7398-7406 ◽  
Author(s):  
Michael F. Maguire ◽  
Rosario Guinea ◽  
Philip Griffin ◽  
Sarah Macmanus ◽  
Robert C. Elston ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Fitness ◽  
Wild Type Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Gag protease cleavage sites (CS) undergo sequence changes during the development of resistance to several protease inhibitors (PIs). We have analyzed the association of sequence variation at the p7/p1 and p1/p6 CS in conjunction with amprenavir (APV)-specific protease mutations following PI combination therapy with APV. Querying a central resistance data repository resulted in the detection of significant associations (P < 0.001) between the presence of APV protease signature mutations and Gag L449F (p1/p6 LP1′F) and P453L (p1/p6 PP5′L) CS changes. In population-based sequence analyses the I50V mutant was invariably linked to either L449F or P453L. Clonal analysis revealed that both CS mutations were never present in the same genome. Sequential plasma samples from one patient revealed a transition from I50V M46L P453L viruses at early time points to I50V M46I L449F viruses in later samples. Various combinations of the protease and Gag mutations were introduced into the HXB2 laboratory strain of HIV-1. In both single- and multiple-cycle assay systems and in the context of I50V, the L449F and P453L changes consistently increased the 50% inhibitory concentration of APV, while the CS changes alone had no measurable effect on inhibitor sensitivity. The decreased in vitro fitness of the I50V mutant was only partially improved by addition of either CS change (I50V M46I L449F mutant replicative capacity ≈ 16% of that of wild-type virus). Western blot analysis of Pr55 Gag precursor cleavage products from infected-cell cultures indicated accumulation of uncleaved Gag p1-p6 in all I50V viruses without coexisting CS changes. Purified I50V protease catalyzed cleavage of decapeptides incorporating the L449F or P453L change 10-fold and 22-fold more efficiently than cleavage of the wild-type substrate, respectively. HIV-1 protease CS changes are selected during PI therapy and can have effects on both viral fitness and phenotypic resistance to PIs.

scholarly journals Role of Cyclin D3 in the Biology of Herpes Simplex Virus 1 ICP0

2001 ◽  
Vol 75 (4) ◽  
pp. 1888-1898 ◽  
Author(s):  
Charles Van Sant ◽  
Pascal Lopez ◽  
Sunil J. Advani ◽  
Bernard Roizman
Keyword(s):  
Cyclin D1 ◽  
Binding Site ◽  
Mutant Virus ◽  
Cyclin D3 ◽  
Wild Type Virus ◽  
Cell Protein ◽  
Cyclin D ◽  
Wild Type ◽  
Human Embryonic Lung

ABSTRACT Earlier reports from this laboratory have shown that the promiscuous transactivator infected-cell protein 0 (ICP0) binds and stabilizes cyclin D3, that the binding site maps to aspartic acid 199 (D199), and that replacement of D199 with alanine abolishes binding and reduces the capacity of the mutant virus to replicate in quiescent cells or to cause mortality in mice infected by a peripheral site. The objective of this report was to investigate the role of cyclin D3 in the biology of ICP0. We report the following results. (i) Wild-type ICP0 activates cyclin D-dependent kinase 4 (cdk4) and stabilizes cyclin D1 although ICP0 does not interact with this cyclin. (ii) The D199A mutant virus (R7914) does not activate cdk4 or stabilize cyclin D1, and neither the wild-type nor the mutant virus activates cdk2. (iii) Early in infection of human embryonic lung (HEL) fibroblasts both wild-type and D199A mutant ICP0s colocalize with PML, and in these cells the ND10 nuclear structures are dispersed. Whereas wild-type ICP0 is transported to the cytoplasm between 3 and 9 h. after infection, ICPO containing the D199A substitution remains quantitatively in the nucleus. (iv) To examine the interaction of ICP0 with cyclin D3, we used a previously described mutant carrying a wild-type ICP0 but expressing cyclin D3 (R7801) and in addition constructed a virus (R7916) that was identical except that it carried the D199A-substituted ICP0. Early in infection with R7801, ICP0 colocalized with cyclin D3 in structures similar to those containing PML. At 3 h after infection, ICP0 was translocated to the cytoplasm whereas cyclin D3 remained in the nucleus. The translocation of ICP0 to the cytoplasm was accelerated in cells expressing cyclin D3 compared with that of ICP0 expressed by wild-type virus. In contrast, ICP0 carrying the D199A substitution remained in the nucleus and did not colocalize with cyclin D3. These studies suggest the following conclusions. (i) ICP0 brings to the vicinity of ND10 cyclin D3 and, in consequence, an activated cdk4. The metabolic events occurring at or near that structure and involving cyclin D3 cause the translocation of ICP0 to the cytoplasm. (ii) In the absence of the cyclin D3 binding site in ICP0, cyclin D3 is not brought to ND10, cyclin D is not stabilized, and the function responsible for the translocation of ICP0 is not expressed, and in quiescent HEL fibroblasts the yields of virus are reduced.

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

2005 ◽  
Vol 79 (15) ◽  
pp. 9492-9502 ◽  
Author(s):  
Luka Čičin-Šain ◽  
Jürgen Podlech ◽  
Martin Messerle ◽  
Matthias J. Reddehase ◽  
Ulrich H. Koszinowski
Keyword(s):  
Fluorescent Protein ◽  
Genetic Recombination ◽  
Viral Fitness ◽  
Host Cells ◽  
Infected Host ◽  
Murine Cytomegalovirus ◽  
Wild Type Virus ◽  
Multiple Infection ◽  
Green Fluorescent Protein Gene ◽  
Multiple Infections

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.

scholarly journals Identification of Amino Acid Residues in BK Virus VP1 That Are Critical for Viability and Growth

2007 ◽  
Vol 81 (21) ◽  
pp. 11798-11808 ◽  
Author(s):  
Aisling S. Dugan ◽  
Megan L. Gasparovic ◽  
Natia Tsomaia ◽  
Dale F. Mierke ◽  
Bethany A. O'Hara ◽  
...  
Keyword(s):  
Amino Acids ◽  
Sialic Acid ◽  
Burst Size ◽  
Point Mutations ◽  
Bk Virus ◽  
Host Cells ◽  
Wild Type Virus ◽  
Wild Type ◽  
Virion Protein ◽  
Virus Propagation

ABSTRACT BK virus (BKV) is a ubiquitous pathogen that establishes a persistent infection in the urinary tract of 80% of the human population. Like other polyomaviruses, the major capsid protein of BKV, virion protein 1 (VP1), is critical for host cell receptor recognition and for proper virion assembly. BKV uses a carbohydrate complex containing α(2,3)-linked sialic acid attached to glycoprotein and glycolipid motifs as a cellular receptor. To determine the amino acids important for BKV binding to the sialic acid portion of the complex, we generated a series of 17 point mutations in VP1 and scored them for viral growth. The first set of mutants behaved identically to wild-type virus, suggesting that these amino acids were not critical for virus propagation. Another group of VP1 mutants rendered the virus nonviable. These mutations failed to protect viral DNA from DNase I digestion, indicating a role for these domains in capsid assembly and/or packaging of DNA. A third group of VP1 mutations packaged DNA similarly to the wild type but failed to propagate. The initial burst size of these mutations was similar to that of the wild type, indicating that there is no defect in the lytic release of the mutated virions. Binding experiments revealed that a subset of the BKV mutants were unable to attach to their host cells. These motifs are likely important for sialic acid recognition. We next mapped these mutations onto a model of BKV VP1 to provide atomic insight into the role of these sites in the binding of sialic acid to VP1.

scholarly journals Competitive Fitness in Coronaviruses Is Not Correlated with Size or Number of Double-Membrane Vesicles under Reduced-Temperature Growth Conditions

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Hawaa M. N. Al-Mulla ◽  
Lauren Turrell ◽  
Nicola M. Smith ◽  
Luke Payne ◽  
Surendranath Baliji ◽  
...  
Keyword(s):  
Membrane Vesicles ◽  
Growth Conditions ◽  
Viral Fitness ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Temperature Growth ◽  
Double Membrane ◽  
Competitive Fitness ◽  
Reduced Temperature

ABSTRACTPositive-stranded viruses synthesize their RNA in membrane-bound organelles, but it is not clear how this benefits the virus or the host. For coronaviruses, these organelles take the form of double-membrane vesicles (DMVs) interconnected by a convoluted membrane network. We used electron microscopy to identify murine coronaviruses with mutations in nsp3 and nsp14 that replicated normally while producing only half the normal amount of DMVs under low-temperature growth conditions. Viruses with mutations in nsp5 and nsp16 produced small DMVs but also replicated normally. Quantitative reverse transcriptase PCR (RT-PCR) confirmed that the most strongly affected of these, the nsp3 mutant, produced more viral RNA than wild-type virus. Competitive growth assays were carried out in both continuous and primary cells to better understand the contribution of DMVs to viral fitness. Surprisingly, several viruses that produced fewer or smaller DMVs showed a higher fitness than wild-type virus at the reduced temperature, suggesting that larger and more numerous DMVs do not necessarily confer a competitive advantage in primary or continuous cell culture. For the first time, this directly demonstrates that replication and organelle formation may be, at least in part, studied separately during infection with positive-stranded RNA virus.IMPORTANCEThe viruses that cause severe acute respiratory syndrome (SARS), poliomyelitis, and hepatitis C all replicate in double-membrane vesicles (DMVs). The big question about DMVs is why they exist in the first place. In this study, we looked at thousands of infected cells and identified two coronavirus mutants that made half as many organelles as normal and two others that made typical numbers but smaller organelles. Despite differences in DMV size and number, all four mutants replicated as efficiently as wild-type virus. To better understand the relative importance of replicative organelles, we carried out competitive fitness experiments. None of these viruses was found to be significantly less fit than wild-type, and two were actually fitter in tests in two kinds of cells. This suggests that viruses have evolved to have tremendous plasticity in the ability to form membrane-associated replication complexes and that large and numerous DMVs are not exclusively associated with efficient coronavirus replication.

scholarly journals Defining the root cause of reduced H1N1 live attenuated influenza vaccine effectiveness: low viral fitness leads to inter-strain competition

npj Vaccines ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Oliver Dibben ◽  
Jonathan Crowe ◽  
Shaun Cooper ◽  
Laura Hill ◽  
Katarzyna E. Schewe ◽  
...  
Keyword(s):  
Influenza Vaccine ◽  
New Caledonia ◽  
Vaccine Effectiveness ◽  
Viral Fitness ◽  
Wild Type Virus ◽  
Significant Protection ◽  
Wild Type ◽  
Virus Shedding ◽  
Live Attenuated Influenza Vaccine ◽  
Strain Competition

AbstractIn the 2013–14 and 2015–16 influenza seasons, reduced vaccine effectiveness (VE) was observed for the H1N1 component of the FluMist quadrivalent live attenuated influenza vaccine (QLAIV) in the USA, leading to loss of Advisory Committee on Immunization Practices recommendation. Here we demonstrate in ferrets that 2015–16A/H1N1pdm09 vaccine strain A/Bolivia/559/2013 (A/BOL13) is outcompeted in trivalent (TLAIV) and QLAIV formulations, leading to reduced protection from wild-type challenge. While monovalent (MLAIV) A/BOL13 provided significant protection from wild-type virus shedding and fever at doses as low as 3.0 log10 fluorescent focus units (FFU), it failed to provide a similar level of protection in TLAIV or QLAIV formulation, even at a 6.0 log10 FFU dose. Conversely, clinically effective H1N1 strain A/New Caledonia/20/1999 provided significant protection in MLAIV, TLAIV, and QLAIV formulations. In conclusion, reduced A/BOL13 replicative fitness rendered it susceptible to inter-strain competition in QLAIV, contributing to its reduced VE in the 2015–16 season.

scholarly journals Glycosylation and disulfide bonding of wild-type SARS-CoV-2 spike glycoprotein

2021 ◽  
Author(s):  
Shijian Zhang ◽  
Eden P. Go ◽  
Haitao Ding ◽  
Saumya Anang ◽  
John C. Kappes ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Neutralizing Antibodies ◽  
Natural Infection ◽  
Etiologic Agent ◽  
Host Cells ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Wild Type ◽  
Spike Glycoprotein ◽  
Sugar Addition

The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19, uses its spike (S) glycoprotein anchored in the viral membrane to enter host cells. The S glycoprotein is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by co-expression of SARS-CoV-2 S, M, E and N proteins contained spike glycoproteins that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to purify the Golgi-modified fraction of a wild-type SARS-CoV-2 S glycoprotein trimer, and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins modified to prevent proteolytic cleavage and to retain a prefusion conformation, more of the wild-type S glycoprotein N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on other characterized S trimer preparations, is predominantly modified in the Golgi compartment by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutralization by soluble ACE2 and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi processing of the native SARS-CoV-2 S glycoprotein carbohydrates and could assist the design of interventions. IMPORTANCE The SARS-CoV-2 coronavirus, which causes COVID-19, uses its spike glycoprotein to enter host cells. The viral spike glycoprotein is the main target of host neutralizing antibodies that help to control SARS-CoV-2 infection and are important for the protection provided by vaccines. The SARS-CoV-2 spike glycoprotein consists of a trimer of two subunits covered with a coat of carbohydrates (sugars). Here, we describe the disulfide bonds that assist the SARS-CoV-2 spike glycoprotein to assume the correct shape, and the composition of the sugar moieties on the glycoprotein surface. We also evaluate the consequences of natural virus variation in O-linked sugar addition and in the cysteine residues involved in disulfide bond formation. This information can expedite the improvement of vaccines and therapies for COVID-19.

scholarly journals Minimising severity of dengue serotype 1 infection by transmissible interfering particles

2020 ◽  
Author(s):  
A. Shausan ◽  
J. Aaskov ◽  
C. Drovandi ◽  
K. Mengersen
Keyword(s):  
Therapeutic Agent ◽  
Antiviral Agent ◽  
Host Cells ◽  
Wild Type Virus ◽  
Parameter Estimates ◽  
Type Virus ◽  
Wild Type ◽  
Significant Difference ◽  
Serotype 1

AbstractTransmissible interfering dengue particles (DENV–TIPs) are engineered dengue virus mutants which are defective and can replicate only with the help of dengue wild–type virus (DENV). In vitro studies have found that when DENV–TIPs and DENV coinfect a cell, they compete for viral genomes and cell proteins for replication and packaging, and DENV–TIPs outperform DENV in this process. Thus, it is hypothesised that DENV–TIPs may be used as a novel therapeutic agent. However, the effectiveness of DENV–TIPs as an antiviral agent is yet to be explored at an epidemiological scale. We present a mathematical model for the replication of DENV and DENV–TIPs as they interact with human host cells, accounting for the effectiveness of DENV–TIPs in blocking DENV from coinfected cells. We fit the model to sequentially measured plasma viral titre data from primary and secondary dengue serotype 1 infected patients in Vietnam. We show that variation in initial DENV load is sufficient to recreate the observed variation between patients. Parameter estimates, differing in primary and secondary infections, do not confirm a significant difference between these two types of infection. We use our model to investigate the potential impact of DENV–TIPs as an antiviral agent. We conclude that, when the effectiveness of DENV–TIPs in inhibiting DENV from coinfected cells is at least 80%, a dose as high as 1012 copies per millilitre of blood is required to reduce duration of infection and peak DENV serotype 1 infection level at any time point of infection. This work provides a quantitative understanding of the relationship between DENV–TIPs levels and their efficiency in clearing dengue viral infection. It will guide future development of mechanistic models of how DENV–TIPs might contribute as an antiviral agent in limiting natural dengue infection.Author summaryInhibition of dengue wild–type virus (DENV) by transmissible interfering dengue particles (DENV–TIPs) is seen in some in vitro studies, and it is hypothesised that DENV–TIPs may be used as a therapeutic agent. However, the efficiency of DENV–TIPs in limiting DENV infection in patients is yet to be explored at an epidemiological scale. Using data collected from dengue serotype 1 infected patients, we model how DENV replicates in an infected patient and how effective DENV–TIPs are in controlling that replication. Our results are of use in the evaluation of DENV–TIPs as a potential antiviral agent.

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