scholarly journals Expression of the Zinc-Finger Antiviral Protein Inhibits Alphavirus Replication

2003 ◽  
Vol 77 (21) ◽  
pp. 11555-11562 ◽  
Author(s):  
Matthew J. Bick ◽  
John-William N. Carroll ◽  
Guangxia Gao ◽  
Stephen P. Goff ◽  
Charles M. Rice ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Sindbis Virus ◽  
Semliki Forest Virus ◽  
Yellow Fever Virus ◽  
Host Protein ◽  
Temperature Sensitive ◽  
Antiviral Protein ◽  
Retroviral Infection ◽  
Virus Mutant ◽  
Simplex Virus

ABSTRACT The rat zinc-finger antiviral protein (ZAP) was recently identified as a host protein conferring resistance to retroviral infection. We analyzed ZAP's ability to inhibit viruses from other families and found that ZAP potently inhibits the replication of multiple members of the Alphavirus genus within the Togaviridae, including Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus. However, expression of ZAP did not induce a broad-spectrum antiviral state as some viruses, including vesicular stomatitis virus, poliovirus, yellow fever virus, and herpes simplex virus type 1, replicated to normal levels in ZAP-expressing cells. We determined that ZAP expression inhibits Sindbis virus replication after virus penetration and entry, but before the amplification of newly synthesized plus strand genomic RNA. Using a temperature-sensitive Sindbis virus mutant expressing luciferase, we further showed that translation of incoming viral RNA is blocked by ZAP expression. Elucidation of the antiviral mechanism by which ZAP inhibits Sindbis virus translation may lead to the development of agents with broad activity against alphaviruses.

scholarly journals Differential Cholesterol Binding by Class II Fusion Proteins Determines Membrane Fusion Properties

2008 ◽  
Vol 82 (18) ◽  
pp. 9245-9253 ◽  
Author(s):  
M. Umashankar ◽  
Claudia Sánchez-San Martín ◽  
Maofu Liao ◽  
Brigid Reilly ◽  
Alice Guo ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Fusion Proteins ◽  
Sindbis Virus ◽  
Semliki Forest Virus ◽  
Yellow Fever Virus ◽  
Class Ii ◽  
Binding Properties ◽  
Functional Differences ◽  
Cholesterol Binding

ABSTRACT The class II fusion proteins of the alphaviruses and flaviviruses mediate virus infection by driving the fusion of the virus membrane with that of the cell. These fusion proteins are triggered by low pH, and their structures are strikingly similar in both the prefusion dimer and the postfusion homotrimer conformations. Here we have compared cholesterol interactions during membrane fusion by these two groups of viruses. Using cholesterol-depleted insect cells, we showed that fusion and infection by the alphaviruses Semliki Forest virus (SFV) and Sindbis virus were strongly promoted by cholesterol, with similar sterol dependence in laboratory and field isolates and in viruses passaged in tissue culture. The E1 fusion protein from SFV bound cholesterol, as detected by labeling with photocholesterol and by cholesterol extraction studies. In contrast, fusion and infection by numerous strains of the flavivirus dengue virus (DV) and by yellow fever virus 17D were cholesterol independent, and the DV fusion protein did not show significant cholesterol binding. SFV E1 is the first virus fusion protein demonstrated to directly bind cholesterol. Taken together, our results reveal important functional differences conferred by the cholesterol-binding properties of class II fusion proteins.

scholarly journals Inhibition of Filovirus Replication by the Zinc Finger Antiviral Protein

2006 ◽  
Vol 81 (5) ◽  
pp. 2391-2400 ◽  
Author(s):  
Stefanie Müller ◽  
Peggy Möller ◽  
Matthew J. Bick ◽  
Stephanie Wurr ◽  
Stephan Becker ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Sindbis Virus ◽  
Time Course ◽  
Ebola Virus ◽  
Leukemia Virus ◽  
Inhibitory Effect ◽  
Marburg Virus ◽  
Moloney Murine Leukemia Virus ◽  
Zinc Finger Motif ◽  
Antiviral Protein

ABSTRACT The zinc finger antiviral protein (ZAP) was recently shown to inhibit Moloney murine leukemia virus and Sindbis virus replication. We tested whether ZAP also acts against Ebola virus (EBOV) and Marburg virus (MARV). Antiviral effects were observed after infection of cells expressing the N-terminal part of ZAP fused to the product of the zeocin resistance gene (NZAP-Zeo) as well as after infection of cells inducibly expressing full-length ZAP. EBOV was inhibited by up to 4 log units, whereas MARV was inhibited between 1 to 2 log units. The activity of ZAP was dependent on the integrity of the second and fourth zinc finger motif, as tested with cell lines expressing NZAP-Zeo mutants. Heterologous expression of EBOV- and MARV-specific sequences fused to a reporter gene suggest that ZAP specifically targets L gene sequences. The activity of NZAP-Zeo in this assay was also dependent on the integrity of the second and fourth zinc finger motif. Time-course experiments with infectious EBOV showed that ZAP reduces the level of L mRNA before the level of genomic or antigenomic RNA is affected. Transient expression of ZAP decreased the activity of an EBOV replicon system by up to 95%. This inhibitory effect could be partially compensated for by overexpression of L protein. In conclusion, the data demonstrate that ZAP exhibits antiviral activity against filoviruses, presumably by decreasing the level of viral mRNA.

scholarly journals Molecular Defects Caused by Temperature-Sensitive Mutations in Semliki Forest Virus nsP1

2008 ◽  
Vol 82 (18) ◽  
pp. 9236-9244 ◽  
Author(s):  
Valeria Lulla ◽  
Dorothea L. Sawicki ◽  
Stanley G. Sawicki ◽  
Aleksei Lulla ◽  
Andres Merits ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Minus Strand ◽  
Nonstructural Proteins ◽  
Mutant Proteins ◽  
Recombinant Viruses ◽  
Mrna Capping

ABSTRACT Alphavirus replicase protein nsP1 has multiple functions during viral RNA synthesis. It catalyzes methyltransferase and guanylyltransferase activities needed in viral mRNA capping, attaches the viral replication complex to cytoplasmic membranes, and is required for minus-strand RNA synthesis. Two temperature-sensitive (ts) mutations in Semliki Forest virus (SFV) were previously identified within nsP1: ts10 (E529D) and ts14 (D119N). Recombinant viruses containing these individual mutations reproduced the features of the original ts strains. We now find that the capping-associated enzymatic activities of recombinant nsP1, containing ts10 or ts14 lesions, were not ts. The mutant proteins and polyproteins also were membrane bound, mutant nsP1 interacted normally with the other nonstructural proteins, and there was no major defect in nonstructural polyprotein processing in the mutants, although ts14 surprisingly displayed slightly retarded processing. The two mutant viruses were specifically defective in minus-strand RNA synthesis at the restrictive temperature. Integrating data from SFV and Sindbis virus, we discuss the domain structure of nsP1 and the relative positioning of and interactions between the replicase proteins. nsP1 is suggested to contain a specific subdomain involved in minus-strand synthesis and interaction with the polymerase nsP4 and the protease nsP2.

scholarly journals nsP4 is a major determinant of alphavirus replicase activity and template selectivity

2021 ◽  
Author(s):  
Laura Sandra Lello ◽  
Koen Bartholomeeusen ◽  
Sainan Wang ◽  
Sandra Coppens ◽  
Rennos Fragkoudis ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Open Reading Frames ◽  
Temperature Sensitive ◽  
Subgenomic Rna ◽  
Positive Strand ◽  
Novel Approach ◽  
Positive Strand Rna ◽  
Chimeric Viruses

Alphaviruses have positive-strand RNA genomes containing two open reading frames (ORFs). The first ORF encodes the non-structural (ns) polyproteins P123 and P1234 that act as precursors for the subunits of the viral RNA replicase (nsP1-nsP4). Processing of P1234 leads to the formation of a negative-strand replicase consisting of nsP4 (RNA polymerase) and P123 components. Subsequent processing of P123 results in a positive-strand replicase. The second ORF encoding the structural proteins is expressed via the synthesis of a subgenomic RNA. Alphavirus replicase is capable of using template RNAs that contain essential cis -active sequences. Here we demonstrate that the replicases of nine alphaviruses, expressed in the form of separate P123 and nsP4 components, are active. Their activity depends on the abundance of nsP4. The match of nsP4 to its template strongly influences efficient subgenomic RNA synthesis. nsP4 of Barmah Forest virus (BFV) formed a functional replicase only with matching P123 while nsP4s of other alphaviruses were compatible also with several heterologous P123s. The P123 components of Venezuelan equine encephalitis virus and Sindbis virus (SINV) required matching nsP4s while P123 of other viruses could form active replicases with different nsP4s. Chimeras of Semliki Forest virus, harboring the nsP4 of chikungunya virus, Ross River virus, BFV or SINV were viable. In contrast, chimeras of SINV, harboring an nsP4 from different alphaviruses, exhibited a temperature-sensitive phenotype. These findings highlight the possibility for formation of new alphaviruses via recombination events and provide a novel approach for the development of attenuated chimeric viruses for vaccination strategies. Importance. A key element of every virus with an RNA genome is the RNA replicase. Understanding the principles of RNA replicase formation and functioning is therefore crucial for understanding and responding to the emergence of new viruses. Reconstruction of the replicases of nine alphaviruses from nsP4 and P123 polyproteins revealed that the nsP4 of the majority of alphaviruses, including the mosquito-specific Eilat virus, could form a functional replicase with P123 originating from a different virus, and the corresponding chimeric viruses were replication-competent. nsP4 also had an evident role in determining the template RNA preference and the efficiency of RNA synthesis. The revealed broad picture of the compatibility of the replicase components of alphaviruses is important for understanding the formation and functioning of the alphavirus RNA replicase and highlights the possibilities for recombination between different alphavirus species.

scholarly journals Multiple nuclear-replicating viruses require the stress-induced protein ZC3H11A for efficient growth

2018 ◽  
Vol 115 (16) ◽  
pp. E3808-E3816 ◽  
Author(s):  
Shady Younis ◽  
Wael Kamel ◽  
Tina Falkeborn ◽  
Hao Wang ◽  
Di Yu ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Nuclear Export ◽  
High Throughput Sequencing ◽  
Rna Binding ◽  
Semliki Forest Virus ◽  
Zinc Finger Protein ◽  
Binding Capacity ◽  
Binding Property ◽  
Nuclear Regions ◽  
Simplex Virus

The zinc finger CCCH-type containing 11A (ZC3H11A) gene encodes a well-conserved zinc finger protein that may function in mRNA export as it has been shown to associate with the transcription export (TREX) complex in proteomic screens. Here, we report that ZC3H11A is a stress-induced nuclear protein with RNA-binding capacity that localizes to nuclear splicing speckles. During an adenovirus infection, the ZC3H11A protein and splicing factor SRSF2 relocalize to nuclear regions where viral DNA replication and transcription take place. Knockout (KO) of ZC3H11A in HeLa cells demonstrated that several nuclear-replicating viruses are dependent on ZC3H11A for efficient growth (HIV, influenza virus, herpes simplex virus, and adenovirus), whereas cytoplasmic replicating viruses are not (vaccinia virus and Semliki Forest virus). High-throughput sequencing of ZC3H11A–cross-linked RNA showed that ZC3H11A binds to short purine-rich ribonucleotide stretches in cellular and adenoviral transcripts. We show that the RNA-binding property of ZC3H11A is crucial for its function and localization. In ZC3H11A KO cells, the adenovirus fiber mRNA accumulates in the cell nucleus. Our results suggest that ZC3H11A is important for maintaining nuclear export of mRNAs during stress and that several nuclear-replicating viruses take advantage of this mechanism to facilitate their replication.

scholarly journals Identification of a Dominant Negative Inhibitor of Human Zinc Finger Antiviral Protein Reveals a Functional Endogenous Pool and Critical Homotypic Interactions

2010 ◽  
Vol 84 (9) ◽  
pp. 4504-4512 ◽  
Author(s):  
Lok Man J. Law ◽  
Owen R. Albin ◽  
John-William N. Carroll ◽  
Christopher T. Jones ◽  
Charles M. Rice ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Zinc Finger ◽  
Viral Genome ◽  
Sindbis Virus ◽  
Mutational Analysis ◽  
Dominant Negative ◽  
Antiviral Protein ◽  
Antiviral Function ◽  
Dominant Negative Inhibitor ◽  
In Cells

ABSTRACT The zinc finger antiviral protein (ZAP) is a host factor with potent antiviral activity when overexpressed in cells. ZAP blocks replication of the prototype alphavirus Sindbis virus (SINV) at a step at or before translation of the incoming viral genome. The mechanism of ZAP anti-SINV activity and the determinants of its antiviral function, however, have not been defined. Here, we have identified a dominant negative inhibitor of human ZAP. Rat ZAP with a cysteine-to-arginine mutation at position 88 (rZAPC88R), previously reported as a nonfunctional form of ZAP, increases SINV growth in cells. These results led us to discover a previously undetectable pool of endogenous functional ZAP within human cells. Investigation of the mechanism of dominant negative inhibition, combined with a comprehensive mutational analysis of the antiviral factor, revealed that homotypic associations are required for ZAP function in limiting SINV propagation.

scholarly journals Poly(Hydroxy)Carboxylates as Selective Inhibitors of Cytomegalovirus and Herpes Simplex Virus Replication

1992 ◽  
Vol 3 (4) ◽  
pp. 215-222 ◽  
Author(s):  
J. Neyts ◽  
R. Snoeck ◽  
P. Wutzler ◽  
M. Cushman ◽  
R. Klöcking ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Replication ◽  
Sindbis Virus ◽  
Semliki Forest Virus ◽  
Viral Envelope ◽  
Host Cells ◽  
Selective Inhibitors ◽  
Simplex Virus ◽  
Hsv 1

Polyhydroxycarboxylates (MW 3800–14000) derived from phenolic (PDP) compounds were found to be selective inhibitors of human cytomegalovirus (CMV), herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), thymidine kinase-deficient (TK−) HSV-1 and vaccinia virus replication at concentrations that are not toxic to the host cells. The PDP compounds were not inhibitory to parainfluenza virus, reovirus, Sindbis virus, or Semliki forest virus. The polycarboxylate aurintricarboxylic acid (ATA) (MW 1149–3336) also proved inhibitory to CMV and HSV replication. The anti-CMV and anti-HSV activities of the ATA polymers increased with increasing molecular weight. The mechanism of anti-CMV activity of both the PDP and ATA series of compounds can be attributed to the inhibition of virion attachment to the cells, probably due to an interaction of these polyanionic compounds with the positively charged domains of the viral envelope glycoproteins.

scholarly journals The alternate triad motif of the poly(ADP-ribose) polymerase-like domain of the human zinc finger antiviral protein is essential for its antiviral activity

2014 ◽  
Vol 95 (4) ◽  
pp. 816-822 ◽  
Author(s):  
Sabine Gläsker ◽  
Maren Töller ◽  
Beate Mareike Kümmerer
Keyword(s):  
Antiviral Activity ◽  
Zinc Finger ◽  
Sindbis Virus ◽  
Antiviral Effect ◽  
Single Amino Acid ◽  
Antiviral Protein ◽  
C Terminus ◽  
Bona Fide ◽  
Rna Knockdown ◽  
Interfering Rna

The human zinc finger antiviral protein (hZAP) gene is spliced to yield a short (hZAP-S) and a long (hZAP-L) isoform. The long isoform possesses a poly(ADP-ribose) polymerase (PARP)-like domain in its C-terminus predicted to be inactive due to alterations in its triad motif compared with bona fide PARPs. Using Sindbis virus as prototype member of alphaviruses we confirmed that hZAP-L is a more potent inhibitor of alphaviruses than hZAP-S. Specific small interfering RNA knockdown of hZAP-L but not hZAP-S demonstrated a role of endogenous hZAP-L in restriction of alphavirus replication. Whilst single amino-acid substitutions in the triad motif of hZAP-L’s PARP-like domain reduced the antiviral activity, exchange of all three triad motif residues to alanine or to the amino acids of active PARPs virtually abolished the antiviral effect. Contrary to previous assumptions, these results indicate an essential function of the PARP-like domain in hZAP-L's antiviral activity.

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