scholarly journals Dominance of a Nonpathogenic Glycoprotein Gene over a Pathogenic Glycoprotein Gene in Rabies Virus

2007 ◽  
Vol 81 (13) ◽  
pp. 7041-7047 ◽  
Author(s):  
Milosz Faber ◽  
Marie-Luise Faber ◽  
Jianwei Li ◽  
Mirjam A. R. Preuss ◽  
Matthias J. Schnell ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Rabies Virus ◽  
Lower Risk ◽  
Rna Synthesis ◽  
Vaccine Candidate ◽  
Neuroblastoma Cells ◽  
Virus Production ◽  
Viral Rna ◽  
Glycoprotein Gene

ABSTRACT The nonpathogenic phenotype of the live rabies virus (RV) vaccine SPBNGAN is determined by an Arg→Glu exchange at position 333 in the glycoprotein, designated GAN. We recently showed that after several passages of SPBNGAN in mice, an Asn→Lys mutation arose at position 194 of GAN, resulting in GAK, which was associated with a reversion to the pathogenic phenotype. Because an RV vaccine candidate containing two GAN genes (SPBNGAN-GAN) exhibits increased immunogenicity in vivo compared to the single-GAN construct, we tested whether the presence of two GAN genes might also enhance the probability of reversion to pathogenicity. Comparison of SPBNGAN-GAN with RVs constructed to contain either both GAN and GAK genes (SPBNGAN-GAK and SPBNGAK-GAN) or two GAK genes (SPBNGAK-GAK) showed that while SPBNGAK-GAK was pathogenic, SPBNGAN-GAN and SPBNGAN-GAK were completely nonpathogenic and SPBNGAK-GAN showed strongly reduced pathogenicity. Analysis of genomic RV RNA in mouse brain tissue revealed significantly lower virus loads in SPBNGAN-GAK- and SPBNGAK-GAN-infected brains than those detected in SPBNGAK-GAK-infected brains, indicating the dominance of the nonpathogenic phenotype determined by GAN over the GAK-associated pathogenic phenotype. Virus production and viral RNA synthesis were markedly higher in SPBNGAN-, SPBNGAK-GAN-, and SPBNGAN-GAK-infected neuroblastoma cells than in the SPBNGAK- and SPBNGAK-GAK-infected counterparts, suggesting control of GAN dominance at the level of viral RNA synthesis. These data point to the lower risk of reversion to pathogenicity of a recombinant RV carrying two identical GAN genes compared to that of an RV carrying only a single GAN gene.

Studies of two temperature-sensitive mutants of Mengo virus

1979 ◽  
Vol 57 (6) ◽  
pp. 902-913 ◽  
Author(s):  
Patrick W. K. Lee ◽  
John S. Colter
Keyword(s):  
Rna Synthesis ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Supporting Evidence ◽  
Structural Polypeptide ◽  
Infected Cells ◽  
Mengo Virus ◽  
In Vitro Stability

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA−ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 °C) to the nonpermissive (39 °C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA− phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 °C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant.Subviral (53 S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 °C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.

scholarly journals An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 547 ◽  
Author(s):  
Silvia Márquez-Jurado ◽  
Aitor Nogales ◽  
Ginés Ávila-Pérez ◽  
Francisco Iborra ◽  
Luis Martínez-Sobrido ◽  
...  
Keyword(s):  
Cdna Clone ◽  
Zika Virus ◽  
Rna Synthesis ◽  
Infectious Clone ◽  
Vero Cells ◽  
Viral Rna ◽  
Single Amino Acid ◽  
Reverse Genetic System ◽  
Infectious Clones

The recent outbreaks of Zika virus (ZIKV), its association with Guillain–Barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat ZIKV disease. In this respect, infectious clones constitute excellent tools to accomplish these goals. However, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. To bypass this problem, several alternative approaches have been applied for the generation of ZIKV clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. Here, we report a simple and novel DNA-launched approach based on the use of a bacterial artificial chromosome (BAC) to generate a cDNA clone of Rio Grande do Norte Natal ZIKV strain. The sequence was identified from the brain tissue of an aborted fetus with microcephaly. The BAC clone was fully stable in bacteria and the infectious virus was efficiently recovered in Vero cells through direct delivery of the cDNA clone. The rescued virus yielded high titers in Vero cells and was pathogenic in a validated mouse model (A129 mice) of ZIKV infection. Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A protein that presented reduced viral RNA synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with ZIKV wild-type. This BAC approach provides a stable and reliable reverse genetic system for ZIKV that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies.

scholarly journals PPEY Motif within the Rabies Virus (RV) Matrix Protein Is Essential for Efficient Virion Release and RV Pathogenicity

2008 ◽  
Vol 82 (19) ◽  
pp. 9730-9738 ◽  
Author(s):  
Christoph Wirblich ◽  
Gene S. Tan ◽  
Amy Papaneri ◽  
Peter J. Godlewski ◽  
Jan Marc Orenstein ◽  
...  
Keyword(s):  
Viral Replication ◽  
Rabies Virus ◽  
Rna Synthesis ◽  
Matrix Protein ◽  
Virus Production ◽  
Wild Type Virus ◽  
Wild Type ◽  
Conserved Sequence ◽  
Virion Release ◽  
The Matrix

ABSTRACT Late (L) domains containing the highly conserved sequence PPXY were first described for retroviruses, and later research confirmed their conservation and importance for efficient budding of several negative-stranded RNA viruses. Rabies virus (RV), a member of the Rhabdoviridae family, contains the sequence PPEY (amino acids 35 to 38) within the N terminus of the matrix (M) protein, but the functions of this potential L-domain in the viral life cycle, viral pathogenicity, and immunogenicity have not been established. Here we constructed a series of recombinant RVs containing mutations within the PPEY motif and analyzed their effects on viral replication and RV pathogenicity. Our results indicate that the first proline at position 35 is the most important for viral replication, whereas P36 and Y38 have a lesser but still noticeable impact. The reduction in viral replication was most likely due to inhibition of virion release, because initially no major impact on RV RNA synthesis was observed. In addition, results from electron microscopy demonstrated that the M4A mutant virus (PPEY→SAEA) displayed a more cell-associated phenotype than that of wild-type RV. Furthermore, all mutations within the PPEY motif resulted in reduced spread of the recombinant RVs as indicated by a reduction in focus size. Importantly, recombinant PPEY L-domain mutants were highly attenuated in mice yet still elicited potent antibody responses against RV G protein that were as high as those observed after infection with wild-type virus. Our data indicate that the RV PPEY motif has L-domain activity essential for efficient virus production and pathogenicity but is not essential for immunogenicity and thus can be targeted to increase the safety of rabies vaccine vectors.

scholarly journals Influenza Virus Nucleoprotein Interacts with Influenza Virus Polymerase Proteins

1998 ◽  
Vol 72 (7) ◽  
pp. 5493-5501 ◽  
Author(s):  
Siddhartha K. Biswas ◽  
Paul L. Boutz ◽  
Debi P. Nayak
Keyword(s):  
Influenza Virus ◽  
Protein Complex ◽  
Rna Synthesis ◽  
Sodium Dodecyl ◽  
Direct Interaction ◽  
Critical Factor ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Replication Assay

ABSTRACT Influenza virus nucleoprotein (NP) is a critical factor in the viral infectious cycle in switching influenza virus RNA synthesis from transcription mode to replication mode. In this study, we investigated the interaction of NP with the viral polymerase protein complex. Using coimmunoprecipitation with monospecific or monoclonal antibodies, we observed that NP interacted with the RNP-free polymerase protein complex in influenza virus-infected cells. In addition, coexpression of the components of the polymerase protein complex (PB1, PB2, or PA) with NP either together or pairwise revealed that NP interacts with PB1 and PB2 but not PA. Interaction of NP with PB1 and PB2 was confirmed by both coimmunoprecipitation and histidine tagging of the NP-PB1 and NP-PB2 complexes. Further, it was observed that NP-PB2 interaction was rather labile and sensitive to dissociation in 0.1% sodium dodecyl sulfate and that the stability of NP-PB2 interaction was regulated by the sequences present at the COOH terminus of NP. Analysis of NP deletion mutants revealed that at least three regions of NP interacted independently with PB2. A detailed analysis of the COOH terminus of NP by mutation of serine-to-alanine (SA) residues either individually or together demonstrated that SA mutations in this region did not affect the binding of NP to PB2. However, some SA mutations at the COOH terminus drastically affected the functional activity of NP in an in vivo transcription-replication assay, whereas others exhibited a temperature-sensitive phenotype and still others had no effect on the transcription and replication of the viral RNA. These results suggest that a direct interaction of NP with polymerase proteins may be involved in regulating the switch of viral RNA synthesis from transcription to replication.

scholarly journals SYNCRIP, a Member of the Heterogeneous Nuclear Ribonucleoprotein Family, Is Involved in Mouse Hepatitis Virus RNA Synthesis

2004 ◽  
Vol 78 (23) ◽  
pp. 13153-13162 ◽  
Author(s):  
Keum S. Choi ◽  
Akihiro Mizutani ◽  
Michael M. C. Lai
Keyword(s):  
Mammalian Cells ◽  
Rna Synthesis ◽  
Rna Binding ◽  
Hepatitis Virus ◽  
Affinity Purification ◽  
Mouse Hepatitis Virus ◽  
Viral Rna ◽  
Negative Strand

ABSTRACT Several cellular proteins, including several heterogeneous nuclear ribonucleoproteins (hnRNPs), have been shown to function as regulatory factors for mouse hepatitis virus (MHV) RNA synthesis as a result of their binding to the 5′ and 3′ untranslated regions (UTRs) of the viral RNA. Here, we identified another cellular protein, p70, which has been shown by UV cross-linking to bind both the positive- and negative-strand UTRs of MHV RNA specifically. We purified p70 with a a one-step RNA affinity purification procedure with the biotin-labeled 5′-UTR. Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry identified it as synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a member of the hnRNP family and localizes largely in the cytoplasm. The p70 was cross-linked to the MHV positive- or negative-strand UTR in vitro and in vivo. The bacterially expressed SYNCRIP was also able to bind to the 5′-UTR of both strands. The SYNCRIP-binding site was mapped to the leader sequence of the 5′-UTR, requiring the UCUAA repeat sequence. To investigate the functional significance of SYNCRIP in MHV replication, we expressed a full-length or a C-terminally truncated form of SYNCRIP in mammalian cells expressing the MHV receptor. The overexpression of either form of SYNCRIP inhibited syncytium formation induced by MHV infection. Furthermore, downregulation of the endogenous SYNCRIP with a specific short interfering RNA delayed MHV RNA synthesis; in contrast, overexpression or downregulation of SYNCRIP did not affect MHV translation. These results suggest that SYNCRIP may be directly involved in MHV RNA replication as a positive regulator. This study identified an additional cellular hnRNP as an MHV RNA-binding protein potentially involved in viral RNA synthesis.

scholarly journals Structural and Functional Analysis of the cis-Acting Elements Required for Plus-Strand RNA Synthesis of Bamboo Mosaic Virus

2005 ◽  
Vol 79 (14) ◽  
pp. 9046-9053 ◽  
Author(s):  
Jen-Wen Lin ◽  
Hsiao-Ning Chiu ◽  
I-Hsuan Chen ◽  
Tzu-Chi Chen ◽  
Yau-Heiu Hsu ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Minus Strand ◽  
Internal Deletion ◽  
Stem Loop ◽  
Cis Acting ◽  
Rna Accumulation

ABSTRACT Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome. The secondary structure of the 3′-terminal sequence of the minus-strand RNA has been predicted by MFOLD and confirmed by enzymatic structural probing to consist of a large, stable stem-loop and a small, unstable stem-loop. To identify the promoter for plus-strand RNA synthesis in this region, transcripts of 39, 77, and 173 nucleotides (Ba-39, Ba-77, and Ba-173, respectively) derived from the 3′ terminus of the minus-strand RNA were examined by an in vitro RNA-dependent RNA polymerase assay for the ability to direct RNA synthesis. Ba-77 and Ba-39 appeared to direct the RNA synthesis efficiently, while Ba-173 failed. Ba-77/Δ5, with a deletion of the 3′-terminal UUUUC sequence in Ba-77, directed the RNA synthesis only to 7% that of Ba-77. However, Ba-77/Δ16 and Ba-77/Δ31, with longer deletions but preserving the terminal UUUUC sequence of Ba-77, restored the template activity to about 60% that of the wild type. Moreover, mutations that changed the sequence in the stem of the large stem-loop interfered with the efficiency of RNA synthesis and RNA accumulation in vivo. The mutant with an internal deletion in the region between the terminal UUUUC sequence and the large stem-loop reduced the viral RNA accumulation in protoplasts, but mutants with insertions did not. Taken together, these results suggest that three cis-acting elements in the 3′ end of the minus-strand RNA, namely, the terminal UUUUC sequence, the sequence in the large stem-loop, and the distance between these two regions, are involved in modulating the efficiency of BaMV plus-strand viral RNA synthesis.

scholarly journals Inter-domain Flexibility of Chikungunya Virus nsP2 Helicase-Protease Differentially Influences Viral RNA Replication and Infectivity

2020 ◽  
pp. JVI.01470-20
Author(s):  
Yee-Song Law ◽  
Sainan Wang ◽  
Yaw Bia Tan ◽  
Orion Shih ◽  
Age Utt ◽  
...  
Keyword(s):  
Chikungunya Virus ◽  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Direct Interaction ◽  
Virus Production ◽  
Rna Replication ◽  
Full Length ◽  
Viral Rna ◽  
Viral Infectivity ◽  
Multifunctional Protein

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for chikungunya fever. Nonstructural protein 2 (nsP2), a multifunctional protein essential for viral replication, has an N-terminal helicase region (nsP2h), which as both nucleotide triphosphatase and RNA triphosphatase activities, as well as a C-terminal cysteine protease region (nsP2p), which is responsible for nonstructural polyprotein processing. The two functional units are connected through a linker of fourteen residues. Although crystal structures of the helicase and protease regions of CHIKV nsP2 have been solved separately, the conformational arrangement of the full-length nsP2 and the biological role of the linker remain elusive. Using the small-angle X-ray scattering (SAXS) method, we demonstrated that the full-length nsP2 is elongated and partially folded in solution. The reconstructed model of the structure of nsP2 contains a flexible inter-domain linker, and there is no direct interaction between the two structured regions. To examine the function of the inter-domain linker, we constructed and characterized a set of CHIKV mutants. The deletion of three or five amino acid residues in the linker region resulted in a modest defect in viral RNA replication and transcription but completely abolished viral infectivity. In contrast, increasing the flexibility of nsP2 by lengthening the inter-domain linker increased both genomic RNA replication and viral infectivity. The enzymatic activities of the corresponding mutant proteins were largely unaffected. This work suggests that increasing the inter-domain flexibility of nsP2 could facilitate the assembly of the replication complex (RC) with increased efficiency and promote virus production.IMPORTANCE: CHIKV nsP2 plays multiple roles in viral RNA replication and virus-host interactions. The helicase and protease regions of nsP2 are connected through a short “linker”. Here, we determined that the conformation of full-length CHIKV nsP2 is elongated and that the protein is flexible in solution. We also highlight the importance of the flexibility of the inter-domain of nsP2 on viral RNA synthesis and infectivity. CHIKV mutants harboring shortened linkers fail to produce infectious virus particles despite showing only relatively mild defects in genomic and subgenomic RNA synthesis. Mutations increasing the length of the inter-domain linker have only mild and generally beneficial impacts on virus replication. Thus, our findings link inter-domain flexibility with the regulation of viral RNA replication and infectivity of the viral genome.

scholarly journals Manganese-Dependent Polioviruses Caused by Mutations within the Viral Polymerase

2003 ◽  
Vol 77 (9) ◽  
pp. 5378-5388 ◽  
Author(s):  
Shane Crotty ◽  
David Gohara ◽  
Devin K. Gilligan ◽  
Sveta Karelsky ◽  
Craig E. Cameron ◽  
...  
Keyword(s):  
Amino Acids ◽  
Virus Production ◽  
Binding Pocket ◽  
Rna Polymerases ◽  
Viral Rna ◽  
Conserved Residues ◽  
Functional Flexibility ◽  
Replicative Polymerases ◽  
Positive Strand Rna

ABSTRACT Viral RNA-dependent RNA polymerases exhibit great sequence diversity. Only six core amino acids are conserved across all polymerases of positive-strand RNA viruses of eukaryotes. While exploring the function of one of these completely conserved residues, asparagine 297 in the prototypic poliovirus polymerase 3Dpol, we identified three viable mutants with noncanonical amino acids at this conserved position. Although asparagine 297 could be replaced by glycine or alanine in these mutants, the viruses exhibited Mn2+-dependent RNA replication and viral growth. All known RNA polymerases and replicative polymerases of bacterial, eukaryotic, and viral organisms are thought to be magnesium dependent in vivo, and therefore these mutant polioviruses may represent the first viruses with a requirement for an alternative polymerase cation. These results demonstrate the extreme functional flexibility of viral RNA-dependent RNA polymerases. Furthermore, the finding that strictly conserved residues in the nucleotide binding pocket of the polymerase can be altered in a manner that supports virus production suggests that drugs targeting this region of the enzyme will still be susceptible to the problem of drug-resistant escape mutants.

scholarly journals Neuronal Cell Surface Molecules Mediate Specific Binding to Rabies Virus Glycoprotein Expressed by a Recombinant Baculovirus on the Surfaces of Lepidopteran Cells

1998 ◽  
Vol 72 (2) ◽  
pp. 1085-1091 ◽  
Author(s):  
Christine Tuffereau ◽  
Jacqueline Benejean ◽  
Anne-Marie Roque Alfonso ◽  
Anne Flamand ◽  
Mark C. Fishman
Keyword(s):  
Cell Surface ◽  
Rabies Virus ◽  
Binding Sites ◽  
Specific Binding ◽  
Neuroblastoma Cells ◽  
Recombinant Baculovirus ◽  
Neuronal Cell ◽  
Previous Treatment ◽  
Double Mutation

ABSTRACT The existence of specific rabies virus (RV) glycoprotein (G) binding sites on the surfaces of neuroblastoma cells is demonstrated.Spodoptera frugiperda (Sf21) cells expressing G of the RV strain CVS (Gcvs-Sf21 cells) bind specifically to neuroblastoma cells of different species but not to any other cell type (fibroblast, myoblast, epithelial, or glioma). Attachment to mouse neuroblastoma NG108-15 cells is abolished by previous treatment of Gcvs-Sf21 cells with anti-G antibody. Substitutions for lysine at position 330 and for arginine at position 333 in RV G greatly reduce interaction between Gcvs-Sf21 cells and NG108-15 cells. These data are consistent with in vivo results: an avirulent RV mutant bearing the same double mutation is not able to infect sensory neurons or motoneurons (P. Coulon, J.-P. Ternaux, A. Flamand, and C. Tuffereau, J. Virol. 72:273–278, 1998) after intramuscular inoculation into a mouse. Furthermore, infection of NG108-15 cells by RV but not by vesicular stomatitis virus leads to a reduction of the number of binding sites at the neuronal-cell surface. Our data strongly suggest that these specific attachment sites on neuroblastoma cells represent a neuronal receptor(s) used by RV to infect certain types of neurons in vivo.

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