Glycosylation Mutants of Dengue Virus NS1 Protein

131

A strain of Tick-borne encephalitis virus designated Zausaev (Za) was isolated in Siberia from a patient who died of a progressive (2-year) form of tick-borne encephalitis 10 years after being bitten by a tick. The complete genomic sequence of this virus was determined, and an attempt was made to correlate the sequence with the biological characteristics of the virus. Phylogenetic analysis demonstrated that this virus belongs to the Siberian subtype of Tick-borne encephalitis virus. Comparison of Za virus with two related viruses, a Far Eastern isolate, Sofjin, and a Siberian isolate, Vasilchenko, revealed differences among the three viruses in pathogenicity for Syrian hamsters, cytopathogenicity for PS cells, plaque morphology, and the electrophoretic profiles of virus-specific nonstructural proteins. Comparative amino acid alignments revealed 10 individual amino acid substitutions in the Za virus polyprotein sequence that were different from those of other tick-borne flaviviruses. Notably, the dimeric form of the Za virus NS1 protein migrated in polyacrylamide gels as a heterogeneous group of molecules with a significantly higher electrophoretic mobility than those of the Sofjin and Vasilchenko viruses. Two amino acid substitutions, T 277 3V and E 279 3G, within the NS1 dimerization domain are probably responsible for the altered oligomerization of Za virus NS1. These studies suggest that the patient from whom Za virus was isolated died due to increased pathogenicity of the latent virus following spontaneous mutagenesis.

Section: Vol 77 2003 Chronic Tick-borne Encephalitis Virus Ns1 Protmentioning

confidence: 73%

Characterization of a Siberian Virus Isolated from a Patient with Progressive Chronic Tick-Borne Encephalitis

Грицун¹,

Фролова

Zhankov

et al. 2003

131

“…However, the lack of sp NS5 mutant viruses and the decreased frequency of recovery suggest that fully functional NS5 is required for virus replication. The lower incidence of temperature sensitivity in NS1 relative to NS5 mutant viruses also suggests that the NS1 structure is either stable at elevated temperatures or protected by dimerization (31,32,40) or direct membrane association (40), neither of which has been shown to occur in NS5.…”

Section: Vol 76 2002mentioning

confidence: 99%

Paired Charge-to-Alanine Mutagenesis of Dengue Virus Type 4 NS5 Generates Mutants with Temperature-Sensitive, Host Range, and Mouse Attenuation Phenotypes

Hanley

Lee

Blaney

et al. 2002

Charge-to-alanine mutagenesis of dengue virus type 4 (DEN4) NS5 gene generated a collection of attenuating mutations for potential use in a recombinant live attenuated DEN vaccine. Codons for 80 contiguous pairs of charged amino acids in NS5 were individually mutagenized to create uncharged pairs of alanine residues, and 32 recombinant mutant viruses were recovered from the 80 full-length mutant DEN4 cDNA constructs. These mutant viruses were tested for temperature-sensitive (ts) replication in both Vero cells and HuH-7 human hepatoma cells. Of the 32 mutants, 13 were temperature sensitive (ts) in both cell lines, 11 were not ts in either cell line, and 8 exhibited a host range (tshr) phenotype. One tshr mutant was ts only in Vero cells, and seven were ts only in HuH-7 cells. Nineteen of the 32 mutants were 10-fold or more restricted in replication in the brains of suckling mice compared to that of wild-type DEN4, and three mutants were approximately 10,000-fold restricted in replication. The level of temperature sensitivity of replication in vitro did not correlate with attenuation in vivo. A virus bearing two pairs of charge-to-alanine mutations was constructed and demonstrated increased temperature sensitivity and attenuation relative to either parent virus. This large set of charge-to-alanine mutations specifying a wide range of attenuation for mouse brain should prove useful in fine-tuning recombinant live attenuated DEN vaccines.Dengue virus (DEN) (genus Flavivirus, family Flaviviridae) is a single-stranded, positive-sense RNA virus with a 10.6-kb genome. The genome organization of DEN viruses is 5Ј-UTR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-UTR-3Ј (UTR, untranslated region; C, capsid; prM, membrane precursor; E, envelope; NS, nonstructural) (6, 34). DEN is comprised of four serotypes, each of which is capable of causing dengue fever (DF) and its more-severe variants, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DEN is transmitted by mosquitoes of the genus Aedes. In the past few decades, unregulated urbanization, modern patterns of travel, cessation of vector control, and other factors have led to a rapid expansion in the geographic range of all four serotypes and the cocirculation of two or more serotypes in many areas (15). Concurrently, the number of reported cases of DHF and DSS has risen dramatically. It is estimated that 50 to 100 million cases of DF and several hundred thousand cases of DHF and DSS occur annually (14, 15). Unfortunately, there is neither a licensed vaccine nor an effective antiviral therapy for the control of DEN infection. Because progression to DHF or DSS is more likely during secondary infection (27, 38), an acceptable dengue vaccine must confer immunity to all four serotypes. To date, attempts to develop tetravalent live-attenuated vaccines via cell culture passage have resulted in uneven attenuation, such that effective immunity is not induced to all four serotypes, and the most immunogenic viruses also tend to be reactogenic (4, 18).We are developing live ...

“…While previous studies with YFV, JEV, and DENV NS1 had established that ablation of N-linked glycans impairs secretion (46,48,52), surface expression of these mutants was not examined. Our results are consistent, however, with a study showing that tunicamycin treatment, which abrogates addition of N-linked glycans, did not alter the plasma membrane expression of DENV NS1 (65).…”

mentioning

confidence: 99%

A Short N-Terminal Peptide Motif on Flavivirus Nonstructural Protein NS1 Modulates Cellular Targeting and Immune Recognition

Youn¹,

Cho

Fremont

et al. 2010

Flavivirus NS1 is a versatile nonstructural glycoprotein, with intracellular NS1 functioning as an essential cofactor for viral replication and cell surface and secreted NS1 antagonizing complement activation. Even though NS1 has multiple functions that contribute to virulence, the genetic determinants that regulate the spatial distribution of NS1 in cells among different flaviviruses remain uncharacterized. Here, by creating a panel of West Nile virus-dengue virus (WNV-DENV) NS1 chimeras and site-specific mutants, we identified a novel, short peptide motif immediately C-terminal to the signal sequence cleavage position that regulates its transit time through the endoplasmic reticulum and differentially directs NS1 for secretion or plasma membrane expression. Exchange of two amino acids within this motif reciprocally changed the cellular targeting pattern of DENV or WNV NS1. For WNV, this substitution also modulated infectivity and antibody-induced phagocytosis of infected cells. Analysis of a mutant lacking all three conserved N-linked glycosylation sites revealed an independent requirement of N-linked glycans for secretion but not for plasma membrane expression of WNV NS1. Collectively, our experiments define the requirements for cellular targeting of NS1, with implications for the protective host responses, immune antagonism, and association with the host cell sorting machinery. These studies also suggest a link between the effects of NS1 on viral replication and the levels of secreted or cell surface NS1.