| 1. |
- Adamiak, Beata, et al.
(författare)
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Herpes simplex virus type 2 glycoprotein g is targeted by the sulfated oligo- and polysaccharide inhibitors of virus attachment to cells
- 2007
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Ingår i: Journal of Virology. - 0022-538X. ; 81:24, s. 13424-13434
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Tidskriftsartikel (refereegranskat)abstract
- Variants of herpes simplex virus type 2 (HSV-2) generated by virus passage in GMK-AH1 cells in the presence of the sulfated oligosaccharide PI-88 were analyzed. Many of these variants were substantially resistant to PI-88 in their initial infection of cells and/or their cell-to-cell spread. The major alteration detected in all variants resistant to PI-88 in the initial infection of cells was a frameshift mutation(s) in the glycoprotein G (gG) gene that resulted in the lack of protein expression. Molecular transfer of the altered gG gene into the wild-type background confirmed that the gG-deficient recombinants were resistant to PI-88. In addition to PI-88, all gG-deficient variants of HSV-2 were resistant to the sulfated polysaccharide heparin. The gG-deficient virions were capable of attaching to cells, and this activity was relatively resistant to PI-88. In addition to having a drug-resistant phenotype, the gG-deficient variants were inefficiently released from infected cells. Purified gG bound to heparin and showed the cell-binding activity which was inhibited by PI-88. Many PI-88 variants produced syncytia in cultured cells and contained alterations in gB, including the syncytium-inducing L792P amino acid substitution. Although this phenotype can enhance the lateral spread of HSV in cells, it conferred no virus resistance to PI-88. Some PI-88 variants also contained occasional alterations in gC, gD, gE, gK, and UL24. In conclusion, we found that glycoprotein gG, a mucin-like component of the HSV-2 envelope, was targeted by sulfated oligo- and polysaccharides. This is a novel finding that suggests the involvement of HSV-2 gG in interactions with sulfated polysaccharides, including cell surface glycosaminoglycans.
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| 2. |
- Agback, Peter, et al.
(författare)
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Multiple Host Factors Interact with the Hypervariable Domain of Chikungunya Virus nsP3 and Determine Viral Replication in Cell-Specific Mode
- 2018
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Ingår i: Journal of Virology. - 0022-538X. ; 92
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Tidskriftsartikel (refereegranskat)abstract
- Alphaviruses are widely distributed in both hemispheres and circulate between mosquitoes and amplifying vertebrate hosts. Geographically separated alphaviruses have adapted to replication in particular organisms. The accumulating data suggest that this adaptation is determined not only by changes in their glycoproteins but also by the amino acid sequence of the hypervariable domain (HVD) of the alphavirus nsP3 protein. We performed a detailed investigation of chikungunya virus (CHIKV) nsP3 HVD interactions with host factors and their roles in viral replication in vertebrate and mosquito cells. The results demonstrate that CHIKV HVD is intrinsically disordered and binds several distinctive cellular proteins. These host factors include two members of the G3BP family and their mosquito homolog Rin, two members of the NAP1 family, and several SH3 domain-containing proteins. Interaction with G3BP proteins or Rin is an absolute requirement for CHIKV replication, although it is insufficient to solely drive it in either vertebrate or mosquito cells. To achieve a detectable level of virus replication, HVD needs to bind members of at least one more protein family in addition to G3BPs. Interaction with NAP1L1 and NAP1L4 plays a more proviral role in vertebrate cells, while binding of SH3 domain-containing proteins to a proline-rich fragment of HVD is more critical for virus replication in the cells of mosquito origin. Modifications of binding sites in CHIKV HVD allow manipulation of the cell specificity of CHIKV replication. Similar changes may be introduced into HVDs of other alphaviruses to alter their replication in particular cells or tissues.IMPORTANCE Alphaviruses utilize a broad spectrum of cellular factors for efficient formation and function of replication complexes (RCs). Our data demonstrate for the first time that the hypervariable domain (HVD) of chikungunya virus nonstructural protein 3 (nsP3) is intrinsically disordered. It binds at least 3 families of cellular proteins, which play an indispensable role in viral RNA replication. The proteins of each family demonstrate functional redundancy. We provide a detailed map of the binding sites on CHIKV nsP3 HVD and show that mutations in these sites or the replacement of CHIKV HVD by heterologous HVD change cell specificity of viral replication. Such manipulations with alphavirus HVDs open an opportunity for development of new irreversibly attenuated vaccine candidates. To date, the disordered protein fragments have been identified in the nonstructural proteins of many other viruses. They may also interact with a variety of cellular factors that determine critical aspects of virus-host interactions.
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| 3. |
- Agback, Peter, et al.
(författare)
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NAP1L1 and NAP1L4 Binding to Hypervariable Domain of Chikungunya Virus nsP3 Protein Is Bivalent and Requires Phosphorylation
- 2021
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Ingår i: Journal of Virology. - 0022-538X .- 1098-5514. ; 95
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Tidskriftsartikel (refereegranskat)abstract
- Chikungunya virus (CHIKV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Within the last 2 decades, CHIKV has expanded its presence to both hemispheres and is currently circulating in both Old and New Worlds. Despite the severity and persistence of the arthritis it causes in humans, no approved vaccines or therapeutic means have been developed for CHIKV infection. Replication of alphaviruses, including CHIKV, is determined not only by their nonstructural proteins but also by a wide range of host factors, which are indispensable components of viral replication complexes (vRCs). Alphavirus nsP3s contain hypervariable domains (HVDs), which encode multiple motifs that drive recruitment of cell- and virus-specific host proteins into vRCs. Our previous data suggested that NAP1 family members are a group of host factors that may interact with CHIKV nsP3 HVD. In this study, we performed a detailed investigation of the NAP1 function in CHIKV replication in vertebrate cells. Our data demonstrate that (i) the NAP1-HVD interactions have strong stimulatory effects on CHIKV replication, (ii) both NAP1L1 and NAP1L4 interact with the CHIKV HVD, (iii) NAP1 family members interact with two motifs, which are located upstream and downstream of the G3BP-binding motifs of CHIKV HVD, (iv) NAP1 proteins interact only with a phosphorylated form of CHIKV HVD, and HVD phosphorylation is mediated by CK2 kinase, and (v) NAP1 and other families of host factors redundantly promote CHIKV replication and their bindings have additive stimulatory effects on viral replication.IMPORTANCE Cellular proteins play critical roles in the assembly of alphavirus replication complexes (vRCs). Their recruitment is determined by the viral nonstructural protein 3 (nsP3). This protein contains a long, disordered hypervariable domain (HVD), which encodes virus-specific combinations of short linear motifs interacting with host factors during vRC assembly. Our study defined the binding mechanism of NAP1 family members to CHIKV HVD and demonstrated a stimulatory effect of this interaction on viral replication. We show that interaction with NAP1L1 is mediated by two HVD motifs and requires phosphorylation of HVD by CK2 kinase. Based on the accumulated data, we present a map of the binding motifs of the critical host factors currently known to interact with CHIKV HVD. It can be used to manipulate cell specificity of viral replication and pathogenesis, and to develop a new generation of vaccine candidates.
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| 4. |
- Agback, Tatiana, et al.
(författare)
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Structural and Functional Characterization of Host FHL1 Protein Interaction with Hypervariable Domain of Chikungunya Virus nsP3 Protein
- 2021
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Ingår i: Journal of Virology. - 0022-538X .- 1098-5514. ; 95
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Tidskriftsartikel (refereegranskat)abstract
- Decades of insufficient control have resulted in unprecedented spread of chikungunya virus (CHIKV) around the globe, and millions have suffered from the highly debilitating disease. Nevertheless, the current understanding of CHIKV-host interactions and adaptability of the virus to replication in mosquitoes and mammalian hosts is still elusive. Our new study shows that four-and-a-half LIM domain protein (FHL1) is one of the host factors that interact with the hypervariable domain (HVD) of CHIKV nsP3. Unlike G3BPs, FHL1 is not a prerequisite of CHIKV replication, and many commonly used cell lines do not express FHL1. However, its expression has a detectable stimulatory effect(s) on CHIKV replication, and Fhl1 knockout (KO) cell lines demonstrate slower infection spread. Nuclear magnetic resonance (NMR)-based studies revealed that the binding site of FHL1 in CHIKV nsP3 HVD overlaps that of another proviral host factor, CD2AP. The structural data also demonstrated that FHL1-HVD interaction is mostly determined by the LIM1 domain of FHL1. However, it does not mirror binding of the entire protein, suggesting that other LIM domains are involved. In agreement with previously published data, our biological experiments showed that interactions of CHIKV HVD with CD2AP and FHL1 have additive effects on the efficiency of CHIKV replication. This study shows that CHIKV mutants with extensive modifications of FHL1or both FHL1and CD2AP-binding sites remain viable and develop spreading infection in multiple cell types. Our study also demonstrated that other members of the FHL family can bind to CHIKV HVD and thus may be involved in viral replication.IMPORTANCE Replication of chikungunya virus (CHIKV) is determined by a wide range of host factors. Previously, we have demonstrated that the hypervariable domain (HVD) of CHIKV nsP3 contains linear motifs that recruit defined families of host proteins into formation of functional viral replication complexes. Now, using NMRbased structural and biological approaches, we have characterized the binding site of the cellular FHL1 protein in CHIKV HVD and defined the biological significance of this interaction. In contrast to previously described binding of G3BP to CHIKV HVD, the FHL1-HVD interaction was found to not be a prerequisite of viral replication. However, the presence of FHL1 has a stimulatory effect on CHIKV infectivity and, subsequently, the infection spread. FHL1 and CD2AP proteins were found to have overlapping binding sites in CHIKV HVD and additive proviral functions. Elimination of the FHL1-binding site in the nsP3 HVD can be used for the development of stable, attenuated vaccine candidates.
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| 5. |
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| 6. |
- Allain, J. P., et al.
(författare)
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Evolutionary rate and genetic drift of hepatitis C virus are not correlated with the host immune response : Studies of infected donor-recipient clusters
- 2000
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Ingår i: Journal of Virology. - 0022-538X .- 1098-5514. ; 74:6, s. 2541-2549
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Tidskriftsartikel (refereegranskat)abstract
- Six donor-recipient clusters of hepatitis C virus (HCV)-infected individuals were studied. For five clusters the period of infection of the donor could be estimated, and for all six clusters the time of infection of the recipients from the donor via blood transfusion was also precisely known. Detailed phylogenetic analyses were carried out to investigate the genomic evolution of the viral quasispecies within infected individuals in each cluster. The molecular clock analysis showed that HCV quasispecies within a patient are evolving at the same rate and that donors that have been infected for longer time tend to have a lower evolutionary rate. Phylogenetic analysis based on the split decomposition method revealed different evolutionary patterns in different donor-recipient clusters. Reactivity of antibody against the first hypervariable region (HVR1) of HCV in donor and recipient sera was evaluated and correlated to the calculated evolutionary rate. Results indicate that anti-HVR1 reactivity was related more to the overall level of humoral immune response of the host than to the HVR1 sequence itself, suggesting that the particular sequence of the HVR1 peptides is not the determinant of reactivity. Moreover, no correlation was found between the evolutionary rate or the heterogeneity of the viral quasispecies in the patients and the strength of the immune response to HVR1 epitopes, Rather, the results seem to imply that genetic drift is less dependent on immune pressure than on the rate of evolution and that the genetic drift of HCV is independent of the host immune pressure.
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