scholarly journals A Conserved Acidic-Cluster Motif in SERINC5 Confers Partial Resistance to Antagonism by HIV-1 Nef

2020 ◽  
Vol 94 (7) ◽  
Author(s):  
Charlotte A. Stoneham ◽  
Peter W. Ramirez ◽  
Rajendra Singh ◽  
Marissa Suarez ◽  
Andrew Debray ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Equine Infectious Anemia Virus ◽  
Mutational Analysis ◽  
Cellular Protein ◽  
Intrinsic Activity ◽  
Antiviral Protein ◽  
Cytoplasmic Loop ◽  
Relative Resistance ◽  
Conserved Sequence ◽  
Hiv 1

ABSTRACT The cellular protein SERINC5 inhibits the infectivity of diverse retroviruses, and its activity is counteracted by the glycosylated Gag (glycoGag) protein of murine leukemia virus (MLV), the S2 protein of equine infectious anemia virus (EIAV), and the Nef protein of human immunodeficiency virus type 1 (HIV-1). Determining the regions within SERINC5 that provide restrictive activity or Nef sensitivity should inform mechanistic models of the SERINC5/HIV-1 relationship. Here, we report that deletion of the conserved sequence EDTEE, which is located within a cytoplasmic loop of SERINC5 and which is reminiscent of an acidic-cluster membrane trafficking signal, increases the sensitivity of SERINC5 to antagonism by Nef, while it has no effect on the intrinsic activity of the protein as an inhibitor of infectivity. These effects correlated with enhanced removal of the ΔEDTEE mutant relative to that of wild-type SERINC5 from the cell surface and with enhanced exclusion of the mutant protein from virions by Nef. Mutational analysis indicated that the acidic residues, but not the threonine, within the EDTEE motif are important for the relative resistance to Nef. Deletion of the EDTEE sequence did not increase the sensitivity of SERINC5 to antagonism by the glycoGag protein of MLV, suggesting that its virologic role is Nef specific. These results are consistent with the reported mapping of the cytoplasmic loop that contains the EDTEE sequence as a general determinant of Nef responsiveness, but they further indicate that sequences inhibitory to as well as supportive of Nef activity reside in this region. We speculate that the EDTEE motif might have evolved to mediate resistance against retroviruses that use Nef-like proteins to antagonize SERINC5. IMPORTANCE Cellular membrane proteins in the SERINC family, especially SERINC5, inhibit the infectivity of retroviral virions. This inhibition is counteracted by retroviral proteins, specifically, HIV-1 Nef, MLV glycoGag, and EIAV S2. One consequence of such a host-pathogen “arms race” is a compensatory change in the host antiviral protein as it evolves to escape the effects of viral antagonists. This is often reflected in a genetic signature, positive selection, which is conspicuously missing in SERINC5. Here we show that despite this lack of genetic evidence, a sequence in SERINC5 nonetheless provides relative resistance to antagonism by HIV-1 Nef.

scholarly journals A conserved acidic cluster motif in SERINC5 confers resistance to antagonism by HIV-1 Nef

2019 ◽  
Author(s):  
Charlotte A. Stoneham ◽  
Peter W. Ramirez ◽  
Rajendra Singh ◽  
Marissa Suarez ◽  
Andrew Debray ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Mutational Analysis ◽  
Cellular Membrane ◽  
Cellular Protein ◽  
Intrinsic Activity ◽  
Antiviral Protein ◽  
Cytoplasmic Loop ◽  
Relative Resistance ◽  
Conserved Sequence ◽  
Hiv 1

AbstractThe cellular protein SERINC5 inhibits the infectivity of diverse retroviruses and is counteracted by the glycoGag protein of MLV, the S2 protein of EIAV, and the Nef protein of HIV-1. Determining regions within SERINC5 that provide restrictive activity or Nef-sensitivity should inform mechanistic models of the SERINC5/HIV-1 relationship. Here, we report that deletion of the highly conserved sequence EDTEE, which is located within a cytoplasmic loop of SERINC5 and is reminiscent of an acidic cluster membrane trafficking signal, increases the sensitivity of SERINC5 to antagonism by Nef while having no effect on the intrinsic activity of the protein as an inhibitor of infectivity. The effects on infectivity correlated with enhanced removal of the ΔEDTEE mutant relative to wild type SERINC5 from the cell surface and with enhanced exclusion of the mutant protein from virions by Nef. Mutational analysis revealed that the acidic residues, but not the threonine, within the EDTEE motif are important for the relative resistance to Nef. Deletion of the EDTEE sequence did not increase the sensitivity of SERINC5 to antagonism by the glycoGag protein of MLV, suggesting that its virologic role is Nef-specific. These results are consistent with the reported mapping of the cytoplasmic loop that contains the EDTEE sequence as a general determinant of Nef-responsiveness, but they further indicate that sequences inhibitory to as well as supportive of Nef-activity reside in this region. We speculate that the EDTEE motif might have evolved to mediate resistance against retroviruses that use Nef-like proteins to antagonize SERINC5.ImportanceCellular membrane proteins in the SERINC family, especially SERINC5, inhibit the infectivity of retroviral virions. This inhibition is counteracted by retroviral proteins, specifically HIV-1 Nef, MLV glycoGag, and EIAV S2. One consequence of such a host-pathogen “arms race” is compensatory change in the host antiviral protein as it evolves to escape the effects of the viral antagonist. This is often reflected in a genetic signature, positive selection, which is conspicuously missing inSERINC5. Here we show that despite this lack of genetic evidence, a sequence in SERINC5 nonetheless provides relative resistance to antagonism by HIV-1 Nef.

Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase

FEBS Letters ◽  
1991 ◽  
Vol 282 (2) ◽  
pp. 231-234 ◽  
Author(s):  
Denise M. Lowe ◽  
Vanita Parmar ◽  
Sharon D. Kemp ◽  
Brendan A. Larder
Keyword(s):  
Reverse Transcriptase ◽  
Mutational Analysis ◽  
Sequence Motifs ◽  
Conserved Sequence ◽  
Conserved Sequence Motifs ◽  
Hiv 1

scholarly journals Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Yadvinder S. Ahi ◽  
Shu Zhang ◽  
Yashna Thappeta ◽  
Audrey Denman ◽  
Amin Feizpour ◽  
...  
Keyword(s):  
Viral Entry ◽  
Equine Infectious Anemia Virus ◽  
Virus Entry ◽  
Cellular Protein ◽  
Negative Effects ◽  
Equine Infectious Anemia ◽  
Anemia Virus ◽  
Leukemia Viruses ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACTGammaretroviruses, such as murine leukemia viruses (MLVs), encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called “glycogag” (glycosylated Gag). MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others. In contrast, however, glycogag was detrimental to MLVs carrying Ebolavirus glycoprotein. Glycogag could be replaced, with respect to viral infectivity, by the unrelated S2 protein of equine infectious anemia virus. We devised an assay for viral entry in which virus particles deliver the Cre recombinase into cells, leading to the expression of a reporter. Data from this assay showed that both the positive and the negative effects of glycogag and S2 upon MLV infectivity are exerted at the level of virus entry. Moreover, transfection of the virus-producing cells with a Serinc5 expression plasmid reduced the infectivity and entry capability of MLV carrying xenotropic MLV Env, particularly in the absence of glycogag. Conversely, Serinc5 expression abrogated the negative effects of glycogag upon the infectivity and entry capability of MLV carrying Ebolavirus glycoprotein. As Serinc5 may influence cellular phospholipid metabolism, it seems possible that all of these effects on virus entry derive from changes in the lipid composition of viral membranes.IMPORTANCEMany murine leukemia viruses (MLVs) encode a protein called “glycogag.” The function of glycogag is not fully understood, but it can assist HIV-1 replication in the absence of the HIV-1 protein Nef under some circumstances. In turn, Nef counteracts the cellular protein Serinc5. Glycogag enhances the infectivity of MLVs with some but not all MLV Env proteins (which mediate viral entry into the host cell upon binding to cell surface receptors). We now report that glycogag acts by enhancing viral entry and that, like Nef, glycogag antagonizes Serinc5. Surprisingly, the effects of glycogag and Serinc5 upon the entry and infectivity of MLV particles carrying an Ebolavirus glycoprotein are the opposite of those observed with the MLV Env proteins. The unrelated S2 protein of equine infectious anemia virus (EIAV) is functionally analogous to glycogag in our experiments. Thus, three retroviruses (HIV-1, MLV, and EIAV) have independently evolved accessory proteins that counteract Serinc5.

scholarly journals A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor

2021 ◽  
Author(s):  
Charlotte A. Stoneham ◽  
Simon Langer ◽  
Paul D. De Jesus ◽  
Jacob M. Wozniak ◽  
John Lapek ◽  
...  
Keyword(s):  
Ascorbate Peroxidase ◽  
Membrane Trafficking ◽  
Protein Trafficking ◽  
Immune Surveillance ◽  
Cellular Membrane ◽  
Cellular Protein ◽  
Wild Type ◽  
Membrane Systems ◽  
Innate Defense ◽  
Hiv 1

AbstractThe HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu’s itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 required PTPN23 but not the retromer subunits. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.Author SummaryVpu triggers the degradation or mis-localization of proteins important to the host’s immune response. Vpu acts as an adaptor, linking cellular protein targets to the ubiquitination and membrane trafficking machinery. Vpu has been localized to various cellular membrane systems. By fusing wild type Vpu and Vpu-mutants to the enzyme ascorbate peroxidase, we defined the cellular proteome in proximity to Vpu and correlated this with the protein’s location. We found that wild type Vpu is proximal to ESCRT proteins, retromer complexes, and sorting and late endosomal proteins. Functionally, we found that the Vpu-mediated degradation of the innate defense protein BST-2 required PTPN23, an ALIX-like protein, consistent with our observation of Vpu’s presence at the limiting membranes of multi-vesicular bodies.

scholarly journals Effect of Small Polyanions on In Vitro Assembly of Selected Members of Alpha-, Beta- and Gammaretroviruses

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 129
Author(s):  
Alžběta Dostálková ◽  
Barbora Vokatá ◽  
Filip Kaufman ◽  
Pavel Ulbrich ◽  
Tomáš Ruml ◽  
...  
Keyword(s):  
Equine Infectious Anemia Virus ◽  
Electron Tomography ◽  
Leukemia Virus ◽  
Virus Like Particles ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Anemia Virus ◽  
Immature Virus ◽  
Hiv 1

The assembly of a hexameric lattice of retroviral immature particles requires the involvement of cell factors such as proteins and small molecules. A small, negatively charged polyanionic molecule, myo-inositol hexaphosphate (IP6), was identified to stimulate the assembly of immature particles of HIV-1 and other lentiviruses. Interestingly, cryo-electron tomography analysis of the immature particles of two lentiviruses, HIV-1 and equine infectious anemia virus (EIAV), revealed that the IP6 binding site is similar. Based on this amino acid conservation of the IP6 interacting site, it is presumed that the assembly of immature particles of all lentiviruses is stimulated by IP6. Although this specific region for IP6 binding may be unique for lentiviruses, it is plausible that other retroviral species also recruit some small polyanion to facilitate the assembly of their immature particles. To study whether the assembly of retroviruses other than lentiviruses can be stimulated by polyanionic molecules, we measured the effect of various polyanions on the assembly of immature virus-like particles of Rous sarcoma virus (RSV), a member of alpharetroviruses, Mason-Pfizer monkey virus (M-PMV) representative of betaretroviruses, and murine leukemia virus (MLV), a member of gammaretroviruses. RSV, M-PMV and MLV immature virus-like particles were assembled in vitro from truncated Gag molecules and the effect of selected polyanions, myo-inostol hexaphosphate, myo-inositol, glucose-1,6-bisphosphate, myo-inositol hexasulphate, and mellitic acid, on the particles assembly was quantified. Our results suggest that the assembly of immature particles of RSV and MLV was indeed stimulated by the presence of myo-inostol hexaphosphate and myo-inositol, respectively. In contrast, no effect on the assembly of M-PMV as a betaretrovirus member was observed.

scholarly journals Functional Replacement and Positional Dependence of Homologous and Heterologous L Domains in Equine Infectious Anemia Virus Replication

2002 ◽  
Vol 76 (4) ◽  
pp. 1569-1577 ◽  
Author(s):  
Feng Li ◽  
Chaoping Chen ◽  
Bridget A. Puffer ◽  
Ronald C. Montelaro
Keyword(s):  
Life Cycle ◽  
Virus Particle ◽  
Matrix Protein ◽  
Particle Production ◽  
Equine Infectious Anemia Virus ◽  
Virus Infectivity ◽  
Gag Protein ◽  
Equine Infectious Anemia ◽  
Anemia Virus ◽  
Hiv 1

ABSTRACT We have previously demonstrated by Gag polyprotein budding assays that the Gag p9 protein of equine infectious anemia virus (EIAV) utilizes a unique YPDL motif as a late assembly domain (L domain) to facilitate release of the budding virus particle from the host cell plasma membrane (B. A. Puffer, L. J. Parent, J. W. Wills, and R. C. Montelaro, J. Virol. 71:6541-6546, 1997). To characterize in more detail the role of the YPDL L domain in the EIAV life cycle, we have examined the replication properties of a series of EIAV proviral mutants in which the parental YPDL L domain was replaced by a human immunodeficiency virus type 1 (HIV-1) PTAP or Rous sarcoma virus (RSV) PPPY L domain in the p9 protein or by proviruses in which the parental YPDL or HIV-1 PTAP L domain was inserted in the viral matrix protein. The replication properties of these L-domain variants were examined with respect to Gag protein expression and processing, virus particle production, and virus infectivity. The data from these experiments indicate that (i) the YPDL L domain of p9 is required for replication competence (assembly and infectivity) in equine cell cultures, including the natural target equine macrophages; (ii) all of the functions of the YPDL L domain in the EIAV life cycle can be replaced by replacement of the parental YPDL sequence in p9 with the PTAP L-domain segment of HIV-1 p6 or the PPPY L domain of RSV p2b; and (iii) the assembly, but not infectivity, functions of the EIAV proviral YPDL substitution mutants can be partially rescued by inclusions of YPDL and PTAP L-domain sequences in the C-terminal region of the EIAV MA protein. Taken together, these data demonstrate that the EIAV YPDL L domain mediates distinct functions in viral budding and infectivity and that the HIV-1 PTAP and RSV PPPY L domains can effectively facilitate these dual replication functions in the context of the p9 protein. In light of the fact that YPDL, PTAP, and PPPY domains evidently have distinct characteristic binding specificities, these observations may indicate different portals into common cellular processes that mediate EIAV budding and infectivity, respectively.

scholarly journals HIV-1 requires Arf6-mediated membrane dynamics to efficiently enter and infect T lymphocytes

2011 ◽  
Vol 22 (8) ◽  
pp. 1148-1166 ◽  
Author(s):  
Laura García-Expósito ◽  
Jonathan Barroso-González ◽  
Isabel Puigdomènech ◽  
José-David Machado ◽  
Julià Blanco ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
T Lymphocytes ◽  
Viral Entry ◽  
Membrane Trafficking ◽  
Membrane Dynamics ◽  
Viral Fusion ◽  
Viral Attachment ◽  
Immunodeficiency Virus ◽  
Vesicular Stomatitis ◽  
Hiv 1

As the initial barrier to viral entry, the plasma membrane along with the membrane trafficking machinery and cytoskeleton are of fundamental importance in the viral cycle. However, little is known about the contribution of plasma membrane dynamics during early human immunodeficiency virus type 1 (HIV-1) infection. Considering that ADP ribosylation factor 6 (Arf6) regulates cellular invasion via several microorganisms by coordinating membrane trafficking, our aim was to study the function of Arf6-mediated membrane dynamics on HIV-1 entry and infection of T lymphocytes. We observed that an alteration of the Arf6–guanosine 5′-diphosphate/guanosine 5′-triphosphate (GTP/GDP) cycle, by GDP-bound or GTP-bound inactive mutants or by specific Arf6 silencing, inhibited HIV-1 envelope–induced membrane fusion, entry, and infection of T lymphocytes and permissive cells, regardless of viral tropism. Furthermore, cell-to-cell HIV-1 transmission of primary human CD4+T lymphocytes was inhibited by Arf6 knockdown. Total internal reflection fluorescence microscopy showed that Arf6 mutants provoked the accumulation of phosphatidylinositol-(4,5)-biphosphate–associated structures on the plasma membrane of permissive cells, without affecting CD4-viral attachment but impeding CD4-dependent HIV-1 entry. Arf6 silencing or its mutants did not affect fusion, entry, and infection of vesicular stomatitis virus G–pseudotyped viruses or ligand-induced CXCR4 or CCR5 endocytosis, both clathrin-dependent processes. Therefore we propose that efficient early HIV-1 infection of CD4+T lymphocytes requires Arf6-coordinated plasma membrane dynamics that promote viral fusion and entry.

A phylogenetically conserved sequence within viral 3' untranslated RNA pseudoknots regulates translation

1993 ◽  
Vol 13 (9) ◽  
pp. 5331-5347
Author(s):  
V Leathers ◽  
R Tanguay ◽  
M Kobayashi ◽  
D R Gallie
Keyword(s):  
Mutational Analysis ◽  
Order Structure ◽  
Primary Sequence ◽  
Base Region ◽  
Suspension Cells ◽  
Conserved Sequence ◽  
Binding Data ◽  
Rna Pseudoknots ◽  
Efficient Level ◽  
Necessary And Sufficient

Both the 68-base 5' leader (omega) and the 205-base 3' untranslated region (UTR) of tobacco mosaic virus (TMV) promote efficient translation. A 35-base region within omega is necessary and sufficient for the regulation. Within the 3' UTR, a 52-base region, composed of two RNA pseudoknots, is required for regulation. These pseudoknots are phylogenetically conserved among seven viruses from two different viral groups and one satellite virus. The pseudoknots contained significant conservation at the secondary and tertiary levels and at several positions at the primary sequence level. Mutational analysis of the sequences determined that the primary sequence in several conserved positions, particularly within the third pseudoknot, was essential for function. The higher-order structure of the pseudoknots was also required. Both the leader and the pseudoknot region were specifically recognized by, and competed for, the same proteins in extracts made from carrot cell suspension cells and wheat germ. Binding of the proteins is much stronger to omega than the pseudoknot region. Synergism was observed between the TMV 3' UTR and the cap and to a lesser extent between omega and the 3' UTR. The functional synergism and the protein binding data suggest that the cap, TMV 5' leader, and 3' UTR interact to establish an efficient level of translation.

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