Biology subjects

Ramirez, P. W.

Publications and source records attributed to Ramirez, P. W..

6 recordsLinked to original sources

bioRxiv2025

A Cluster of Acidic Residues in the Cytoplasmic Domain of SARS-CoV-2 Spike is Required for Virion-Incorporation and Infectivity

Abstract/SummaryLike all coronaviruses, the infectivity of SARS-CoV-2 virus particles (virions) requires incorporation of the Spike glycoprotein. Yet, the mechanisms that support the virion-incorporation of Spike are incompletely defined. We noted an unusual feature of human sarbecovirus Spike proteins: their cytoplasmic domains (CDs) contain a stretch of acidic amino acids (DEDDSE). This sequence resembles a cluster of acidic residues, or acidic cluster (AC) motif, found in the cytoplasmic domain of the cellular endoprotease Furin. In Furin, the acidic cluster acts as a protein sorting signal, supporting its intracellular localization at the trans-Golgi network (TGN). We tested the contribution of the acidic cluster motif in the Spike CD to protein interactions and to the infectivity of SARS-CoV-2. We used virus-like particles (VLPs) as a model for viral "infection" (transduction). The SARS-CoV2 VLPs were produced by co-expressing Spike (S), Membrane (M), Envelope (E) and Nucleocapsid (N) proteins and deliver an RNA encoding luciferase to target cells expressing the ACE2 receptor. Remarkably, when all five acidic residues of the DEDDSE sequence were replaced with alanines, the VLPs were rendered non-infectious. The N-terminal DE residues provided most of the activity of the acidic cluster. These virologi-cally-impaired Spike mutants were able to reach the cell surface and induce the formation of syncytia, indicating that they are fusogenic and capable of anterograde traffic through the biosynthetic pathway to the plasma membrane. Despite this, they failed to efficiently incorporate into virions. We observed acidic cluster motif-dependent interactions of the Spike CD with several cellular proteins that could potentially support its role in virion-incorporation, including the ERM proteins Ezrin, Radixin, and Moesin; the retromer subunit Vps35, and the medium subunits of the clathrin adaptor complexes AP1 and AP2. While the key cofactor and mechanism of action remains to be defined, this region of acidic residues in the Spike CD appears to be a novel determinant of SARS-CoV-2 infectivity.

molecular biology↗

bioRxiv2024

Electromagnetic waves destabilize the SARS-CoV-2 Spike protein and reduce SARS-CoV-2 Virus-Like Particle (SC2-VLP) infectivity

Infection and transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to pose a global public health concern. Using electromagnetic waves represents an alternative strategy to inactivate pathogenic viruses such as SARS-CoV-2 and reduce overall transmission. However, whether electromagnetic waves reduce SARS-CoV-2 infectivity is unclear. Here, we adapted a coplanar waveguide (CPW) to identify electromagnetic waves that could neutralize SARS-CoV-2 virus-like particles (SC2-VLPs). Treatment of SC2-VLPs, particularly at frequencies between 2.5-3.5 GHz at an electric field of 400 V/m for 2 minutes, reduced infectivity. Exposure to a frequency of 3.1 GHz decreased the binding of SC2-VLPs to antibodies directed against the Spike S1 subunit receptor binding domain (RBD). These results suggest that electromagnetic waves alter the conformation of Spike, thereby reducing viral attachment to host cell receptors. Overall, this data provides proof-of-concept in using electromagnetic waves for sanitation and prevention efforts to curb the transmission of SARS-CoV-2 and potentially other pathogenic enveloped viruses.

microbiology↗

bioRxiv2020

Nef enhances HIV-1 replication and infectivity independently of SERINC3 and SERINC5 in CEM T cells

The lentiviral nef gene encodes several discrete activities aimed at co-opting or antagonizing cellular proteins and pathways to defeat host defenses and maintain persistent infection. Primary functions of Nef include downregulation of CD4 and MHC class-I from the cell surface, disruption or mimicry of T-cell receptor signaling, and enhancement of viral infectivity by counteraction of the host antiretroviral proteins SERINC3 and SERINC5. In the absence of Nef, SERINC5 incorporates into virions and inhibits viral fusion with target cells, decreasing infectivity. However, whether Nefs counteraction of SERINC5 is the cause of its positive influence on viral growth-rate in CD4-positive T cells is unclear. Here, we utilized CRISPR/Cas9 to knockout SERINC3 and SERINC5 in a leukemic CD4-positive T cell line (CEM) that displays relatively robust nef-related infectivity and growth-rate phenotypes. As previously reported, viral replication was attenuated in CEM cells infected with HIV-1 lacking Nef (HIV-1{Delta}Nef). This attenuated growth-rate phenotype was observed regardless of whether the coding regions of the serinc3 or serinc5 genes were intact. Moreover, knockout of serinc3 or serinc5 failed to restore the infectivity of HIV1{Delta}Nef virions produced from infected CEM cells. Taken together, our results corroborate a similar study using another T-lymphoid cell line (MOLT-3) and indicate that the antagonism of SERINC3 and SERINC5 cannot fully explain the virology of HIV-1 lacking Nef.

microbiology↗

bioRxiv2020

Structural Basis of CD4 Downregulation by HIV-1 Nef

The HIV-1 protein Nef suppresses multiple immune surveillance mechanisms to promote viral pathogenesis1. Individuals infected with HIV-1 encoding defective nef genes do not develop AIDS for decades2,3. A key target of Nef is the cellular receptor CD4. Although essential for viral entry into host cells, CD4 is problematic for the virus later in its replication cycle: CD4 disrupts processing of the viral glycoprotein, Env, inhibiting infectivity4; it interferes with the release of new virions5,6; and it causes vulnerability to superinfection, causing premature cell death and limiting viral productivity7. Furthermore, binding of CD4 to Env exposes otherwise-concealed Env epitopes, rendering infected cells more susceptible to antibody-dependent cellular cytotoxicity and virus particles more susceptible to neutralizing antibodies8-10. HIV-1 has evolved strategies to mitigate these problems. Newly synthesized CD4 is targeted in the endoplasmic reticulum by the viral Vpu protein for proteasomal degradation11. Surface-expressed CD4, in contrast, is targeted by Nef for endocytosis and lysosomal degradation12-15. Nefs effect on CD4 involves hijacking of clathrin adaptor complex 2 (AP2)-dependent endocytosis16,17. Although how Nef associates with a part of the tetrameric AP2 is understood18, a complete understanding of the interaction, especially how CD4 is sequestered by Nef into a complex with AP2, has remained elusive. Here, we present a high-resolution crystal structure that describes the underlying mechanism. An intricate combination of conformational changes occurs in both Nef and AP2 to enable CD4 binding and downregulation. Strikingly, a pocket on Nef previously identified as crucial for recruiting class I MHC is also responsible for recruiting CD4, revealing a potential approach to inhibit two of Nefs activities and sensitize the virus to immune clearance

biochemistry↗

bioRxiv2020

Sensitivity to Monoclonal Antibody 447-52D and an Open Env Trimer Conformation Correlate Poorly with Inhibition of HIV-1 Infectivity by SERINC5

The host protein SERINC5 inhibits the infectivity of HIV-1 virions in an Env-dependent manner and is counteracted by Nef. The conformation of the Env trimer reportedly correlates with sensitivity to SERINC5. Here, we tested the hypothesis that the "open" conformation of the Env trimer revealed by sensitivity to the V3-loop specific antibody 447-52D directly correlates with sensitivity to SERINC5. Of five Envs tested, SF162 was the most sensitive to neutralization by 447-52D, but it was not the most sensitive to SERINC5; instead the Env of LAI was substantially more sensitive to SERINC5 than all the other Envs. Mutational opening of the trimer by substitution of two tyrosines that mediate interaction between the V2 and V3 loops sensitized the Envs of JRFL and LAI to 447-52D as previously reported, but only BaL was sensitized to SERINC5. These data suggest that trimer "openness" is not sufficient for sensitivity to SERINC5.

molecular biology↗

bioRxiv2019

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

The 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 {Delta}EDTEE 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.\n\nImportanceCellular 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 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.

microbiology↗