Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity.

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.

Relative resistance of patient-derived envelope sequences to SERINC5-mediated restriction of HIV-1 infectivity.

IMPORTANCE: Pathogenesis of HIV-1 is enhanced through several viral-encoded proteins that counteract a range of host restriction molecules. HIV-1 Nef counteracts the cell membrane protein SERINC5 by downregulating it from the cell surface, thereby enhancing virion infectivity. Some subtype B reference Envelope sequences have shown the ability to bypass SERINC5 infectivity restriction independent of Nef. However, it is not clear if and to what extent circulating HIV-1 strains can exhibit resistance to SERINC5 restriction. Using a panel of Envelope sequences isolated from 50 Tanzanians infected with non-B HIV-1 subtypes, we show that the lentiviral reporters pseudotyped with patient-derived Envelopes have reduced sensitivity to SERINC5 and that this sensitivity differed among viral subtypes. Moreover, we found that SERINC5 sensitivity within patient-derived Envelopes can be modulated by separate regions, highlighting the complexity of viral/host interactions.

Dissecting Naturally Arising Amino Acid Substitutions at Position L452 of SARS-CoV-2 Spike.

Mutations at spike protein L452 are recurrently observed in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC), including omicron lineages. It remains elusive how amino acid substitutions at L452 are selected in VOC. Here, we characterized all 19 possible mutations at this site and revealed that five mutants expressing the amino acids Q, K, H, M, and R gained greater fusogenicity and pseudovirus infectivity, whereas other mutants failed to maintain steady-state expression levels and/or pseudovirus infectivity. Moreover, the five mutants showed decreased sensitivity toward neutralization by vaccine-induced antisera and conferred escape from T cell recognition. Contrary to expectations, sequence data retrieved from the Global Initiative on Sharing All Influenza Data (GISAID) revealed that the naturally occurring L452 mutations were limited to Q, M, and R, all of which can arise from a single nucleotide change. Collectively, these findings highlight that the codon base change mutational barrier is a prerequisite for amino acid substitutions at L452, in addition to the phenotypic advantages of viral fitness and decreased sensitivity to host immunity. IMPORTANCE In a span of less than 3 years since the declaration of the coronavirus pandemic, numerous SARS-CoV-2 variants of concern have emerged all around the globe, fueling a surge in the number of cases and deaths that caused severe strain on the health care system. A major concern is whether viral evolution eventually promotes greater fitness advantages, transmissibility, and immune escape. In this study, we addressed the differential effect of amino acid substitutions at a frequent mutation site, L452 of SARS-CoV-2 spike, on viral antigenic and immunological profiles and demonstrated how the virus evolves to select one amino acid over the others to ensure better viral infectivity and immune evasion. Identifying such virus mutation signatures could be crucial for the preparedness of future interventions to control COVID-19.

Aromatic Side Chain at Position 412 of SERINC5 Exerts Restriction Activity toward HIV-1 and Other Retroviruses.

The host transmembrane protein SERINC5 is incorporated into viral particles and restricts infection by certain retroviruses. However, what motif of SERINC5 mediates this process remains elusive. By conducting mutagenesis analyses, we found that the substitution of phenylalanine with alanine at position 412 (F412A) resulted in a >75-fold reduction in SERINC5's restriction function. The F412A substitution also resulted in the loss of SERINC5's function to sensitize HIV-1 neutralization by antibodies recognizing the envelope's membrane proximal region. A series of biochemical analyses revealed that F412A showed steady-state protein expression, localization at the cellular membrane, and incorporation into secreted virus particles to a greater extent than in the wild type. Furthermore, introduction of several amino acid mutations at this position revealed that the aromatic side chains, including phenylalanine, tyrosine, and tryptophan, were required to maintain SERINC5 functions to impair the virus-cell fusion process and virion infectivity. Moreover, the wild-type SERINC5 restricted infection of lentiviruses pseudotyped with envelopes of murine leukemia viruses, simian immunodeficiency virus, and HIV-2, and F412A abrogated this function. Taken together, our results highlight the importance of the aromatic side chain at SERINC5 position 412 to maintain its restriction function against diverse retrovirus envelopes. IMPORTANCE The host protein SERINC5 is incorporated into progeny virions of certain retroviruses and restricts the infectivity of these viruses or sensitizes the envelope glycoprotein to a class of neutralizing antibodies. However, how and which part of SERINC5 engages with the diverse array of retroviral envelopes and exerts its antiretroviral functions remain elusive. During mutagenesis analyses, we eventually found that the single substitution of phenylalanine with alanine, but not with tyrosine or tryptophan, at position 412 (F412A) resulted in the loss of SERINC5's functions toward diverse retroviruses, whereas F412A showed steady-state protein expression, localization at the cellular membrane, and incorporation into progeny virions to a greater extent than the wild type. Results suggest that the aromatic side chain at position 412 of SERINC5 plays a critical role in mediating antiviral functions toward various retroviruses, thus providing additional important information regarding host and retrovirus interaction.

Immune targeting of HIV-1 reservoir cells: a path to elimination strategies and cure.

Successful approaches for eradication or cure of HIV-1 infection are likely to include immunological mechanisms, but remarkably little is known about how human immune responses can recognize and interact with the few HIV-1-infected cells that harbour genome-intact viral DNA, persist long term despite antiretroviral therapy and represent the main barrier to a cure. For a long time regarded as being completely shielded from host immune responses due to viral latency, these cells do, on closer examination with single-cell analytic techniques, display discrete footprints of immune selection, implying that human immune responses may be able to effectively engage and target at least some of these cells. The failure to eliminate rebound-competent virally infected cells in the majority of persons likely reflects the evolution of a highly selected pool of reservoir cells that are effectively camouflaged from immune recognition or rely on sophisticated approaches for resisting immune-mediated killing. Understanding the fine-tuned interplay between host immune responses and viral reservoir cells will help to design improved interventions that exploit the immunological vulnerabilities of HIV-1 reservoir cells.

Factors Associated with Neutralizing Antibody Responses following 2-Dose and 3rd Booster Monovalent COVID-19 Vaccination in Japanese People Living with HIV.

People living with HIV (PLWH) could be at risk of blunted immune responses to COVID-19 vaccination. We investigated factors associated with neutralizing antibody (NAb) responses against SARS-CoV-2 and variants of concern (VOCs), following two-dose and third booster monovalent COVID-19 mRNA vaccination in Japanese PLWH. NAb titers were assessed in polyclonal IgG fractions by lentiviral-based pseudovirus assays. Overall, NAb titers against Wuhan, following two-dose vaccination, were assessed in 82 PLWH on treatment, whereby 17/82 (20.73%) were classified as low-NAb participants. Within the low-NAb participants, the third booster vaccination enhanced NAb titers against Wuhan and VOCs, albeit to a significantly lower magnitude than the rest. In the multivariate analysis, NAb titers against Wuhan after two-dose vaccination correlated with age and days since vaccination, but not with CD4+ count, CD4+/CD8+ ratio, and plasma high-sensitivity C-Reactive protein (hsCRP). Interestingly, an extended analysis within age subgroups revealed NAb titers to correlate positively with the CD4+ count and negatively with plasma hsCRP in younger, but not older, participants. In conclusion, a third booster vaccination substantially enhances NAb titers, but the benefit may be suboptimal in subpopulations of PLWH exhibiting low titers at baseline. Considering clinical and immune parameters could provide a nuanced understanding of factors associated with vaccine responses in PLWH.

Evaluation of Neutralizing Activity against Omicron Subvariants in BA.5 Breakthrough Infection and Three-Dose Vaccination Using a Novel Chemiluminescence-Based, Virus-Mediated Cytopathic Assay.

Neutralizing potency of humoral immune responses induced by prior infection or vaccination is vital for protecting of individuals and population against severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). However, the emergence of viral variants that can evade neutralization by vaccine- or infection-induced immunity is a significant public health threat and requires continuous monitoring. Here, we have developed a novel scalable chemiluminescence-based assay for assessing SARS-CoV-2-induced cytopathic effect to quantify the neutralizing activity of antisera. The assay leverages the correlation between host cell viability and ATP levels in culture to measure the cytopathic effect on target cells induced by clinically isolated, replication-competent, authentic SARS-CoV-2. With this assay, we demonstrate that the recently arisen Omicron subvariants BQ.1.1 and XBB.1 display a significant decrease in sensitivity to neutralization by antibodies elicited from breakthrough infections with Omicron BA.5 and from receipt of three doses of mRNA vaccines. Thus, this scalable neutralizing assay provides a useful platform to assess the potency of acquired humoral immunity against newly emerging SARS-CoV-2 variants. IMPORTANCE The ongoing global pandemic of SARS-CoV-2 has emphasized the importance of neutralizing immunity in protecting individuals and populations against severe respiratory illness. In light of the emergence of viral variants with the potential to evade immunity, continuous monitoring is imperative. A virus plaque reduction neutralization test (PRNT) is a "gold standard" assay for analyzing neutralizing activity for authentic viruses that form plaques, like influenza virus, dengue virus, and SARS-CoV-2. However, this method is labor intensive and is not efficient for performing large-scale neutralization assays on patient specimens. The assay system established in this study allows for the detection of a patient's neutralizing activity by simply adding an ATP detection reagent, providing a simple evaluation system for neutralizing activity of antisera as an alternative to the plaque reduction method. Our extended analysis of the Omicron subvariants highlights their increasing capability to evade neutralization by both vaccine- and infection-induced humoral immunity.

The SARS-CoV-2 Omicron BA.1 spike G446S mutation potentiates antiviral T-cell recognition.

Although the Omicron variant of the SARS-CoV-2 virus shows resistance to neutralizing antibody, it retains susceptibility to the cellular immune response. Here we characterize vaccine-induced T cells specific for various SARS-CoV-2 variants and identified HLA-A*24:02-restricted CD8+ T cells that strongly suppress Omicron BA.1 replication in vitro. Mutagenesis analyses revealed that a G446S mutation, located just outside the N-terminus of the cognate epitope, augmented TCR recognition of this variant. In contrast, no enhanced suppression of replication is observed against cells infected with the prototype, Omicron BA.2, and Delta variants that express G446. The enhancing effect of the G446S mutation is lost when target cells are treated with inhibitors of tripeptidyl peptidase II, a protein that mediates antigen processing. These ex vivo analysis and in vitro results demonstrate that the G446S mutation in the Omicron BA.1 variant affects antigen processing/presentation and potentiates antiviral activity by vaccine-induced T cells, leading to enhanced T cell recognition towards emerging variants.

The SARS-CoV-2 spike S375F mutation characterizes the Omicron BA.1 variant.

Recent studies have revealed the unique virological characteristics of Omicron, particularly those of its spike protein, such as less cleavage efficacy in cells, reduced ACE2 binding affinity, and poor fusogenicity. However, it remains unclear which mutation(s) determine these three virological characteristics of Omicron spike. Here, we show that these characteristics of the Omicron spike protein are determined by its receptor-binding domain. Of interest, molecular phylogenetic analysis revealed that acquisition of the spike S375F mutation was closely associated with the explosive spread of Omicron in the human population. We further elucidated that the F375 residue forms an interprotomer pi-pi interaction with the H505 residue of another protomer in the spike trimer, conferring the attenuated cleavage efficiency and fusogenicity of Omicron spike. Our data shed light on the evolutionary events underlying the emergence of Omicron at the molecular level.

Phenotypic and Genotypic Co-receptor Tropism Testing in HIV-1 Epidemic Region of Tanzania Where Multiple Non-B Subtypes Co-circulate.

HIV human immunodeficiency virus type I (HIV-1) entry inhibitor potency is dependent on viral co-receptor tropisms and thereby tropism determination is clinically important. However, phenotypic tropisms of HIV-1 non-B subtypes have been poorly investigated and the genotypic prediction algorithms remain insufficiently validated. To clarify this issue, we recruited 52 treatment-naïve, HIV-1-infected patients in Tanzania, where multiple HIV-1 non-B subtypes co-circulate. Sequence analysis of 93 infectious envelope clones isolated from their plasma viral RNA revealed the co-circulation of subtypes A1, C, D, and inter-subtype recombinant forms (isRFs). Phenotypic tropism assays revealed that lentivirus reporters pseudotyped with 75 (80.6%) and 5 (5.4%) envelope clones could establish infection toward U87.CD4 cells expressing CCR5 (R5) and CXCR4 (X4), respectively; whereas the remaining 13 (14%) clones could infect both cells. Genotypic analyses by widely used algorithms including V3 net charge, Geno2pheno, WebPSSM, and PhenoSeq showed that almost all phenotypic X4-tropic clones and only 15 of 75 phenotypic R5-tropic clones were concordantly predicted. However, the remaining 60 phenotypic R5-tropic clones were discordantly predicted by at least one algorithm. In particular, 2 phenotypic R5-tropic clones were discordantly predicted by all algorithms tested. Taken together, the results demonstrate the limitation of currently available genotypic algorithms for predicting co-receptor inference among co-circulating multiple non-B subtypes and emerging isRFs. Also, the phenotypic tropism dataset presented here could be valuable for retraining of the widely used genotypic prediction algorithms to enhance their performance.

SARS-CoV-2 D614G spike mutation increases entry efficiency with enhanced ACE2-binding affinity.

The causative agent of the COVID-19 pandemic, SARS-CoV-2, is steadily mutating during continuous transmission among humans. Such mutations can occur in the spike (S) protein that binds to the ACE2 receptor and is cleaved by TMPRSS2. However, whether S mutations affect SARS-CoV-2 cell entry remains unknown. Here, we show that naturally occurring S mutations can reduce or enhance cell entry via ACE2 and TMPRSS2. A SARS-CoV-2 S-pseudotyped lentivirus exhibits substantially lower entry than that of SARS-CoV S. Among S variants, the D614G mutant shows the highest cell entry, as supported by structural and binding analyses. Nevertheless, the D614G mutation does not affect neutralization by antisera against prototypic viruses. Taken together, we conclude that the D614G mutation increases cell entry by acquiring higher affinity to ACE2 while maintaining neutralization susceptibility. Based on these findings, further worldwide surveillance is required to understand SARS-CoV-2 transmissibility among humans.

SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity.

Many SARS-CoV-2 variants with naturally acquired mutations have emerged. These mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has been investigated, sensitivity to human leukocyte antigen (HLA)-restricted cellular immunity remains largely unexplored. Here, we demonstrate that two recently emerging mutations in the receptor-binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429 and B.1.617) and Y453F (in B.1.1.298), confer escape from HLA-A24-restricted cellular immunity. These mutations reinforce affinity toward the host entry receptor ACE2. Notably, the L452R mutation increases spike stability, viral infectivity, viral fusogenicity, and thereby promotes viral replication. These data suggest that HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes and that a further threat of the SARS-CoV-2 pandemic is escape from cellular immunity.

Impaired ability of Nef to counteract SERINC5 is associated with reduced plasma viremia in HIV-infected individuals.

HIV-1 Nef plays an essential role in enhancing virion infectivity by antagonizing the host restriction molecule SERINC5. Because Nef is highly polymorphic due to the selective forces of host cellular immunity, we hypothesized that certain immune-escape polymorphisms may impair Nef's ability to antagonize SERINC5 and thereby influence viral fitness in vivo. To test this hypothesis, we identified 58 Nef polymorphisms that were overrepresented in HIV-infected patients in Japan sharing the same HLA genotypes. The number of immune-associated Nef polymorphisms was inversely correlated with the plasma viral load. By breaking down the specific HLA allele-associated mutations, we found that a number of the HLA-B*51:01-associated Y120F and Q125H mutations were most significantly associated with a reduced plasma viral load. A series of biochemical experiments showed that the double mutations Y120F/Q125H, but not either single mutation, impaired Nef's ability to antagonize SERINC5 and was associated with decreasing virion infectivity and viral replication in primary lymphocytes. In contrast, other Nef functions such as CD4, CCR5, CXCR4 and HLA class I downregulation and CD74 upregulation remained unchanged. Taken together, our results suggest that the differential ability of Nef to counteract SERINC5 by naturally occurring immune-associated mutations was associated with the plasma viral load in vivo.