Influenza virus matrix protein (M1), a critical protein required for virus assembly and budding, is presumed to interact with viral glycoproteins on the outer side and viral ribonucleoprotein on the inner side. However, because of the inherent membrane-binding ability of M1 protein, it has been difficult to demonstrate the specific interaction of M1 protein with hemagglutinin (HA) or neuraminidase (NA), the influenza virus envelope glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and floatation in sucrose gradients, we observed that the membrane-bound M1 protein expressed alone or coexpressed with heterologous Sendai virus F was totally TX-100 soluble but the membrane-bound M1 protein expressed in the presence of HA and NA was predominantly detergent resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of HA facilitated binding of M1 to detergent-resistant membranes. Analysis of the membrane association of M1 in the early and late phases of the influenza virus infectious cycle revealed that the interaction of M1 with mature glycoproteins which associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of membrane-bound M1. Immunofluorescence analysis by confocal microscopy also demonstrated that, in influenza virus-infected cells, a fraction of M1 protein colocalized with HA and associated with the HA in transit to the plasma membrane via the exocytic pathway. Similar results for colocalization were obtained when M1 and HA were coexpressed and HA transport was blocked by monensin treatment. These studies indicate that both HA and NA interact with influenza virus M1 and that HA associates with M1 via its cytoplasmic tail and transmembrane domain.Influenza viruses, enveloped RNA viruses containing singlestranded, segmented RNA of negative polarity, assemble and bud from the plasma membrane of virus-infected cells into the outside environment. Complete virions are usually not observed inside the cell in the productive infectious cycle. Furthermore, in polarized epithelial cells, influenza viruses bud asymmetrically, i.e., predominantly from the apical plasma membrane (30). For virus budding to occur, two processes are obligatory (27). Firstly, all viral structural components, namely, the matrix protein (M1), the viral nucleocapsid (viral ribonucleoprotein [vRNP]) containing vRNA, nucleoprotein (NP), polymerase proteins (PB1, PB2, and PA), and NS2 (NEP) as well as the viral envelope containing the host lipids and three transmembrane proteins (hemagglutinin [HA], neuraminidase [NA], and M2) must be transported and targeted either individually or as complex subviral components to the assembly site at the plasma membrane. Secondly, these viral proteins and/or subviral components must interact with each other to initiate the budding processes leading to morphogenesis of virus particles and release of virions.Influenza virus M1, the most abundant protein in the virus particle,...

CD8+ class I–restricted cytotoxic T lymphocytes (CTLs) usually incompletely suppress HIV-1 in vivo, and while analogous partial suppression induces antiretroviral drug-resistance mutations, epitope escape mutations are inconsistently observed. However, escape mutation depends on the net balance of selective pressure and mutational fitness costs, which are poorly understood and difficult to study in vivo. Here we used a controlled in vitro system to evaluate the ability of HIV-1 to escape from CTL clones, finding that virus replicating under selective pressure rapidly can develop phenotypic resistance associated with genotypic changes. Escape varied between clones recognizing the same Gag epitope or different Gag and RT epitopes, indicating the influence of the T cell receptor on pressure and fitness costs. Gag and RT escape mutations were monoclonal intra-epitope substitutions, indicating limitation by fitness constraints in structural proteins. In contrast, escape from Nef-specific CTL was more rapid and consistent, marked by a polyclonal mixture of epitope point mutations and upstream frameshifts. We conclude that incomplete viral suppression by CTL can result in rapid emergence of immune escape, but the likelihood is strongly determined by factors influencing the fitness costs of the particular epitope targeted and the ability of responding CTL to recognize specific epitope variants.

Although the use of chimeric antigen receptors (CARs) based on single-chain antibodies for gene immunotherapy of cancers is increasing due to promising recent results, the earliest CAR therapeutic trials were done for HIV-1 infection in the late 1990s. This approach utilized a CAR based on human CD4 as a binding domain and was abandoned for a lack of efficacy. The growing number of HIV-1 broadly neutralizing antibodies (BNAbs) offers the opportunity to generate novel CARs that may be more active and revisit this modality for HIV-1 immunotherapy. We used sequences from seven well-defined BNAbs varying in binding sites and generated single-chain-antibody-based CARs. These CARs included 10E8, 3BNC117, PG9, PGT126, PGT128, VRC01, and X5. Each novel CAR exhibited conformationally relevant expression on the surface of transduced cells, mediated specific proliferation and killing in response to HIV-1-infected cells, and conferred potent antiviral activity (reduction of viral replication in log 10 units) to transduced CD8 ؉ T lymphocytes. The antiviral activity of these CARs was reproducible but varied according to the strain of virus. These findings indicated that BNAbs are excellent candidates for developing novel CARs to consider for the immunotherapeutic treatment of HIV-1. R ecent years have seen a surge in immunotherapeutic approaches for treating malignancy, including numerous promising human trials of chimeric antigen receptor (CAR) gene therapy to generate tumor-specific T cells, based on the importance of CD8 ϩ T lymphocytes (CTLs) in tumor surveillance and malignant cell clearance through cytotoxicity. The general approach has been to identify monoclonal antibodies that bind a tumor cell surface antigen and use a single-chain version of the antibody as an artificial T cell receptor by genetic fusion to the CD3 chain signaling domain. As opposed to native T cell receptors (TCRs), CARs have the advantage of being major histocompatibility complex (MHC) unrestricted and therefore broadly applicable across human individuals and are also unaffected by tumor cell immune evasion through MHC downregulation. IMPORTANCE While chimeric antigen receptors (CARsNotably, one of the earliest tested clinical applications of CARs was for the treatment of HIV-1 infection. In 1994, Roberts et al. designed two virus-specific CARs using CD4 or a single-chain antibody as the binding domain for recombinant gp120 on the surface of cells (1), and these CARs were shown subsequently to have the direct capacity to kill HIV-1-infected cells and suppress viral replication at levels similar to those of HIV-1-specific CTL clones isolated from infected persons (2). Based on these data, the CD4-based CAR, consisting of the CD4 extracellular and transmembrane domains fused to the CD3 intracellular signaling domain (CD4 Ϫ ), was advanced to clinical trials starting in the late 1990s, using retroviral transduction of autologous peripheral blood T lymphocytes and reinfusion. Unfortunately, this effort was abandoned after these trials showed...

Cytotoxic T lymphocytes (CTLs) are crucial for immune control of viral infections. "Functional avidity," defined by the sensitizing dose of exogenously added epitope yielding half-maximal CTL triggering against uninfected target cells (SD 50 ), has been utilized extensively as a measure of antiviral efficiency. However, CTLs recognize infected cells via endogenously produced epitopes, and the relationship of SD 50 to antiviral activity has never been directly revealed. We elucidate this relationship by comparing CTL killing of cells infected with panels of epitope-variant viruses to the corresponding SD 50 for the variant epitopes. This reveals a steeply sigmoid relationship between avidity and infected cell killing, with avidity thresholds (defined as the SD 50 required for CTL to achieve 50% efficiency of infected cell killing [KE 50 ]), below which infected cell killing rapidly drops to none and above which killing efficiency rapidly plateaus. Three CTL clones recognizing the same viral epitope show the same KE 50 despite differential recognition of individual epitope variants, while CTLs recognizing another epitope show a 10-fold-higher KE 50 , demonstrating epitope dependence of KE 50 . Finally, the ability of CTLs to suppress viral replication depends on the same threshold KE 50 . Thus, defining KE 50 values is required to interpret the significance of functional avidity measurements and predict CTL efficacy against virus-infected cells in pathogenesis and vaccine studies.

Sendai virus matrix protein (M protein) is critically important for virus assembly and budding and is presumed to interact with viral glycoproteins on the outer side and viral nucleocapsid on the inner side. However, since M protein alone binds to lipid membranes, it has been difficult to demonstrate the specific interaction of M protein with HN or F protein, the Sendai viral glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and flotation in sucrose gradients, we report that the membrane-bound M protein expressed alone or coexpressed with heterologous glycoprotein (influenza virus HA) was totally TX-100 soluble but the membrane-bound M protein coexpressed with HN or F protein either individually or together was predominantly detergent-resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of F protein facilitated binding of M protein to detergent-resistant membranes. Analysis of the membrane association of M protein in the early and late phases of the Sendai virus infectious cycle revealed that the interaction of M protein with mature glycoproteins that associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of the membrane-bound M protein. Immunofluorescence analysis by confocal microscopy also demonstrated that in Sendai virus-infected cells, a fraction of M protein colocalized with F and HN proteins and that some M protein also became associated with the F and HN proteins while they were in transit to the plasma membrane via the exocytic pathway. These studies indicate that F and HN interact with M protein in the absence of any other viral proteins and that F associates with M protein via its cytoplasmic tail and transmembrane domain.