Domenico Tortorella

Enhancement of Zika virus pathogenesis by preexisting antiflavivirus immunity

One antibody for all and all antibodies for one Antibodies against related flavi-viruses such as dengue (DENV) and West Nile (WNV) can cross-react with Zika virus (ZIKV) and could thereby increase disease severity. Bardina et al. tested whether DENV and WNV antibodies from humans, or even yellow fever vaccination, could enhance ZIKV infection. In a mouse model, low titers of DENV and WNV antibodies enhanced ZIKV viremia, especially in the spinal cord and testes, whereas high titers remained protective. Generally, WNV antibodies were less disease-enhancing than DENV antibodies, and, in macaques, yellow fever vaccination had very little effect. Science, this issue p. 175 Antibodies against dengue and West Nile viruses cross-react with anti–Zika virus antibodies to enhance infection and fever in mice. Zika virus (ZIKV) is spreading rapidly into regions around the world where other flaviviruses, such as dengue virus (DENV) and West Nile virus (WNV), are endemic. Antibody-dependent enhancement has been implicated in more severe forms of flavivirus disease, but whether this also applies to ZIKV infection is unclear. Using convalescent plasma from DENV- and WNV-infected individuals, we found substantial enhancement of ZIKV infection in vitro that was mediated through immunoglobulin G engagement of Fcγ receptors. Administration of DENV- or WNV-convalescent plasma into ZIKV-susceptible mice resulted in increased morbidity—including fever, viremia, and viral loads in spinal cord and testes—and increased mortality. Antibody-dependent enhancement may explain the severe disease manifestations associated with recent ZIKV outbreaks and highlights the need to exert great caution when designing flavivirus vaccines.

Antigen presentation subverted: Structure of the human cytomegalovirus protein US2 bound to the class I molecule HLA-A2

Many persistent viruses have evolved the ability to subvert MHC class I antigen presentation. Indeed, human cytomegalovirus (HCMV) encodes at least four proteins that down-regulate cell-surface expression of class I. The HCMV unique short (US)2 glycoprotein binds newly synthesized class I molecules within the endoplasmic reticulum (ER) and subsequently targets them for proteasomal degradation. We report the crystal structure of US2 bound to the HLA-A2/Tax peptide complex. US2 associates with HLA-A2 at the junction of the peptide-binding region and the α3 domain, a novel binding surface on class I that allows US2 to bind independently of peptide sequence. Mutation of class I heavy chains confirms the importance of this binding site in vivo. Available data on class I-ER chaperone interactions indicate that chaperones would not impede US2 binding. Unexpectedly, the US2 ER-luminal domain forms an Ig-like fold. A US2 structure-based sequence alignment reveals that seven HCMV proteins, at least three of which function in immune evasion, share the same fold as US2. The structure allows design of further experiments to determine how US2 targets class I molecules for degradation.

Signal peptide peptidase is required for dislocation from the endoplasmic reticulum

Human cytomegalovirus (HCMV) prevents the display of class I major histocompatibility complex (MHC) peptide complexes at the surface of infected cells as a means of escaping immune detection. Two HCMV-encoded immunoevasins, US2 and US11, induce the dislocation of class I MHC heavy chains from the endoplasmic reticulum membrane and target them for proteasomal degradation in the cytosol. Although the outcome of the dislocation reactions catalysed is similar, US2 and US11 operate differently: Derlin-1 is a key component of the US11 but not the US2 pathway. So far, proteins essential for US2-dependent dislocation have not been identified. Here we compare interacting partners of wild-type US2 with those of a dislocation-incompetent US2 mutant, and identify signal peptide peptidase (SPP) as a partner for the active form of US2. We show that a decrease in SPP levels by RNA-mediated interference inhibits heavy-chain dislocation by US2 but not by US11. Our data implicate SPP in the US2 pathway and indicate the possibility of a previously unknown function for this intramembrane-cleaving aspartic protease in dislocation from the endoplasmic reticulum.

A glycosylated type I membrane protein becomes cytosolic when peptide: N‐glycanase is compromised

The human cytomegalovirus‐encoded glycoprotein US2 catalyzes proteasomal degradation of Class I major histocompatibility complex (MHC) heavy chains (HCs) through dislocation of the latter from the endoplasmic reticulum (ER) to the cytosol. During this process, the Class I MHC HCs are deglycosylated by an N‐glycanase‐type activity. siRNA molecules designed to inhibit the expression of the light chain, β2‐microglobulin, block the dislocation of Class I MHC molecules, which implies that US2‐dependent dislocation utilizes correctly folded Class I MHC molecules as a substrate. Here we demonstrate it is peptide: N‐glycanase (PNGase or PNG1) that deglycosylates dislocated Class I MHC HCs. Reduction of PNGase activity by siRNA expression in US2‐expressing cells inhibits deglycosylation of Class I MHC HC molecules. In PNGase siRNA‐treated cells, glycosylated HCs appear in the cytosol, providing the first evidence for the presence of an intact N‐linked type I membrane glycoprotein in the cytosol. N‐glycanase activity is therefore not required for dislocation of glycosylated Class I MHC molecules from the ER.

Human Cytomegalovirus US2 Endoplasmic Reticulum-Lumenal Domain Dictates Association with Major Histocompatibility Complex Class I in a Locus-Specific Manner

ABSTRACT The human cytomegalovirus-encoded US2 glycoprotein targets endoplasmic reticulum-resident major histocompatibility complex (MHC) class I heavy chains for rapid degradation by the proteasome. We demonstrate that the endoplasmic reticulum-lumenal domain of US2 allows tight interaction with class I molecules encoded by the HLA-A locus. Recombinant soluble US2 binds properly folded, peptide-containing recombinant HLA-A2 molecules in a peptide sequence-independent manner, consistent with US2s ability to broadly downregulate class I molecules. The physicochemical properties of the US2/MHC class I complex suggest a 1:1 stoichiometry. These results demonstrate that US2 does not require additional cellular proteins to specifically interact with soluble class I molecules. Binding of US2 does not significantly alter the conformation of class I molecules, as a soluble T-cell receptor can simultaneously recognize class I molecules associated with US2. The lumenal domain of US2 can differentiate between the products of distinct class I loci, as US2 binds several HLA-A locus products while being unable to bind recombinant HLA-B7, HLA-B27, HLA-Cw4, or HLA-E. We did not observe interaction between soluble US2 and either recombinant HLA-DR1 or recombinant HLA-DM. The substrate specificity of US2 may help explain the presence in human cytomegalovirus of multiple strategies for downregulation of MHC class I molecules.

Neutralizing antibodies against previously encountered influenza virus strains increase over time: a longitudinal analysis.

Antigenic variation promotes neutralizing antibodies to both seasonal and pandemic influenza A strains in humans. Only a Matter of Time In the fast-paced world of scientific research, most conclusions are drawn on few—or single—time points or with animal models that may accelerate—but not always accurately reproduce—disease progression. Longitudinal analyses are rare. Indeed, this is the case in influenza virus research, where it is thought that previous infections influence the outcome of subsequent infections, but little is actually known about how sequential exposures to antigenically diverse viruses affect the antibody response to influenza A viruses. Now, Miller et al. use samples obtained over a 20-year period from 40 individuals involved in the Framingham Heart Study to look at antibody titers to seasonal and pandemic influenza strains over time. The authors find longitudinal increases in neutralizing antibodies to previously encountered seasonal and pandemic flu strains. This increase was not age-dependent because it was observed against strains encountered later in life as well as in earlier exposures. Titers to the more conserved hemagglutinin stalk domain increased modestly as well, but no neutralizing antibody titer increase was observed against a more antigenically stable virus (human cytomegalovirus). These results suggest that antigenic variation may drive the hierarchical humoral immune response to influenza strains. The contribution of antigenic variation to antibody titers to the conserved stalk region supports the pursuit of vaccine strategies that increase exposure to antigenically diverse strains of influenza. Antigenic diversity shapes immunity in distinct and unexpected ways. This is particularly true of the humoral response generated against influenza A viruses. Although it is known that immunological memory developed against previously encountered influenza A virus strains affects the outcome of subsequent infections, exactly how sequential exposures to antigenically variant viruses shape the humoral immune response in humans remains poorly understood. To address this important question, we performed a longitudinal analysis of antibody titers against various pandemic and seasonal strains of influenza virus spanning a 20-year period (1987 to 2008) with samples from 40 individuals (birth dates, 1917 to 1952) obtained from the Framingham Heart Study. Longitudinal increases in neutralizing antibody titers were observed against previously encountered pandemic H2N2, H3N2, and H1N1 influenza A virus strains. Antibody titers against seasonal strains encountered later in life also increased longitudinally at a rate similar to that against their pandemic predecessors. Titers of cross-reactive antibodies specific to the hemagglutinin stalk domain were also investigated because they are influenced by exposure to antigenically diverse influenza A viruses. These titers rose modestly over time, even in the absence of major antigenic shifts. No sustained increase in neutralizing antibody titers against an antigenically more stable virus (human cytomegalovirus) was observed. The results herein describe a role for antigenic variation in shaping the humoral immune compartment and provide a rational basis for the hierarchical nature of antibody titers against influenza A viruses in humans.

Visualization of the ER-to-cytosol dislocation reaction of a type I membrane protein

The human cytomegalovirus gene products US2 and US11 induce proteasomal degradation of MHC class I heavy chains. We have generated an enhanced green fluorescent protein–class I heavy chain (EGFP–HC) chimeric molecule to study its dislocation and degradation in US2‐ and US11‐expressing cells. The EGFP–HC fusion is stable in control cells, but is degraded rapidly in US2‐ or US11‐expressing cells. Proteasome inhibitors induce in a time‐dependent manner the accumulation of EGFP–HC molecules in US2‐ and US11‐expressing cells, as assessed biochemically and by cytofluorimetry of intact cells. Pulse–chase analysis and subcellular fractionation show that EGFP–HC proteins are dislocated from the endoplasmic reticulum and can be recovered as deglycosylated fluorescent intermediates in the cytosol. These results raise the possibility that dislocation of glycoproteins from the ER may not require their full unfolding.

The Human Cytomegalovirus US10 Gene Product Delays Trafficking of Major Histocompatibility Complex Class I Molecules

ABSTRACT Human cytomegalovirus (HCMV) US10 encodes a glycoprotein that binds to major histocompatibility complex (MHC) class I heavy chains. While expression of US10 delays the normal trafficking of MHC class I molecules out of the endoplasmic reticulum, US10 does not obviously facilitate or inhibit the action of two other HCMV-encoded MHC class I binding proteins, US2 and US11.

The cytomegalovirus-encoded chemokine receptor US28 promotes intestinal neoplasia in transgenic mice.

US28 is a constitutively active chemokine receptor encoded by CMV (also referred to as human herpesvirus 5), a highly prevalent human virus that infects a broad spectrum of cells, including intestinal epithelial cells (IECs). To study the role of US28 in vivo, we created transgenic mice (VS28 mice) in which US28 expression was targeted to IECs. Expression of US28 was detected in all IECs of the small and large intestine, including in cells expressing leucine rich repeat containing GPCR5 (Lgr5), a marker gene of intestinal epithelial stem cells. US28 expression in IECs inhibited glycogen synthase 3β (GSK-3β) function, promoted accumulation of β-catenin protein, and increased expression of Wnt target genes involved in the control of the cell proliferation. VS28 mice showed a hyperplastic intestinal epithelium and, strikingly, developed adenomas and adenocarcinomas by 40 weeks of age. When exposed to an inflammation-driven tumor model (azoxymethane/dextran sodium sulfate), VS28 mice developed a significantly higher tumor burden than control littermates. Transgenic coexpression of the US28 ligand CCL2 (an inflammatory chemokine) increased IEC proliferation as well as tumor burden, suggesting that the oncogenic activity of US28 can be modulated by inflammatory factors. Together, these results indicate that expression of US28 promotes development of intestinal dysplasia and cancer in transgenic mice and suggest that CMV infection may facilitate development of intestinal neoplasia in humans.

Protein Unfolding Is Not a Prerequisite for Endoplasmic Reticulum-to-Cytosol Dislocation

We examined the effects of protein folding on endoplasmic reticulum (ER)-to-cytosol transport (dislocation) by exploiting the well-characterized dihydrofolate reductase (DHFR) domain. DHFR retains the capacity to bind folate analogues in the lumen of microsomes and in the ER of intact cells, upon which it acquires a conformation resistant to proteinase K digestion. Here we show that a Class I major histocompatibility complex heavy chain fused to DHFR is still recognized by the human cytomegalovirus-encoded glycoproteins US2 and US11, resulting in dislocation of the fusion protein from the ERin vitro and in vivo. A folded state of the DHFR domain does not impair dislocation of Class I MHC heavy chainsin vitro or in living cells. In fact, a slight acceleration of the dislocation of DHFR heavy chain fusion was observed in vitro in the presence of a folate analogue. These results suggest that one or more of the channels used for dislocation can accommodate polypeptides that contain a tightly folded domain of considerable size. Our data raise the possibility that the Sec61 channel can be modified to accommodate a folded DHFR domain for dislocation, but not for translocation into the ER, or that a channel altogether distinct from Sec61 is used for dislocation.