TAP-translocated peptides specifically bind proteins in the endoplasmic reticulum,including gp96, protein disulfide isomerase and calreticulin

The endoplasmic reticulum (ER) membrane-embedded transporter associated with antigen processing (TAP) associates with peptides in the cytosol and translocates these into the ER lumen. Here, MHC class I molecules bind a subset of these peptides and the remainder is either removed or degraded, or may be retained in the ER in association with other proteins. We have visualized peptide-binding proteins in the ER using radioactive peptides with a photoreactive group. Besides TAP, two proteins were identified as gp96 and protein disulfide isomerase (PDI). Calreticulin, previously found in complex with TAP, only binds glycosylated peptides. In addition, two as yet unidentified, ER luminal glycoproteins (gp120 and gp170) were visualized. The effects of peptide size and sequence on binding to the ER-resident proteins were studied by using partially degenerated peptides with photoreactive side chains. All identified proteins were able to bind peptides within the size range of peptides translocated by TAP, from 8 to more than 20 amino acids. Whereas PDI associated with all peptides tested, gp96 and gp120 showed a clear sequence preference for non-charged amino acids at positions 2 and 9 in 9mer peptides. Thus various ER proteins, other than the MHC class I heterodimer and TAP, are able to interact with peptides albeit with a different substrate selectivity.     

Translocation of long peptides by transporters associated with antigen processing (TAP)

The major histocompatibility complex (MHC)-encoded transporters associated with antigen processing (TAP) translocate peptides from the cytosol into the lumen of the endoplasmic reticulum (ER) where they associate with MHC class I molecules. The length of class I-binding peptides is usually 8–11 amino acids, but examples of significantly longer peptides have been described. The preferred lengths and upper and lower size limits for peptides translocated by TAP have not been determined in detail because in the currently used test systems, peptides are subject to proteolytic degradation. In the present study, three sets of individual peptides or partially randomized peptide libraries ranging between 6 and 40 residues were used that contained a radiolabeled tyrosine and a consensus sequence for ER-specific N-glycosylation at opposite ends, thus ensuring that only nondegraded peptides were monitored in the transport/glycosylation assay. For three different transporters, rat TAP1/2^a, rat TAP1/2^u and hTAP, the most efficient ATP-dependent transport was observed for peptides with 8–12 amino acids. Hexamers and longer peptides of up to 40 amino acids were also translocated, albeit less efficiently. For two of the three sets of peptides analyzed, rat TAP1/2^a showed a less stringent length selection than rat TAP1/2^u and human TAP. The superior transport of the decamer of the TNKT . Y series was not due to faster degradation or less efficient glycosylation of shorter or longer length variants. A binding assay with TAP-containing microsomes revealed a high affinity for the radiolabeled decamer (K_D = 580 nM), while other length variants were clearly inferior in their binding affinities. Thus, TAP binds and preferentially translocates peptides with a length suitable for binding to MHC class I molecules, but peptides that are considerably longer may also be substrates. About 10⁵ peptide binding sites per cell equivalent of microsomes were determined, providing an estimate for the number of TAP complexes in the ER membrane.     

The interface between tapasin and MHC class I: identification of amino acid residues in both proteins that influence their interaction

Prior to the binding of antigenic peptide, a complex of chaperone proteins associates with the Major Histocompatibility Complex (MHC) class I heavy chain/β₂m heterodimer. Although each dornain of the MHC class I heavy chain contains amino acid resid uses that influence chaperone binding, there are several pieces of evidence that point to an interaction between the MHC clas 1α2/α3 domains and tapasin. In egard to the site on tapasin involved in the tapasin/MHC interface, we have found that a particular region of tapasin (containing amino acid residues 334–342) is necessary for the binding of tapasin to the MHC class I heavy chain. Our results also indicate that amino acids in this region of tapasin also affect the proportion of MHC class I open forms expressed at the cell surface and MHC class I egress from the endoplasmic reticulurn. Based on these results and those obtained by other laboratories, a model for MHC class I/tapasin interaction is proposed.     

Major histocompatibility complex class I presentation of ovalbumin peptide 257–264 from exogenous sources: protein context influences the degree of TAP-independent presentation

Peritoneal macrophages from C57BL/6 mice process antigens from bacteria or coated on polystyrene beads for presentation by major histocompatibility complex (MHC) class I molecules. To investigate this antigen processing pathway, peritoneal macrophages from homozygous TAP1^−/− mice, which lack the transporter associated with antigen processing (TAP) and are defective in presenting endogenous antigens on MHC class I, were used. TAP1^−/− or C57BL/6 macrophages were co-incubated with either bacteria or polystyrene beads containing the 257–264 epitope from ovalbumin [OVA(257–264)], which binds the mouse class I molecule K^b. The source of the OVA(257–264) epitope was either the Crl-OVA(257–264) (Crl-OVA) fusion protein, the maltose binding protein (MBP)-Crl-OVA fusion protein, native OVA or bacterial recombinant OVA (rOVA); Crl-OVA, MBP-Crl-OVA and rOVA were each expressed in bacteria, and Crl-OVA and MBP-Crl-OVA purified from bacterial lysates and native egg OVA were coated onto polystyrene beads. The data reveal that peritoneal macrophages from C57BL/6 and TAP1^−/− mice can process bacteria expressing Crl-OVA, MBP-Crl-OVA and rOVA as well as beads coated with native OVA, purified Crl-OVA, and purified MBP-Crl-OVA and present OVA(257–264) for recognition by OVA(257–264)/K^b-specific T hybridoma cells, albeit with different relative processing efficiencies. The processing efficiency of TAP1^−/− macrophages co-incubated with bacteria or beads containing Crl-OVA or MBP-Crl-OVA was reduced approximately three to five times compared to C57BL/6 macrophages, but OVA(257–264) was presented 100 times less efficiently when the source of OVA(257–264) was full-length OVA. Chloroquine inhibition studies showed a differential requirement for acidic compartments in C57BL/6 versus TAP1^−/− macrophages, which also depended upon the source of the OVA (257–264) epitope (Crl-OVA versus full-length OVA). These data suggest that TAP1^−/− and C57BL/6 macrophages may process Crl-OVA and full-length OVA in different cellular compartments and that the protein context of the OVA(257–264) epitope influences the extent of TAP-independent processing for MHC class I presentation.     

Down-regulation of the antigen processing machinery is linked to a loss of inflammatory response in colorectal cancer

Antitumor inflammatory response is known to inhibit tumor growth in colorectal carcinoma. The density and functionality of tumor-infiltrating lymphocytes (TIL) is regulated by the antigen processing machinery through regulator proteins such as transporters associated with antigen processing (TAP) and major histocompatibility complex (MHC) class I antigen. We aimed to investigate the in vivo association of those factors and their impact on prognosis in colorectal cancer. TAP1, TAP2 and MHC class I antigen expression, inflammatory infiltrate and TIL (CD4⁺, CD8⁺, and CD20⁺) were assessed by immunohistochemistry in 336 sporadic colorectal carcinomas. The factors were correlated with each other and with clinic-pathological parameters and patient outcome. We found TAP1 and TAP2 expression to be significantly associated with MHC class I antigen expression (TAP1: r = 0.363, P < .001; TAP2: r = 0.393, P < .001). Increased density of CD8⁺ TIL was predominantly found in TAP1, TAP2 and MHC class I antigen–positive cases. Increased density of CD4⁺ TIL was linked with TAP1 and TAP2, but not with MHC class I antigen. High CD4⁺ and CD8⁺ cell count but not TAP1, TAP2 and MHC class I antigen expression had favorable prognostic impact in colorectal cancer (P = .003 and P = .003, respectively). In conclusion, our data show that the expression of key components of the antigen processing machinery is tightly linked to the density of TIL, which are positive prognostic factors in colorectal cancer in vivo. This implies that modulation of these factors may help to enhance antitumor inflammatory response which in turn may improve patient prognosis.     

Characterization of the binding profile of peptide to transporter associated with antigen processing (TAP) using Gaussian process regression

Although MHC–peptide binding is the most selective event in epitope presentation process, the protein fragments generated by proteasomal cleavage require to be recognized by transporter associated with antigen processing (TAP) and translocated from cytosol to endoplasmic reticulum before they can be loaded into the ligand-binding groove of MHC. In this article, we report the use of a new and powerful machine learning tool called Gaussian process (GP) to model the linear and nonlinear relationships between the sequence pattern and binding affinity of peptide to TAP, and to explain the physicochemical properties and structural implications underlying the specific recognition and association of peptide with TAP. The resulting statistics are compared systematically with those obtained by sophisticated PLS, ANN and SVM. Results show that: (i) Nonlinear methods such as the ANN and GP perform much better than the linear PLS. (ii) GP is capable of handling both linearity- and nonlinearity-hybrid relationship and thus exhibits a good performance relative to other two nonlinear methods. (iii) Investigation of the GP model shows that the P1, P2, P3 and P9 of peptide are the most important positions that dominate TAP–peptide recognition, P5 contributes slightly to the peptide binding, whereas P4, P6, P7 and P8 can only exert very limited potency on the binding. (iv) Diverse properties cast remarkable effects on the interaction between TAP and peptide. In particular, hydrophobility, electronic property and hydrogen bond contribute most significantly to the binding affinity of TAP–peptide association.     

Transporter associated with antigen processing (TAP) 1 gene polymorphisms in patients with hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is a lung inflammatory disease caused by the inhalation of a variety of antigens. Previous studies support the role of the major histocompatibility complex (MHC) class II genes in the susceptibility to develop HP. However, the putative role of other MHC loci has not been elucidated. Transporters associated with antigen processing (TAP) genes are located within the MHC class II region and play an important role transporting peptides across the endoplasmic reticulum membrane for MHC class I molecules assembly. The distribution of single nucleotide polymorphisms (SNPs) in TAP1 genes was analyzed in 73 hypersensitivity pneumonitis (HP) patients and 58 normal subjects. We found a significant association of the allele Gly-637 (GGC) (p=0.00004, OR=27.30, CI=3.87-548.04) and the genotypes Asp-637/Gly-637 (p=0.01, OR=16.0, CI=2.19-631.21), Pro-661/Pro-661 (p=0.006, OR=11.30, CI=2.28-75.77) with HP. A significant decrease in the frequency of the allele Pro-661 (CCA) (p=0.008, OR=0.06, CI=0-0.45), the genotype Asp-637/Asp-637 (p=0.01, OR=0.17, 95% CI=0.05-0.58) and the haplotype [Val-333 (GTC), Val-458 (GTG), Gly-637 (GGC), Pro-661 (CCA)] was detected in HP patients compared with controls (p=0.002, OR=0.07, CI=0.0-0.57). These findings suggest that TAP1 gene polymorphisms are related to HP risk, and highlight the importance of the MHC in the development of this disease.     

Major histocompatibility complex class II-restricted presentation of secreted and endoplasmic reticulum resident antigens requires the invariant chains and is sensitive to lysosomotropic agents

We have tested the involvement of the invariant chains (Ii) p31 and p41 in the presentation of peptides derived from hen egg lysozyme (HEL) constructs targeted to different intracellular compartments within transfected fibroblasts. The endogenous HEL constructs were either present in the cytosol (HELc), secreted (HELs), or linked to the mammalian (KDEL C-terminal sequence that causes retention of HEL in the endoplasmic reticulum (ER)/pre-Golgi recycling compartment (HELr). Using Ii-negative antigen-presenting cells, the presentation of HELr to a HEL 46-61 specific T cell hybridoma was far less efficient than the presentation of the HELs. High levels of Ii expression enhanced drastically the presentation of the HEL 46-61 determinant derived from both HELr and HELs. HELr and HELs presentation was fully sensitive to lysosomotropic agents such as chloroquine, indicating that the formation of complexes between major histocompatibility complex (MHC) class II molecules and determinants derived from endogenous antigens entering the secretory pathway is taking place in an acidic compartment. The degradation and dissociation of Ii might be a prerequisite for the efficient presentation of endogenously derived determinants by MHC class II molecules, as for the presentation of most exogenous antigens. All our results are compatible with the notion that endogenous molecules being translocated into the lumen of the ER could be presented by class II molecules through a processing pathway involving an acidic compartment in which Ii chains dissociate from class II molecules.     

Potential limitations in using minor histocompatibility antigen-specific cytotoxic T cells for targeting solid tumor cells

We have shown previously that KIAA0223, a gene encoding a minor histocompatibility antigen, HA-1, whose expression was believed to be restricted to the hematopoietic cells, is aberrantly expressed in some solid tumor cell lines. However, its significance in tumor immunity needs to be determined. Cytotoxic activity of HA-1(H)-specific cytotoxic T lymphocytes (CTLs) was assessed against solid tumor cell lines expressing KIAA0223 using (51)Cr release assays. Five of seven cell lines were lysed when HLA-A*0201 was adequately expressed. One of the two CTL-resistant cell lines became susceptible after treatment with IFN-gamma and TNF-alpha, while the other was lysed only after pulsing with HA-1(H) peptide. In most cell lines tested, HA-1(H) peptide was properly generated and presented for recognition by the CTL. However, impaired antigen processing and presentation observed in this study may result in escape from CTL recognition in vivo, as well as in vitro, as observed in this study.     

Stress protein (hsp73)-mediated,TAP-independent processing of endogenous,truncated SV 40 large T antigen for Db-restricted peptide presentation

Transporter associated with antigen processing (TAP)-competent and TAP-deficient cell lines were transfected with expression plasmids encoding either the wild-type (wt) large tumor antigen (T-Ag) of SV40, or a truncated cytoplasmic variant (cT-Ag) of this viral protein. Stable expression of comparable levels of both forms of the viral protein was observed in different transfectants. The truncated cT-Ag variant, but not the wtT-Ag was stably associated with the con-stitutively expressed, cytosolic heat shock protein (hsp)73 chaperone. Two D^b-binding peptides and one K^b-binding peptide of T-Ag were presented to cytotoxic T lymphocyte lines (CTLL) by TAP-competent transfectants expressing either wtT-Ag or cT-Ag. TAP-deficient transfectants expressing the wtT-Ag did not present any of these epitopes to CTLL. In contrast, TAP-deficient transfectants expressing the truncated hsp73-associated cT-Ag, presented the two D^b-binding epitopes, but not the K^b-binding T-Ag epitope to CTLL. Regurgitation of peptides by transfectants was not detectable. The described data indicate that a pool of post-Golgi D^b molecules is available for 2–3 h in TAP-deficient transfectants for loading with peptides released during endolysosomal processing of hsp73-associated, endogenous antigen.     

In the absence of major histocompatibility complex class II molecules,invariant chain is translocated to late endocytic compartments by autophagy

It has been suggested that the cytoplasmic amino-terminal tail of invariant chain (Ii) contains a sorting signal that directs trafficking of the major histocompatibility complex (MHC) class II: Ii oligomeric complex to endocytic compartments. This model is based, in part, on the observation that in the absence of MHC class II molecules, Ii is detectable in lysosomal structures, a phenotype that is dependent on an intact NH₂ terminus. However, the route by which Ii gains access to endosomal compartments in the absence of class II molecules remains uncertain. Here we report a mechanism that localizes Ii in lysosomal compartments independently of class II. We show that murine Ii can be detected by immunofluorescence within late endocytic compartments of stably transfected Ltk^? mouse fibroblasts. Immunochemical studies indicate that degradation of Ii in these cells is sensitive to the lysosomotropic agent ammonium chloride, yet the majority of Ii that undergoes this apparent lysosomal degradation is sensitive to the enzyme endoglycosidase H. This finding suggests that Ii may reach the lysosomal compartment by a route that bypasses the Golgi complex. Consistent with this possibility, we found that in contrast to Ii which is complexed to class II molecules, transport of free Ii to lysosomes is prevented by 3-methyladenine, an inhibitor of the autophagic pathway of protein degradation, a process which involves direct transport from the endoplasmic reticulum to lysosomes. These data suggest the route of transport that leads to endosomal localization of Ii in the absence of class II is distinct from that taken when expressed with class II. This forces a re-evaluation of the concept that the cytosolic tail of Ii contains a dominant Golgi-to-endosomal sorting signal.     

Genes encoded in the major histocompatibility complex affecting the generation of peptides for TAP transport

The B cell line 721.174 has lost the ability to present intracellular antigens to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL). This phenotype results from a homozygous deletion in the MHC that includes the peptide transporter genes TAP1 and TAP2, and the proteasome subunits LMP2 and LMP7. Recent work has shown that such cells transfected with TAP genes load their class I molecules with endogenous peptides, and present several viral epitopes to class I-restricted CTL. These data implied that the LMP2 and LMP7 genes were not required for the presentation of most epitopes through class I molecules. By contrast, while confirming the previous reports, we have identified several epitopes that appear to require genes in the MHC in addition to the TAP for their presentation. Further analysis localizes the defect to proteolysis in the cytosol. In one case, presentation could be partially restored by re-expression of full-length LMP7. Control experiments with LMP7, from which the putative pro-region had been removed, failed to restore presentation, and this lack of effect correlated with failure of the shortened LMP7 to incorporate into the proteasome. These results suggest a role for LMP7 in the generation of a viral epitope, but leave open the possibility that additional genes within the .174 deletion are required for full restoration of antigen presentation.     

Calreticulin maintains the low threshold of peptide required for efficient antigen presentation

Calreticulin (CRT) plays a critical role in MHC class I antigen processing and elicits peptide-specific CD8(+) T cell responses against tumours when administered with peptides. However, how CRT contributes to class I antigen processing and the mechanism of its adjuvant effect in anti-tumour responses, remain to be elucidated. Here we show that reduced class I expression in CRT deficient cells can be restored by the direct delivery of peptides into the ER or by incubation at low temperature. CRT deficient cells exhibited a TAP-deficient phenotype in terms of class I assembly, without loss of TAP expression or functionality. Furthermore, a higher concentration of antigen in the cytosol is required for specific T cell stimulation, suggesting that CRT has a functional role in the maintenance of the low peptide concentration threshold required in the ER for efficient antigen presentation. In the absence of CRT, ERp57 is up-regulated, which indicates that they collaborate with each other in class I antigen processing.     

Three hundred twenty-six genetic variations in genes encoding nine members of ATP-binding cassette, subfamily B (ABCB/MDR/TAP), in the Japanese population

We screened DNAs from 48 Japanese individuals for single-nucleotide polymorphisms (SNPs) in nine genes encoding components of ATP-binding cassette subfamily B (ABCB/MDR/TAP) by directly sequencing the entire applicable genomic regions except for repetitive elements. This approach identified 297 SNPs and 29 insertion/deletion polymorphisms among the nine genes. Of the 297 SNPs, 50 were identified in the ABCB1 gene, 14 in TAP1, 35 in TAP2, 48 in ABCB4, 13 in ABCB7, 21 in ABCB8, 21 in ABCB9, 13 in ABCB10, and 82 in ABCB11. Thirteen were located in 5′ flanking regions, 237 in introns, 37 in exons, and 10 in 3′ flanking regions. These variants may contribute to investigations of possible correlations between genotypes and disease-susceptibility phenotypes or responsiveness to drug therapy. Received: September 18, 2001 / Accepted: November 2, 2001     

DM influences the abundance of major histocompatibility complex class II alleles with low affinity for class II-associated invariant chain peptides via multiple mechanisms

DM catalyses class II-associated invariant chain peptide (CLIP) release, edits the repertoire of peptides bound to major histocompatibility complex (MHC) class II molecules, affects class II structure, and thereby modulates binding of conformation-sensitive anti-class II antibodies. Here, we investigate the ability of DM to enhance the cell surface binding of monomorphic antibodies. We show that this enhancement reflects increases in cell surface class II expression and total cellular abundance, but notably these effects are selective for particular alleles. Evidence from analysis of cellular class II levels after cycloheximide treatment and from pulse-chase experiments indicates that DM increases the half-life of affected alleles. Unexpectedly, the pulse-chase experiments also revealed an early effect of DM on assembly of these alleles. The allelically variant feature that correlates with susceptibility to these DM effects is low affinity for CLIP; DM-dependent changes in abundance are reduced by invariant chain (CLIP) mutants that enhance CLIP binding to class II. We found evidence that DM mediates rescue of peptide-receptive DR0404 molecules from inactive forms in vitro and evidence suggesting that a similar process occurs in cells. Thus, multiple mechanisms, operating along the biosynthetic pathway of class II molecules, contribute to DM-mediated increases in the abundance of low-CLIP-affinity alleles.     

TAP‐mediated processing of exoerythrocytic antigens is essential for protection induced with radiation‐attenuated Plasmodium sporozoites

MHC class I dependent CD8⁺ T cells are essential for protection induced by radiation‐attenuated Plasmodium sporozoites (RAS) in murine malaria models. Apart from the mechanism of activation of CD8⁺ T cells specific for the circumsporozoite protein, the major sporozoite antigen (Ag), CD8⁺ T cells specific for other exoerythrocytic Ags that have been shown to mediate protection have not been thoroughly investigated. Specifically, mechanisms of processing and presentation of exoerythrocytic Ags, which includes liver stage (LS) Ags, remain poorly understood. We hypothesize that as exogenous proteins, LS Ags are processed by mechanisms involving either the TAP‐dependent phagosomal‐to‐cytosol or TAP‐independent vacuolar pathway of cross‐presentation. We used TAP‐deficient mice to investigate whether LS Ag mediated induction of naïve CD8⁺ T cells and their recall during sporozoite challenge occur by the TAP‐dependent or TAP‐independent pathways. On the basis of functional attributes, CD8⁺ T cells were activated via the TAP‐independent pathway during immunizations with Plasmodium berghei RAS; however, IFN‐γ⁺CD8⁺ T cells previously induced by P. berghei RAS in TAP‐deficient mice failed to be recalled against sporozoite challenge and the mice became parasitemic. On the basis of these observations, we propose that TAP‐associated Ag processing is indispensable for sterile protection induced with P. berghei RAS.     

Peptide trimming by endoplasmic reticulum aminopeptidases: Role of MHC class I binding and ERAP dimerization

Presentation of short peptides, produced through intracellular proteolysis, by MHC class I molecules (MHC-I) is the basis of adaptive immune surveillance and responses by cytolytic CD8⁺ T lymphocytes. In the principal pathway of peptide processing for MHC-I that operates in all nucleated cells, MHC-I-binding peptides are produced through stepwise proteolysis starting with source protein degradation by cytosolic proteasome complexes. Among the fraction of proteasome products reaching the lumen of the endoplasmic reticulum, a significant proportion is thought to have a length exceeding that adapted to MHC class I binding and requires N-terminal trimming. This is carried out by one murine and two human endoplasmic reticulum aminopeptidases, the ERAP enzymes. While the critical role of ERAP for producing a ligandome optimized for MHC-I is well documented, it remains unclear how this is mechanistically achieved. In this review, we will discuss the evidence supporting the alternative “MHC template” and “molecular ruler” models that have been proposed to explain how ERAP activity adapts to the ligand requirements of MHC-I. We will also review evidence for dimerization of the two human ERAP enzymes and its potential functional relevance.     

Further characterization of hla homozygous typing cell lines at the LMP2 polymorphic codon 60 by an ARMS typing method

LMP2 is a subunit of the 20S proteasome within the cellular cytosolic compartment that is thought to cleave proteins into approximately 9 amino acid long oligopeptides. It is hypothesized that changes in the low molecular mass protease (LMP) gene sequence may alter the activity or specificity in which the LMP genes cleave peptides. Currently, the typing method for LMP2 involves polymerase chain reaction (PCR), restriction enzyme digestion, and gel electrophoresis. To help reduce the cost and cumbersomeness of this method, a new typing method was adapted for the LMP2 gene. To establish this new amplification refractory mutation system (ARMS) typing method, primers have been defined, amplification conditions optimized, and control cell lines sequenced to validate testing parameters. Results are listed for selected 10th and 11th International Histocompatibility Workshop homozygous cell lines.     

Antigen presentation in SARS-CoV-2 infection: the role of class I HLA and ERAP polymorphisms

Given the highly polymorphic nature of Human Leukocyte Antigen (HLA) molecules, it is not surprising that they function as key regulators of the host immune response to almost all invading pathogens, including SARS-CoV-2, the etiological agent responsible for the recent COVID-19 pandemic. Several correlations have already been established between the expression of a specific HLA allele/haplotype and susceptibility/progression of SARS-CoV-2 infection and new ones are continuously emerging. Protective and harmful HLA variants have been described in both mild and severe forms of the disease, but considering the huge amount of existing variants, the data gathered in such a brief span of time are to some extent confusing and contradictory. The aim of this mini-review is to provide a snap-shot of the main findings so far collected on the HLA-SARS-CoV-2 interaction, so as to partially untangle this intricate yarn. As key factors in the generation of antigenic peptides to be presented by HLA molecules, ERAP1 and ERAP2 role in SARS-CoV-2 infection will be revised as well.