Sequence comparison between the extracellular domain of M2 protein human and avian influenza A virus provides new information for bivalent influenza vaccine design

To prevent the human and economic losses caused by human and avian influenza viruses, it is necessary to prepare safe bivalent influenza vaccines. Recent studies found that human influenza vaccines based on the extracellular domain of influenza M2 protein (M2e) induced broad-spectrum protective immunity in various antigen constructs. A prerequisite for using the M2e protein as a bivalent influenza vaccine component was to find out the sequence differences between human and non-human (avian or swine) influenza M2e proteins. Here, we completed such a comparison using 716 influenza M2e sequences available in Genbank. The results found one region on M2e protein consistent with host restriction specificities: PIRNEWGCRCN, PTRNGWECKCS and PIRNGWECRCN (aa10-20; the human, avian and swine specific M2e sequence, respectively). Interestingly, the comparison result was then validated by immunoblotting and enzyme-linked immunosorbent assay. The monoclonal antibody against the EVETPIRN sequence (aa6-13) of human M2e protein could weakly recognize avian M2e proteins bearing the EVETPTRN sequence (aa6-13) but failed to recognize avian M2e proteins bearing the EVETLTRN sequence (aa6-13). The data in this study provided useful information in the race to develop bivalent influenza vaccines against avian and human influenza A virus infection in human beings.     

Localization of the HIV-1 gp120 Conformational Epitope Recognized by Virus-Neutralizing Monoclonal Antibodies 2G12

A phage peptide library was used to select peptides interacting with virus-neutralizing monoclonal antibodies (mAb) 2G12 which recognize a discontinuous surface epitope of HIV-1 gp120. With the published X-ray data, gp120 regions involved in the antigenic determinant were predicted. Binding with mAb 2G12 was ascribed to Thr297, Phe383, Tyr384, Arg419, Ile240, Thr415, Leu416, Pro417, Lys421, and Trp112. Though distant in the gp120 sequence, these residues are close in space and form the 2G12 epitope on the gp120 surface.     

从噬菌体随机肽库中筛选流感病毒神经氨酸酶的抑制多肽

目的:从噬菌体呈现12肽库中筛选与流感病毒神经氨酸酶特异性结合的肽。方法:以甲三型流感病毒裂解疫苗原液为靶分子,经过3轮生物淘选,从噬菌体随机肽库中筛选与之结合的噬菌体。用ELISA方法鉴定噬菌体克隆与靶分子的结合力,用荧光方法测定噬菌体克隆对流感病毒A/Sydney/5/97(H3N2)神经氨酸酶的抑制活性。对筛选到的阳性克隆进行DNA序列测定并推导出相应的氨基酸序列。结果:经过3轮筛选后,42个噬菌体克隆与靶分子有高度亲和力,23个噬菌体克隆对流感病毒A/Sydney/5/97(H3N2)神经氨酸酶有抑制活性。对27个噬菌体克隆的测序结果表明,分别有10个和2个克隆的序列是一致的,其氨基酸序列分别为KSLSRHDHIHHH和WPRHHHSASVQT。结论:通过噬菌体肽库筛选到抑制流感病毒神经氨酸酶的12肽,为进一步研究对流感病毒神经氨酸酶有抑制活性的分子药物奠定了基础。     

Characterization of two distinct major histocompatibility complex class I Kk-restricted T-cell epitopes within the influenza A/PR/8/34 virus hemagglutinin.

Cytotoxic T-lymphocyte (CTL) clones specific for the influenza A/PR/8/34 virus hemagglutinin (HA) were isolated by priming CBA mice with a recombinant vaccinia virus expressing the HA molecule. The epitopes recognized by two of these clones, which were CD8+, Kk restricted, and HA subtype specific, were defined by using a combination of recombinant vaccinia viruses expressing HA fragments and synthetic peptides. One epitope is in the HA1 subunit at residues 259 to 266 (numbering from the initiator methionine), amino acid sequence FEANGNLI, and the other epitope is in the HA2 subunit at residues 10 to 18 (numbering from the amino terminus of the HA2 subunit), sequence IEGGWTGMI. These two peptides are good candidates for naturally processed HA epitopes presented during influenza infection, as they are the same length (eight and nine residues) as other naturally processed viral peptides presented to CTL. A comparison of the sequences of these two new epitopes with those of the three previously published Kk-restricted T-cell epitopes showed some homology among all of the epitopes, suggesting a binding motif. In particular, an isoleucine residue at the carboxy-terminal end is present in all of the epitopes. On the basis of this homology, we predicted that the Kk-restricted epitope in influenza virus nucleoprotein, previously defined as residues 50 to 63, was contained within residues 50 to 57, sequence SDYEGRLI. This shorter peptide was found to sensitize target cells at a 200-fold lower concentration than did nucleoprotein residues 50 to 63 when tested with a CTL clone, confirming the alignment of Kk-restricted epitopes.     

用噬菌体随机肽库筛选SARS冠状病毒S蛋白单克隆抗体2C5的模拟表位

SARS-CoVS蛋白特异的单克隆抗体2C5具有病毒中和作用。以单克隆抗体2C5为筛选靶分子,筛选噬菌体展示随机7肽库。经三轮淘洗后随机挑选20个噬菌体克隆进行ELISA分析和序列测定。在10个ELISAOD值大于0.2的阳性噬菌体克隆中,有8个噬菌体克隆展示有共同的7肽序列TPEQQFT。展示有该序列的噬菌体克隆能竞争抑制SARS-CoVS蛋白抗原与单抗2C5的结合。结果表明TPEQQFT为单克隆抗体2C5的模拟表位。该结果可对进一步研究S蛋白结构与功能和设计SARS疫苗有一定的参考意义。     

The Magnitude and Specificity of Influenza A Virus-Specific Cytotoxic T-Lymphocyte Responses in Humans Is Related to HLA-A and -B Phenotype

The repertoire of human cytotoxic T-lymphocytes (CTL) in response to influenza A viruses has been shown to be directed towards multiple epitopes, with a dominant response to the HLA-A2-restricted M1(58-66) epitope. These studies, however, were performed with peripheral blood mononuclear cells (PBMC) of individuals selected randomly with respect to HLA phenotype or selected for the expression of one HLA allele without considering an influence of other HLA molecules. In addition, little information is available on the influence of HLA makeup on the overall CTL response against influenza viruses. Here, the influenza A virus-specific CTL response was investigated in groups of HLA-A and -B identical individuals. Between groups the individuals shared two or three of the four HLA-A and -B alleles. After in vitro stimulation of PBMC with influenza virus, the highest CTL activity was found in HLA-A2(+) donors. A similar pattern was observed for the precursor frequency of virus-specific CTL (CTLp) ex vivo, with a higher CTLp frequency in HLA-A2-positive donors than in HLA-A2-negative donors, which were unable to recognize the immunodominant M1(58-66) epitope. In addition, CTL activity and frequency of CTLp for the individual influenza virus epitopes were determined. The frequency of CTLp specific for the HLA-B8-restricted epitope NP(380-388) was threefold lower in HLA-B27-positive donors than in HLA-B27-negative donors. In addition, the frequency of CTLp specific for the HLA-A1-restricted epitope NP(44-52) was threefold higher in HLA-A1-, -A2-, -B8-, and -B35-positive donors than in other donors, which was confirmed by measuring the CTL activity in vitro. These findings indicate that the epitope specificity of the CTL response is related to the phenotype of the other HLA molecules. Furthermore, the magnitude of the influenza virus-specific CTL response seems dependent on the HLA-A and -B phenotypes.     

Influenza PR8 HA-specific Fab fragments produced by phage display methods

Anti-influenza hemagglutinin (HA) Fabs were isolated from a phage display library using purified HA of influenza virus A/Puerto Rico/8/34 (PR8; H1N1) as an antigen. Four Fab clones displaying a 25-50-fold higher binding signal to PR8 HA than the control were selected for further analysis and comparison with anti-PR8 monoclonal antibody (mAb). All four Fabs and mAb recognized the PR8 HA under non-reducing conditions but rarely bound to reduced PR8 HA. Inhibition of influenza virus infection on MDCK cells was observed with Fab1 and mAb in a dose-dependent manner while Fab3 and 4 exhibited only a partial inhibitory effect. Moreover, Fab1 clone and mAb exhibited cross-reactivity with the A/Peking/262/95 (A/Peking; H1N1) strain. The inhibitory effects of mAb on both influenza strains were more potent than Fab1, which is likely attributed to its higher affinity for the antigen. SPR analyses, in fact, revealed that Fab1 and mAb have K_D of 1.5 × 10⁻⁸ and 3.2 × 10⁻⁹ M, respectively. These results strongly suggest that phage library-derived Fabs can be readily prepared and that such HA-specific Fabs with inhibitory action on influenza infection may be used to treat influenza patients.     

A survey of furin substrate specificity using substrate phage display.

The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.     

Consequences of suboptimal priming are apparent for low-avidity T-cell responses

The emergence of the novel reassortant A(H1N1)-2009 influenza virus highlighted the threat to the global population posed by an influenza pandemic. Pre-existing CD8(+) T-cell immunity targeting conserved epitopes provides immune protection against newly emerging strains of influenza virus, when minimal antibody immunity exists. However, the occurrence of mutations within T-cell antigenic peptides that enable the virus to evade T-cell recognition constitutes a substantial issue for virus control and vaccine design. Recent evidence suggests that it might be feasible to elicit CD8(+) T-cell memory pools to common virus mutants by pre-emptive vaccination. However, there is a need for a greater understanding of CD8(+) T-cell immunity towards commonly emerging mutants. The present analysis focuses on novel and immunodominant, although of low pMHC-I avidity, CD8(+) T-cell responses directed at the mutant influenza D(b)NP(366) epitope, D(b)NPM6A, following different routes of infection. We used a C57BL/6J model of influenza to dissect the effectiveness of the natural intranasal (i.n.) versus intraperitoneal (i.p.) priming for generating functional CD8(+) T cells towards the D(b)NPM6A epitope. In contrast to comparable CD8(+) T-cell responses directed at the wild-type epitopes, D(b)NP(366) and D(b)PA(224), we found that the priming route greatly affected the numbers, cytokine profiles and TCR repertoire of the responding CD8(+) T cells directed at the D(b)NPM6A viral mutant. As the magnitude, polyfunctionality, and T-cell repertoire diversity are potential determinants of the protective efficacy of CD8(+) T-cell responses, our data have implications for the development of vaccines to combat virus mutants.     

Self-assembly polymerization enhances the immunogenicity of influenza M2e peptide

The extracellular domain of Influenza M2 protein (M2e) was considered as a promising target for universal influenza vaccine development. Several M2e-based influenza vaccines have been developed and many of them used a mutant M2e peptide, in which the two conserved cysteine residues were substituted by serine residues. In this paper, we compared the antigenicity and immunogenicity of wild type and cysteine-mutant M2e peptides. We found that the cysteine substitution slightly affected the antigenicity of M2e epitope, but greatly reduced the immunogenicity of M2e peptide. The cysteine substitution also disabled the M2e peptide from inducing protection against influenza virus challenge in mice. Further analysis revealed that the immunogenicity of M2e peptide was enhanced by the self-assembly of the peptide through inter-peptide disulfide bonds. These results provide new information to improve the design of M2e-based vaccines against potential influenza pandemics.     

Activated protein C-protein C inhibitor complex formation: characterization of a neoepitope provides evidence for extensive insertion of the reactive center loop

Protein C inhibitor, a serine proteinase inhibitor (serpin), is the physiologically most important inhibitor of activated protein C. We have made a monoclonal antibody (M36) that binds with equally high affinity to an epitope present in activated protein C-protein C inhibitor complexes and cleaved loop-inserted protein C inhibitor. Insertion of a synthetic N-acetylated tetradecapeptide (corresponding to residues P1-P14 of the reactive center loop) into beta-sheet A of the uncleaved inhibitor also exposed the epitope. The antibody had no apparent affinity for native uncleaved inhibitor or for the free peptide. Synthetic P1-P14 analogues, with Arg P13 or Ala P9 substituted to the residues found in mouse protein C inhibitor (Thr and Ile, respectively), were also inserted in beta-sheet A. The Arg P13/Thr substitution led to a greatly impaired reactivity with the antibody, whereas the Ala P9/Ile mutation resulted in a modest loss of reactivity with the antibody. These results indicate that complex formation leads to insertion of the reactive center loop in beta-sheet A from Arg P14 and presumably beyond Ala P9. Moreover, to the best of our knowledge, this is the first instance where the neoepitope of a complexation-specific monoclonal antibody has been localized to the loop-inserted part of beta-sheet A, the part of the serpin where the complexation-induced conformational change is most conspicuous.     

Synthetic Long Peptide Influenza Vaccine Containing Conserved T and B Cell Epitopes Reduces Viral Load in Lungs of Mice and Ferrets

Currently licensed influenza vaccines mainly induce antibodies against highly variable epitopes. Due to antigenic drift, protection is subtype or strain-specific and regular vaccine updates are required. In case of antigenic shifts, which have caused several pandemics in the past, completely new vaccines need to be developed. We set out to develop a vaccine that provides protection against a broad range of influenza viruses. Therefore, highly conserved parts of the influenza A virus (IAV) were selected of which we constructed antibody and T cell inducing peptide-based vaccines. The B epitope vaccine consists of the highly conserved HA2 fusion peptide and M2e peptide coupled to a CD4 helper epitope. The T epitope vaccine comprises 25 overlapping synthetic long peptides of 26-34 amino acids, thereby avoiding restriction for a certain MHC haplotype. These peptides are derived from nucleoprotein (NP), polymerase basic protein 1 (PB1) and matrix protein 1 (M1). C57BL/6 mice, BALB/c mice, and ferrets were vaccinated with the B epitopes, 25 SLP or a combination of both. Vaccine-specific antibodies were detected in sera of mice and ferrets and vaccine-specific cellular responses were measured in mice. Following challenge, both mice and ferrets showed a reduction of virus titers in the lungs in response to vaccination. Summarizing, a peptide-based vaccine directed against conserved parts of influenza virus containing B and T cell epitopes shows promising results for further development. Such a vaccine may reduce disease burden and virus transmission during pandemic outbreaks.     

Detailed characterization of an anti-factor IX monoclonal antibody that neutralizes the prolonged ox brain prothrombin time of hemophilia B(M) by synthetic peptides

In a previous study, we prepared a monoclonal antibody (MoAb) to coagulation factor IX (FIX), designated 65-10, which interfered with the activation of FIX by the activated factor XI/Ca(2+) and neutralized the prolonged ox brain prothrombin time of hemophilia B(M) [11,12]. The location of the epitope on the FIX for 65-10 MoAb is (168) Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr-Arg-Val-Val(182) [21]. In this paper, we studied in more detail an epitope on FIX using the systematic substitution of different amino acids at each residue of the epitope peptides and the influence of the epitope peptide on the prolonged ox brain prothrombin time of the hemophilia B(M) plasma of 65-10 MoAb. In the replacement set of amino acids, peptides showing low or no reactivity to 65-10 were (175)Phe --> Asp, Glu, Gly, Lys, Arg, Thr, Val, (176)Asn --> Asp, Glu, Phe, Ile, Lys, Leu, Pro, Val, Tyr, (177)Asp --> Cys, Glu, Phe, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, and (178) Phe --> Pro. These results imply that a hydrophobic molecule of (175) Phe, a hydrophilic molecule of (176)Asn, and a negative charge molecule of (177)Asp were important to the epitope. The 65-10 MoAb antibody neutralized the prolonged ox brain prothrombin time of hemophilia B(M) Nagoya 2 ((180)Arg -->Trp) and Kashihara ((181)Val --> Phe) as well as B(M) Kiryu ((313)Val --> Asp) and Niigata ((390)Ala --> Val). This reaction was inhibited by preincubation with a (168) Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr-Arg-Val-Val(182) peptide conjugated with bovine serum albumin (BSA). 65-10 MoAb that has been useful in detailing epitopes will be useful for qualitative analysis of hemophilia B(M).     

禽流感病毒H5N1全基因组克隆与分析

为明确广东地区分离的一株禽流感病毒H5N1的遗传背景,建立流感病毒反向遗传的平台。对该株禽流感病毒进行了空斑纯化与组织细胞培养,检测其在MDCK细胞中的增殖特性;利用H5N1病毒通用引物,通过RT-PCR对该病毒全基因组的8条片段进行全长克隆及测序分析;将H5N1的8条全长基因组片段分别插入反向遗传通用载体中,构建禽流感病毒H5N1的感染性克隆。结果表明,该H5N1毒株在MDCK细胞中可不依赖胰酶进行有效增殖与复制,可使MDCK细胞出现典型细胞病变,具有高致病性禽流感病毒的细胞增殖特征。RT-PCR克隆得到该H5N1毒株的PB2、PB1、PA、HA、NP、NA、M和NS八条全长片段,经测序分析确认该毒株的基因序列,其内部编码序列出现多处突变,其中HA连接肽为多个连续碱性氨基酸,表明该毒株可不依赖胰酶进行有效复制,与细胞培养结果一致,未出现抗药性的遗传突变。PCR与测序证明,插入H5N1八个全长基因组片段的载体序列完全正确,表明成功构建了该毒株的感染性克隆。为明确病毒遗传信息,建立流感病毒反向遗传的平台,为进一步研究禽流感病毒相关疫苗提供了研究基础。     

A mutation on influenza C virus M1 protein affects virion morphology by altering the membrane affinity of the protein

Reverse genetics has been documented for influenza A, B, and Thogoto viruses belonging to the family Orthomyxoviridae. We report here the reverse genetics of influenza C virus, another member of this family. The seven viral RNA (vRNA) segments of C/Ann Arbor/1/50 were expressed in 293T cells from cloned cDNAs, together with nine influenza C virus proteins. At 48 h posttransfection, the infectious titer of the culture supernatant was determined to be 2.51 x 10(3) 50% egg infectious doses/ml, which is lower than the number of influenza C virus-like particles (VLPs) (10(6)/ml) generated using the same system. By generating influenza C VLPs containing a given vRNA segment, we showed that each of the vRNA segments was similarly synthesized in the plasmid-transfected cells but that some segments were less efficiently incorporated into the VLPs. This finding leads us to speculate that the differences in incorporation efficiency into VLPs between segments might be a reason for the inefficient production of infectious viruses. Second, we generated a mutant recombinant virus, rMG96A, which possesses an Ala-->Thr mutation at residue 24 of the M1 protein, a substitution demonstrated to be involved in the morphology (filamentous or spherical) of the influenza C VLPs. As expected, rMG96A exhibited a spherical morphology, whereas recombinant wild-type of C/Ann Arbor/1/50, rWT, exhibited a mainly filamentous morphology. Membrane flotation analysis of the cells infected with rWT or rMG96A revealed a difference in the ratio of membrane-associated M1 proteins, suggesting that the affinity of M1 protein to the cell membrane is a determinant for virion morphology.     

N-terminus of M2 protein could induce antibodies with inhibitory activity against influenza virus replication

New influenza vaccines have been designed based on the fact that the extracellular domain of M2 protein (M2e) is nearly invariant in all influenza A strains. To clarify which exact region of M2e could induce antibodies with inhibitory activities against influenza virus replication, four overlapping peptides covering M2e were synthesized and then coupled to the carrier protein bovine serum albumin through the cysteine of the peptides. After a vaccination course, all these four peptide vaccines could induce high levels of rabbit antibodies with predefined peptide specificity (antibody dilution: 1:6400-1:25600). Besides, the anti-N-terminal antibodies (AS2) reacted strongly with M2e, and reacted weakly with the middle part and C-terminus of M2e. The MDCK assay for cytopathic effect proved that antibodies recognizing the N-terminus of M2e could obviously inhibit replication of influenza A virus (A/wuhan/359/95) and influenza B virus (B/wuhan/321/99) in vitro in a dose-dependent manner, while antibodies recognizing the middle part and the C-terminus of M2e did not show such significant inhibitory activities. Sequence analysis indicates that the first nine N-terminal amino acid residues of M2e are extremely conservative. Just this region containing the first nine amino acid residues could induce antibodies with inhibitory activity against influenza A and influenza B virus replication, suggesting that the N-terminus of M2e may contain an epitope that could induce inhibitory antibodies against influenza virus replication in vitro.     

Isolation and comparative characterization of Ki-67 equivalent antibodies from the HuCAL phage display library

It has been shown that a repetitive motif with the sequence FKEL(F) within the Ki-67 antigen (pKi-67) serves as an epitope for the Ki-67 antibody and equivalent clones. However, no direct correlation between reactivity towards Ki-67 epitopes and reactivity in formalin-fixed paraffin-embedded (FFPE) tissue could be found. In this study our aim was the isolation and characterization of new monoclonal Ki-67 equivalent antibodies in an in vitro approach. To select pKi-67 reactive phage antibodies, we used a large naive Fab-phage library (Human Combinatorial Antibody Library; HuCAL). We implemented a panning strategy against two different overlapping peptides, both containing the 'FKELF' epitope. ELISA screening of randomly picked phage antibody clones after the third selection round yielded six highly reactive clones against the 'FKELF' epitope, of which five were found to be reactive in FFPE tissue, showing a Ki-67 equivalent staining pattern. Substitutional epitope analysis on peptide arrays of the new recombinant pKi-67 binders and of the established murine clones Ki-67, Mib-1 and Mib-5 were carried out to compare their fine specificities. The results suggest that the lysine residue in the epitope is critical for recognition of Ki-67 antigen in FFPE tissue.     

Fine specificity analysis of human influenza-specific cloned cell lines

Influenza-specific human-T-cell clones, proliferating in the presence of virus-infected cells with restriction by class II molecules and displaying class II-restricted CTL activity or specific helper activity in antibody synthesis, have been analyzed for antigenic specificities. All of them were obtained by in vitro stimulation against influenza A/Texas virus. In all cases the virus specificity appeared identical in cytolytic and proliferative responses. Three of the clones were broadly cross-reactive, recognizing all or almost all type A influenza strains. The three remaining clones were subtype specific when tested with human strains and recognized the surface glycoproteins of influenza virus. One of these lines reacted with an epitope of the neuraminidase N2 while the other two recognized the hemagglutinin H3. By using a large panel of mammalian and avian influenza strains, it can be demonstrated that hemagglutinin-specific human T cells can recognize a cross-reacting determinant shared by H3 and H4 subtypes of hemagglutinin which has never been detected with antibodies.     

The role of the endoplasmic reticulum in antigen processing. N-glycosylation of influenza hemagglutinin abrogates CD4+ cytotoxic T cell recognition of endogenously processed antigen.

Evidence is presented for an endogenous route of Ag processing for CD4+ T cell recognition of influenza hemagglutinin that requires obligatory traffic of de novo synthesized hemagglutinin across the lumen of the endoplasmic reticulum for processing in a cytosolic compartment. I-Ad-restricted T cell clones that recognize synthetic peptides corresponding to two distinct antigenic regions of the HA1 subunit, HA1 56-76 and HA1 177-199, are cytotoxic and, dependent on epitope specificity can recognize endogenously processed Ag and lyse class II+ target cells infected with a recombinant vaccinia-X31 HA virus. HA1 56-76 specific T cell clones fail to recognize (target cells infected with) influenza X31 viruses, containing a single residue change, HA1 63 Asp----Asn that introduces an oligosaccharide attachment site: Asp63Cys64Thr65. Recognition is restored, however, by tunicamycin treatment of mutant virus infected target cells. Inasmuch as N-glycosylation of nascent hemagglutinin polypeptides occurs in the lumen of the endoplasmic reticulum, this indicates a route of endogenous processing for hemagglutinin, requiring transport across the endoplasmic reticulum, which has been confirmed by the failure of CD4+ T cells to recognize a recombinant VACC-hemagglutinin virus in which the same single residue change, HA1 63 Asp----Asn has been introduced by site directed mutagenesis.