The conserved carboxyl terminus of human parainfluenza virus type 2 V protein plays an important role in virus growth

Our previous results have shown that some residues of V protein-specific domain in human parainfluenza virus type 2 (hPIV2) are essential not only for STAT protein degradation but also for promoting virus growth. Here, we demonstrated that the virus growth of these recombinant hPIV2s (rPIV2) expressing mutated V proteins were improved in HeLa cell transiently expressing the wild-type V protein, but not in the cells constitutively expressing it. Consequently, we identified the region of the V protein that is essential for its oligomerization and for complex formation with NP protein. We also identified a host protein, AlP1/Alix, involved in apoptosis and efficient budding of several enveloped viruses as an interacting partner of the V and NP proteins. Depletion of AIP1/Alix by small interfering RNA suppressed virus growth. These data suggest that the conserved carboxyl terminus of the V protein plays an important role in virus growth.     

Incomplete replication of human parainfluenza virus type 4 in LLC-MK2 cells and in L929 cells

Human parainfluenza virus type 4A (hPIV-4A) and type 4B (hPIV-4B) were tested for their ability to replicate in the monkey kidney LLC-MK2 cell line (MK2 cells) and the murine L929 cell line (L929 cells). These cells are normally non-permissive for replication of hPIV-4; however, treatment with acetylated trypsin led to virus replication in MK2 cells, but was less effective for L929 cells. Endogenously produced interferon (IFN) played no role in virus replication in L929 cells. Synthesis of virus-specific polypeptides was suppressed in L929 cells. WhereasNP-mRNA and HN-mRNA were detected in MK2 cells, no HN-mRNA was detected in L929 cells. These results indicate that hPIV-4 can infect both MK2 cells and L929 cells. In MK2 cells, when protease exists in the extracellular medium, hPIV-4 exhibits multistep growth. In L929 cells, however, the cause of incomplete replication might be lack of other unknown factors. Received: 10 January 2000     

Identification of RNA-binding regions on the P and V proteins of human parainfluenza virus type 2

We have shown that the P and V proteins of human parainfluenza virus type 2 (hPIV-2) bind to genomic RNA by using Northwestern blot analysis. To identify the RNA-binding regions on the P and V proteins, we used a set of deletion mutants produced in Escherichia coli. One region required for the RNA-binding was found in the P–V common domain (aa 1–82). Others were found in the P protein-specific region (aa 249–354) and the V protein-specific region (aa 176–225). In addition, we have shown that substitutions of some basic residues with alanines in these regions abrogate RNA-binding by the P or V proteins. Intriguingly, the P and V proteins of hPIV2 can selectively bind to the viral RNA under our experimental conditions.     

Binding of the V proteins to the nucleocapsid proteins of human parainfluenza type 2 virus

Interaction of the nucleocapsid (NP) and V proteins of human parainfluenza type 2 virus (HPIV-2) was investigated using a transient expression system. When the NP proteins were co-expressed with the V proteins, some of the NP proteins were translocated into the nuclei. These findings suggest that the NP protein interact with the V proteins. We examined the interaction of the NP proteins and the P, V proteins or deletion mutants of V protein using immunofluorescence and co-immunoprecipitation plus Western blotting analyses, and showed that the V proteins of HPIV-2 bind to the NP proteins and that the N-terminal domain of V protein interacts directly with the NP proteins. When the NP proteins were co-expressed with the V proteins or the N-terminal fragments (aa 1–46), the NP proteins were detected diffusely in the nuclei of the transfected cells, and were also detected in cytoplasmic inclusions. The NP and V proteins were co-localized in the nuclei or cytoplasm. Futhermore, the NP proteins were co-precipitated with the P, V, and V(1–164) proteins by a specific antibody. The P proteins interact more closely with the NP proteins than do the V proteins. These findings indicate that the V proteins have the ability to bind the NP proteins. Received: 4 January 1996     

Development of a real-time RT-PCR assay for detection and quantitation of parainfluenza virus 3

A TaqMan-based real-time RT-PCR assay was developed to detect and quantify human parainfluenza virus 3 (PIV3). Two sets of primer–probe pairs were designed based on the nucleotide (nt) sequence of the nucleocapsid (N) gene. The primer–probe pairs were derived from the 3′ end of the N gene (set 1) and the 5′ region of the gene (set 2), respectively. Using real-time RT-PCR, the sensitivity of set 1 was determined to be about 9 copies of PIV3 genome, while the sensitivity of set 2 was about 93 copies of PIV3 genome. Set 1 was chosen for subsequent experiments. This primer–probe pair detected PIV3, but not any of several other respiratory viruses, indicating that the assay is PIV3 specific. For clinical evaluation, the assay was employed to test 80 nasopharyngeal aspirates from children with respiratory symptoms. The results confirmed the presence of PIV3 in 12 specimens previously identified as positive by culture confirmation, and showed all of which contained more than 100 copies of PIV3 genome. In addition, the method also detected PIV3 genomes in specimens found negative by culture confirmation, indicating the value of this RT-PCR assay. These data thus demonstrate the application of the real-time RT-PCR assay for the detection and quantification of PIV3 in clinical specimens.     

Characterization of naturally occurring parainfluenza virus type 2 (hPIV-2) variants

BACKGROUND: Human parainfluenza viruses (hPIV) are respiratory pathogens responsible for upper and lower respiratory tract infections. In most labs, the clinical diagnosis of hPIV is routinely done using techniques based on the detection of viral antigens such as immunofluorescence assay or/and viral isolation. STUDY DESIGN: Five hPIV-2 isolated from respiratory samples exhibited unusual phenotypic and antigenic characteristics. These isolates showed important syncytial cytopathic effect and failed to react with one specific monoclonal antibody. These variant strains were subsequently compared with hPIV-2 prototype strain by cellular and molecular techniques. RESULTS: Both variant and prototype strains showed similar growth kinetics. Observation of plaque formation and syncytia assay indicated a more important fusogenic activity for the variant strains. Sequencing of fusion (F) and hemagglutinin-neuraminidase (HN) genes showed differences between the "atypical" hPIV-2 isolates and the Greer hPIV-2 prototype strain. These differences were analyzed with molecular modelling and structure prediction soft wares. A potential new glycosylation site in HN, in addition to minor changes observed in the predicted structure for the variant strains could explain their antigenic variation. Genetic changes in the fusion peptide and the cleavage site of F could also explain the difference observed in the fusion activity. CONCLUSIONS: Continuous global viral surveillance is essential to monitor antigenic changes that may occur in nature particularly with regards to the implementation of diagnostic assays. The differences observed in F and HN between the prototype strain and clinical hPIV-2 variants could also provide new data for the analysis of Paramyxovirus fusion mechanisms and their pathogenesis.     

Comparison of differing cytopathic effects in human airway epithelium of parainfluenza virus 5 (W3A), parainfluenza virus type 3, and respiratory syncytial virus

Parainfluenza virus 5 (PIV5) infects a wide range of animals including dogs, pigs, cats, and humans; however, its association with disease in humans remains controversial. In contrast to parainfluenza virus 3 (PIV3) or respiratory syncytial virus (RSV), PIV5 is remarkably non-cytopathic in monolayer cultures of immortalized epithelial cells. To compare the cytopathology produced by these viruses in a relevant human tissue, we infected an in vitro model of human ciliated airway epithelium and measured outcomes of cytopathology. PIV5, PIV3 and, RSV all infected ciliated cells, and PIV5 and PIV3 infection was dependent on sialic acid residues. Only PIV5-infected cells formed syncytia. PIV5 infection resulted in a more rapid loss of infected cells by shedding of infected cells into the lumen. These studies revealed striking differences in cytopathology of PIV5 versus PIV3 or RSV and indicate the extent of cytopathology determined in cell-lines does not predict events in differentiated airway cells.     

Analysis of nucleotides 13-96 of the human parainfluenza virus type 3 antigenomic promoter reveals positive- and negative-acting replication elements

During replication of human parainfluenza virus type 3 (HPIV3), the 96-nucleotide antigenomic promoter (AGP) of HPIV3 directs the synthesis of genomic RNA. Previous work showed that nucleotides 1-12 were critical in promoting replication of an HPIV3 minireplicon, but nucleotides 13-96 were not investigated. In this study, the role of nucleotides 13-96 in AGP function was analyzed by creating and assaying mutations in an HPIV3 minireplicon. A replication promoting element known as promoter element II (nt 79-96) was confirmed in the HPIV3 AGP. Additionally, nucleotides 13-39 were found to constitute an additional positive-acting cis-element. However, detailed analysis of the 13-39 element revealed a complicated control element with both stimulatory and repressing elements. Specifically, nucleotides 21-28 were shown to repress RNA replication, while flanking sequences had a stimulatory effect.     

Attenuating mutations in the P/C gene of human parainfluenza virus type 1 (HPIV1) vaccine candidates abrogate the inhibition of both induction and signaling of type I interferon (IFN) by wild-type HPIV1

Recombinant human parainfluenza virus type 1 (HPIV1) and mutants containing point and deletion (Delta) mutations in the P/C gene (r-CDelta10-15HNT553A, r-CR84G, r-CF170S and r-CDelta170), which have previously been evaluated as HPIV1 vaccine candidates, were evaluated for their effect on the type I interferon (IFN) response in vitro. HPIV1 wt infection inhibited the IFN response by inhibiting IFN regulatory factor-3 (IRF-3) activation and IFN production in A549 cells and IFN signaling in Vero cells. In contrast, r-CR84G, r-CF170S and r-CDelta170 were defective for inhibition of IRF-3 activation and IFN production and r-CF170S and r-CDelta170 did not inhibit IFN signaling. Thus, HPIV1 antagonizes the IFN response at both the level of induction and signaling, and antagonism at both levels was disrupted by mutations in the P/C gene. Because CF170S affects C and not P, the anti-IFN function can be attributed to the C proteins. These data, in the context of previous in vivo studies, suggest that the loss of antagonism of the IFN response at both the level of induction and signaling, observed with the P/C mutants, r-CF170S and r-CDelta170, was necessary for significant attenuation in African green monkeys (AGMs).     

The prolonged culture of human immunodeficiency virus type 1 in primary lymphocytes increases its sensitivity to neutralization by soluble CD4

Primary strains of human immunodeficiency virus type 1 (HIV-1) are known to adapt to replication in cell lines in vitro by becoming sensitive to soluble CD4 (sCD4) and neutralizing antibodies (NAb). T-cell lines favor isolation of variants that use CXCR4 as a co-receptor, while primary isolates predominantly use CCR5. We have now studied how a primary R5 isolate, CC1/85, adapts to prolonged replication in primary human peripheral blood mononuclear cells (PBMC). After 19 passages, a variant virus, CCcon.19, had increased sensitivity to both sCD4 and NAb b12 that binds to a CD4-binding site (CD4BS)-associated epitope, but decreased sensitivity to anti-CD4 antibodies. CCcon.19 retains the R5 phenotype, its resistance to other NAbs was unaltered, its sensitivity to various entry inhibitors was unchanged, and its ability to replicate in macrophages was modestly increased. We define CCcon.19 as a primary T-cell adapted (PTCA) variant. Genetic sequence analysis combined with mutagenesis studies on clonal, chimeric viruses derived from CC1/85 and the PTCA variant showed that the most important changes were in the V1/V2 loop structure, one of them involving the loss of an N-linked glycosylation site. Monomeric gp120 proteins expressed from CC1/85 and the PTCA variant did not differ in their affinities for sCD4, suggesting that the structural consequences of the sequence changes were manifested at the level of the native, trimeric Env complex. Overall, the adaptation process probably involves selection for variants with higher CD4 affinity and hence greater fusion efficiency, but this also involves the loss of some resistance to neutralization by agents directed at or near to the CD4BS. The loss of neutralization resistance is of no relevance under in vitro conditions, but NAbs would presumably be a counter-selection pressure against such adaptive changes in vivo, at least when the humoral immune response is intact.     

登革病毒4型E蛋白的表达与多克隆抗体的制备

 目的 在原核细胞中表达登革病毒4型E蛋白及E蛋白III区,分别以两种蛋白免疫家兔,获得可检测登革病毒的多克隆抗体。方法 将登革病毒4型E蛋白与E蛋白III区编码序列克隆到pET-32a（+）质粒中,分别构建两种蛋白的表达载体,以IPTG诱导其在Rosetta细胞中的大量表达,并进行SDS-PAGE检测。分别以两种蛋白免疫家兔,制备出针对E蛋白与E蛋白III区的多克隆抗体,并对其进行Western blot鉴定。结果 登革病毒4型E蛋白与E蛋白III区在Rosetta细胞中以包涵体的形式大量表达;利用所制备的多克隆抗体对登革病毒进行检测,出现了预期的条带。结论 本研究制备获得的多克隆抗体可用于登革病毒的检测,为登革病毒相关研究奠定了基础。     

A survey of respiratory syncytial virus and parainfluenza virus type 3 neutralising and immunoprecipitating antibodies in relation to Paget disease

The aetiology of Paget disease of bone has not been established but certain features have suggested involvement of a parainfluenzalike virus. To seek further evidence of the possible role of paramyxoviruses in Paget disease we have surveyed the presence of neutralising and immunoprecipitating antibodies to both respiratory syncytial virus and parainfluenza virus type 3 in the sera of patients attending a bone disease clinic. These two viruses were implicated by the sporadic observation of viral antigen in individual nuclei of osteoclasts in Paget disease bone lesions. A total of 315 samples were obtained from 177 patients attending the clinic during 1 year. Thirty-six of the patients had confirmed Paget disease and the remainder other conditions. All sera possessed neutralising activity to both viruses. The mean titres for each virus were similar in patients with Paget disease and those with other conditions whether matched or not. In the case of respiratory syncytial virus the neutralising titres were distributed closer to the mean in the Paget group and showed little variation in repeat samples taken over periods of up to 1 year in contrast to the greater variability of the control group. The antigenic specificity of 20 age- and sex-matched sera from each group was examined by immunoprecipitation. No significant differences were observed between Paget and non-Paget patients. These results do not provide confirmation of involvement of either virus in Paget disease, but the serological data suggest that persistent infection with respiratory syncytial virus can occur.     

Relationship between type specific antibodies to herpes simplex virus and type specificity in human sera

A quantitative analysis was carried out on the relationship between type specific neutralizing antibodies to herpes simplex virus and the type specificity, using a mutual absorbing procedure. Forty-two samples were selected among human sera, on the basis of type specificity expressed by the value of II/I index. All sera with values of less than 90 of II/I index contained only type 1 specific antibody, while those with values over 111 contained only type 2 specific antibody. When the values were between 90 and 110, type 1 specific antibody was present in all, and type 2 specific antibody was present in some serum samples.     

人类免疫缺陷病毒1型云南株外膜蛋白原核表达克隆的构建

目的为研究我国云南１型人类免疫缺陷病毒（Ｈｕｍａｎｉｍｍｕｎｏｄｅｆｉｃｉｅｎｃｙｖｉｒｕｓｔｙｐｅ１，ＨＩＶ－１）流行株外膜蛋白（ｇｐ１２０）的有关抗原表位。方法采用套式聚合酶链式反应，以来自云南流行区ＨＩＶ－１感染者的外周血单核巨噬细胞基因组为模板，进行云南流行株外膜蛋白基因（ｅｎｖ）片段的扩增，并将ｅｎｖ基因片段与原核表达载体ｐＢＶ２２０进行重组，构建成质粒ｐＹＮｅｎｖ并在大肠杆菌（Ｅ．ｃｏｌｉＤＨ１０ｂ）中获得表达。采用限制性内切酶分析进行重组质粒的鉴定。结果含重组质粒的宿主菌经３０℃２０小时培养后，转入４２℃培养５小时，经ＳＤＳ－ＰＡＧＥ蛋白电泳分析有重组蛋白的表达。经Ｗｅｓｔｅｒｎｂｌｏｔ反应证实，该重组蛋白可与来自该流行区的ＨＩＶ－１感染者血清（含多克隆抗体）发生特异反应。结论该重组膜蛋白可作为抗原用于ＨＩＶ－１膜蛋白抗体的检测，并为进一步研究ＨＩＶ－１ｇｐ１２０的病理机制奠定了基础。     

Ⅰ型人类免疫缺陷病毒包膜糖蛋白CD4结合位点修饰诱导小鼠体液免疫反应的实验研究

目的 探讨Ⅰ型人类免疫缺陷病毒(HIV-1)包膜糖蛋白(Env) gp120的CD4结合位点(CD4BS)核心区第423、425和431位氨基酸三联突变ING/MKE对其诱导体液免疫反应的影响.方法 构建HIV-1原代毒株06044包膜gp120 ING/MKE三联突变表达载体pcT22-06044 gp120T-ING/MKE(gp120T-ING/MKE),在体外转染的HEK293T细胞表达三聚化野生型gp120Twt蛋白和突变体gp120T-ING/MKE蛋白.免疫BALB/c小鼠后检测结合抗体、中和抗体和骨髓抗原特异性浆细胞.结果 获得真核表达载体gp120T-ING/MKE.转染后,在293T细胞培养上清中检测到了三聚化重组蛋白.末次免疫后14 d,gp120Twt和gp120T-ING/MKE免疫血清中结合抗体滴度都大于1∶1 000,但两组血清抗体滴度无显著差异.突变体组骨髓特异性浆细胞分泌水平和非特异浆细胞水平均低于野生型gp120免疫组.野生型和突变体免疫诱导血清抗体的中和活性均不强.结论 HIV-1包膜蛋白CD4结合区423、425和431位三联突变没有改善gp120蛋白的免疫原性.