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Paramyxovirus genomes, like that of human parainfluenza virus type 2 (hPIV2), have lengths of precisely multiples-of-six nucleotides (“rule of six”), where each nucleoprotein subunit (NP) binds exactly six nucleotides. Ten residues of its RNA binding groove contact the genome RNA; but only one, Q202, directly contacts a nucleotide base. The mutation of NP(Q202) leads to two phenotypes: the ability of the viral polymerase to replicate minigenomes with defective bipartite promoters where NP(wt) is inactive, and the inability to rescue rPIV2 carrying this point mutation by standard means. The absence of an rPIV2 NP(Q202A) prevented further study of the latter phenotype. By extensive and repeated cocultivation of transfected cells, an rPIV2 carrying this mutation was finally recovered, and this virus was apparently viable due to the presence of an additional NP mutation (I35L). Our results suggest that these two phenotypes are due to separate effects of the Q202 mutation, and that the problematic rescue phenotype may be due to the inability of the transfected cell to incorporate viral nucleocapsids during virus budding. IMPORTANCE Paramyxovirus genomes are contained within a noncovalent homopolymer of its nucleoprotein (NP) and form helical nucleocapsids (NC) whose 3′ ends contain the promoters for the initiation of viral RNA synthesis. This work suggests that these NC 3′ ends may play another role in the virus life cycle via their specific interaction with virus-modified cell membranes needed for the incorporation of viral NCs into budding virions.

We previously reported that human parainfluenza virus type 2 (hPIV-2) promoted RhoA activation and subsequent filamentous actin (F-actin) formation. Actin-binding proteins, such as profilin and cofilin, are involved in the regulation of F-actin formation by RhoA signaling. In the present study, we identified profilin2 as a key molecule that is involved in hPIV-2-induced F-actin formation. Immunoprecipitation assays demonstrated that hPIV-2 V protein binds to profilin2 but not to profilin1. Mutation of Trp residues within C-terminal region of V protein abolished the binding capacity to profilin2. Depletion of profilin2 resulted in the inhibition of hPIV-2-induced F-actin formation and the suppression of hPIV-2 growth. Overexpression of wild type V but not Trp-mutated V protein reduced the quantity of actin co-immunoprecipitated with profilin2. Taken together, these results suggest that hPIV-2 V protein promotes F-actin formation by affecting actin-profilin2 interaction through its binding to profilin2.

Tight junctions enable epithelial cells to form physical barriers that act as an innate immune defense against respiratory infection. However, the involvement of tight junction molecules in paramyxovirus infections, which include various respiratory pathogens, has not been examined in detail. Human parainfluenza virus type 2 (hPIV2) infects airway epithelial cells and causes respiratory illness. In the present study, we found that hPIV2 infection of cultured cells induces expression of claudin-1 (CLDN1), an essential component of tight junctions. This induction seemed to be intrinsically restricted by V, an accessory protein that modulates various host responses, to enable efficient virus propagation. By generating CLDN1 over-expressing and knockout cell lines, we showed that CLDN1 is involved in the restriction of hPIV2 spread via cell-to-cell contact. Taken together, we identified CLDN1 an inhibitory factor for hPIV2 dissemination, and that its V protein acts to counter this.

Intracellular iron concentration is tightly controlled for cell viability. It is known to affect the growth of several viruses, but the molecular mechanisms are not well understood. We found that iron chelators inhibit growth of human parainfluenza virus type 2 (hPIV-2). Furthermore, infection with hPIV-2 alters ferritin localization from granules to a homogenous distribution within cytoplasm of iron-stimulated cells. The V protein of hPIV-2 interacts with ferritin heavy chain 1 (FTH1), a ferritin subunit. It also binds to nuclear receptor coactivator 4 (NCOA4), which mediates autophagic degradation of ferritin, so-called ferritinophagy. V protein consequently interferes with interaction between FTH1 and NCOA4. hPIV-2 growth is inhibited in FTH1 knockdown cell line where severe hPIV-2-induced apoptosis is shown. In contrast, NCOA4 knockdown results in the promotion of hPIV-2 growth and limited apoptosis. Our data collectively suggest that hPIV-2 V protein inhibits FTH1-NCOA4 interaction and subsequent ferritinophagy. This iron homeostasis modulation allows infected cells to avoid apoptotic cell death, resulting in effective growth of hPIV-2. IMPORTANCE hPIV-2 V protein interferes with interaction between FTH1 and NCOA4 and inhibits NCOA4-mediated ferritin degradation, leading to the inhibition of iron release to the cytoplasm. This iron homeostasis modulation allows infected cells to avoid apoptotic cell death, resulting in effective growth of hPIV-2.

Asymptomatic patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection must be quickly identified and isolated to prevent the spread of the virus. The number of asymptomatic healthy people is completely unknown because they remain untested. Detection of specific SARS-CoV-2 antibodies has been widely accepted as a diagnostic test, and an immunochromatographic test, which is simpler and relatively cheaper than other methods, is becoming the gold standard for identifying healthy people who had been infected with SARS-CoV-2 in the past. In this study, 1,528 volunteers who worked at a particular hospital were subjected to an immunochromatographic IgG test for SARS-CoV-2 to determine the ratio of asymptomatic people. Only 12 volunteers (0.79%) were IgG+, with no significant background differences in the sex, age, profession, experiences of working at the emergency department or caring for coronavirus disease 2019 patients. If this IgG+ ratio was to be extrapolated to Wakayama city’s population, 2,780 out of 3,54,063 people may be asymptomatic for SARS-CoV-2. The results imply that anyone may get infected with SARS-CoV-2 but remain asymptomatic.

Background Coronavirus disease (COVID-19) is a growing concern worldwide. Approximately 5% of COVID-19 cases require intensive care. However, the optimal treatment for respiratory failure in COVID-19 patients is yet to be determined. Case presentation A 79-year-old man with severe acute respiratory distress syndrome due to COVID-19 was admitted to our intensive care unit. Prone ventilation was effective in treating the patient's hypoxemia. Furthermore, the patient received lung protective ventilation with a tidal volume of 6-8 mg/kg (predicted body weight). However, the patient's respiratory failure did not improve and he died 16 days after admission because of multiple organ failure. Serial chest computed tomography revealed a change from ground-glass opacity to consolidation pattern in both lungs. Conclusions We report a protracted case of COVID-19 in a critically ill patient in Japan. Although prone ventilation could contribute to treating hypoxemia, its efficacy in preventing mortality from COVID-19 is unknown.

Cavin proteins have important roles in the formation of caveolae in lipid raft microdomains. Pulse-chase experiments of cells infected with human parainfluenza virus type 2 (hPIV-2) showed decreased proteasomal degradation of Cavin3. Overexpression of hPIV-2 V protein alone was sufficient to inhibit Cavin3 degradation. Immunoprecipitation analysis revealed that V protein bound to Cavin3. Trp residues within C-terminal region of V protein, as well as the N-terminal region of Cavin3, are important for V-Cavin3 interaction. Cavin3 knockdown suppressed hPIV-2 growth without affecting its entry, replication, transcription, or translation. Higher amounts of Cavin3 were observed in V protein-overexpressing cells than in control cells in lipid raft microdomains. Our data collectively suggest that hPIV-2 V protein binds to and stabilizes Cavin3, which in turn facilitates assembly and budding of hPIV-2 in lipid raft microdomains.

The unusual ability of a human parainfluenza virus type 2 (hPIV2) nucleoprotein point mutation (NPQ202A) to strongly enhance minigenome replication was found to depend on the absence of a functional, internal element of the bipartite replication promoter (CRII). This point mutation allows relatively robust CRII-minus minigenome replication in a CRII-independent manner, under conditions in which NPwt is essentially inactive. The nature of the amino acid at position 202 apparently controls whether viral RNA-dependent RNA polymerase (vRdRp) can, or cannot, initiate RNA synthesis in a CRII-independent manner. By repressing genome synthesis when vRdRp cannot correctly interact with CRII, gln202 of N, the only residue of the RNA-binding groove that contacts a nucleotide base in the N-RNA, acts as a gatekeeper for wild-type (CRII-dependent) RNA synthesis. This ensures that only hexamer-length genomes are replicated, and that the critical hexamer phase of the cis-acting mRNA editing sequence is maintained.

Hazara nairovirus (HAZV) is a trisegmented RNA virus most closely related to Crimean-Congo hemorrhagic fever virus (CCHFV) in the order Bunyavirales. The terminal roughly 20 nucleotides (nt) of its genome ends are highly complementary, similar to those of other segmented negative-strand RNA viruses (sNSV), and act as promoters for RNA synthesis. These promoters contain two elements: the extreme termini of both strands (promoter element 1 [PE1]) are conserved and virus specific and are found bound to separate sites on the polymerase surface in crystal structures of promoter-polymerase complexes. The following sequences (PE2) are segment specific, with the potential to form double-stranded RNA (dsRNA), and the latter aspect is also important for promoter activity. Nairovirus genome promoters differ from those of peribunyaviruses and arenaviruses in that they contain a short single-stranded region between the two regions of complementarity. Using a HAZV minigenome system, we found the single-stranded nature of this region, as well as the potential of the following sequence to form dsRNA, is essential for reporter gene expression. Most unexpectedly, the sequence of the PE2 dsRNA appears to be equally important for promoter activity. These differences in sNSV PE2 promoter elements are discussed in light of our current understanding of the initiation of RNA synthesis. IMPORTANCE A minigenome system for HAZV, closely related to CCHFV, was used to study its genome replication. HAZV genome ends, like those of other sNSV, such as peribunyaviruses and arenaviruses, are highly complementary and serve as promoters for genome synthesis. These promoters are composed of two elements: the extreme termini of both 3′ and 5′ strands that are initially bound to separate sites on the polymerase surface in a sequence-specific fashion and the following sequences with the potential to anneal but whose sequence is not important. Nairovirus promoters differ from the other sNSV cited in that they contain a short single-stranded RNA (ssRNA) region between the two elements. The single-stranded nature of this region is an essential element of the promoter, whereas its sequence is unimportant. The sequence of the following complementary region is unexpectedly also important, a possible rare example of sequence-specific dsRNA recognition.

Receptor destruction has been considered one of the mechanisms of homologous Sendai virus (SeV) interference. However, direct evidence of receptor destruction upon virus infection and its relevance to interference is missing. To investigate a precise mechanism of homologous interference, we established SeV persistently infected cells. The persistently infected cells inhibited superinfection by homologous SeV but supported replication of human parainfluenza virus 2 (hPIV2) and influenza A virus (IAV). We confirmed that SeV particles could not attach to or penetrate the infected cells and that the hemagglutinin-neuraminidase (HN) protein of SeV was involved in the interference. Lectin blot assays showed that the α2,3-linked sialic acids were specifically reduced in the SeV-infected cells, but the level of α2,6-linked sialic acids had not changed. As infection with IAV removed both α2,3- and α2,6-linked sialic acids, especially α2,3-linked sialic acids, IAV-infected cells inhibited superinfection of SeV. These results provide concrete evidence that destruction of the specific SeV receptor, α2,3-linked sialic acids, is relevant to homologous interference by SeV. IMPORTANCE Viral interference is a classically observed phenomenon, but the precise mechanism is not clear. Using SeV interference, we provide concrete evidence that reduction of the α2,3-linked sialic acid receptor by the HN of SeV is closely related with viral interference. Since SeV infection resulted in decrease of only α2,3-linked sialic acids, IAV, which also utilized α2,6-linked sialic acids to initiate infection, superinfected the SeV-infected cells. In contrast, SeV could not superinfect the IAV-infected cells because both α2,3- and α2,6-linked sialic acids were removed. These results indicate that receptor destruction critically contributes to viral interference.

Virus-specific interaction between the attachment protein (HN) and the fusion protein (F) is prerequisite for the induction of membrane fusion by parainfluenza viruses. This HN-F interaction presumably is mediated by particular amino acids in the HN stalk domain and those in the F head domain. We found in the present study, however, that a simian virus 41 (SV41) F-specific chimeric HPIV2 HN protein, SCA, whose cytoplasmic, transmembrane, and stalk domains were derived from the SV41 HN protein, could not induce cell-cell fusion of BHK-21 cells when coexpressed with an SV41 HN-specific chimeric PIV5 F protein, no. 36. Similarly, a headless form of the SV41 HN protein failed to induce fusion with chimera no. 36, whereas it was able to induce fusion with the SV41 F protein. Interestingly, replacement of 13 amino acids of the SCA head domain, which are located at or around the dimer interface of the head domain, with SV41 HN counterparts resulted in a chimeric HN protein, SCA-RII, which induced fusion with chimera no. 36 but not with the SV41 F protein. More interestingly, retroreplacement of 11 out of the 13 amino acids of SCA-RII with the SCA counterparts resulted in another chimeric HN protein, IM18, which induced fusion either with chimera no. 36 or with the SV41 F protein, similar to the SV41 HN protein. Thus, we conclude that the F protein specificity of the HN protein that is observed in the fusion event is not solely defined by the primary structure of the HN stalk domain. IMPORTANCE It is appreciated that the HN head domain initially conceals the HN stalk domain but exposes it after the head domain has bound to the receptors, which allows particular amino acids in the stalk domain to interact with the F protein and trigger it to induce fusion. However, other regulatory roles of the HN head domain in the fusion event have been ill defined. We have shown in the current study that removal of the head domain or amino acid substitutions in a particular region of the head domain drastically change the F protein specificity of the HN protein, suggesting that the ability of a given HN protein to interact with an F protein is defined not only by the primary structure of the HN stalk domain but also by its conformation. This notion seems to account for the unidirectional substitutability among rubulavirus HN proteins in triggering noncognate F proteins.

It has been reported that dual or multiple viruses can coinfect epithelial cells of the respiratory tract. However, little has been reported on in vitro interactions of coinfected viruses. To explore how coinfection of different viruses affects their biological property, we examined growth of influenza A virus (IAV) and human parainfluenza virus type 2 (hPIV2) during coinfection of Vero cells. We found that IAV growth was enhanced by coinfection with hPIV2. The enhanced growth of IAV was not reproduced by coinfection with an hPIV2 mutant with reduced cell fusion activity, or by ectopic expression of the V protein of hPIV2. In contrast, induction of cell fusion by ectopic expression of the hPIV2 HN and F proteins augments IAV growth. hPIV2 coinfection supported IAV growth in cells originated from the respiratory epithelium. The enhancement correlated closely with cell fusion ability of hPIV2 in those cells. These results indicate that cell fusion induced by hPIV2 infection is beneficial to IAV replication and that enhanced viral replication by coinfection with different viruses can modify their pathological consequences.

Human parainfluenza virus type 2 phosphoprotein (P) is an essential component of viral polymerase. The P gene encodes both P and accessory V proteins by a specific gene editing mechanism. Therefore, the N-terminal 164 amino acids of P protein are common to V protein. Interestingly, while P protein is located in the cytoplasm, V protein is found mainly in the nucleus. Using deletion mutants, we show the presence of a nuclear localization signal (NLS) in the P/V common domain, and a nuclear export signal (NES) in the C-terminal P specific region. The NLS region makes a complex with importin α5 or 7. In the presence of leptomycin B, P protein is retained in the nucleus, indicating that it contains a CRM1-dependent NES. We identified the NLS (65PVKPRRKK72) and the NES (225IIELLKGLDL234) using β-galactosidase fusion proteins. Moreover, nucleocytoplasmic shuttling of P protein appears to be important for efficient viral polymerase activity.

We previously demonstrated that human parainfluenza virus type 2 (hPIV-2) induces RhoA activation, which promotes its growth. RhoA controls the equilibrium between globular and filamentous actin (F-actin). We found that F-actin formation is induced by wild type (wt) hPIV-2 infection, and that inhibition of F-actin formation by cytochalasin D decreases hPIV-2 growth. In wt RhoA-expressing cells, F-actin formation occurs and hPIV-2 growth is promoted. Overexpression of T19N RhoA, a dominant negative (DN) form of RhoA, inhibits hPIV-2-induced F-actin formation, and suppresses hPIV-2 growth. Immunoprecipitation assays reveal that hPIV-2 V protein binds only to DN RhoA, and this interaction requires its C-terminal Trp residues. F-actin formation is not observed during infection of recombinant hPIV-2 expressing Trp-mutated V protein (VW178H/W182E/W192A). Overexpression of V protein, but not that of VW178H/W182E/W192A, causes F-actin formation. Our results suggest that hPIV-2 V protein enhances hPIV2 growth through RhoA-induced F-actin formation, by selectively binding to inactive RhoA.

Membrane fusion by the parainfluenza viruses is induced by virus-specific functional interaction between the attachment protein (HN) and the fusion (F) protein. This interaction is thought to be mediated by transient contacts between particular amino acids in the HN stalk domain and those in the F head domain. However, we recently reported that replacement of specified amino acids at or around the dimer interface of the HN head domain remarkably affected the F protein specificity. We then intended to further investigate this issue in the present study and revealed that the HPIV2 HN protein can be converted to an SV41 HN-like protein by substituting at least nine amino acids in the HPIV2 HN head domain with the SV41 HN counterparts in addition to the replacement of the stalk domain, indicating that specified amino acids in the HN head domain play very important roles in determining the specificity of the HN-F interaction. On the other hand, we previously reported that the PIV5 F protein can be converted to an SV41 F-like protein by replacing 21 amino acids in the head domain of the PIV5 F protein with those of the SV41 F protein. We then intended to further investigate this issue in the present study and found that replacement of 15 amino acids in the stalk domain in addition to the replacement of the 21 amino acids in the head domain of the PIV5 F protein resulted in creation of a more SV41 F-like protein, indicating that specified amino acids in the F stalk domain play important roles in determining the specificity of the HN-F interaction. These results suggest that the conformations of the HN stalk domain and the F head domain are dependent on the structures of the HN head domain and the F stalk domain, respectively. Presumably, the conformations of the former domains, which are considered directly involved in the HN-F interaction, can be modified by subtle changes in the structure of the latter domains, resulting in an altered specificity for the interacting partners.

The multifunctional V protein of human parainfluenza virus type 2 (hPIV2) plays important roles in controlling viral genome replication, inhibiting the host interferon response and promoting virus growth. We screened a yeast two-hybrid library using V protein as bait to identify host factors that are important for other functions of V. One of several positive clones isolated from HeLa cell-derived cDNA library encodes caspase-1. We found that the C-terminal region of V interacts with the C-terminal region of caspase-1 in mammalian cells. Moreover, the V protein repressed caspase-1 activity and the formation of interleukin-1β (IL-1β) in a dose-dependent manner. IL-1β secretion induced by wild-type hPIV2 infection in human monocytic THP-1 cells was significantly lower than that induced by recombinant hPIV2 lacking V protein or having a mutant V. These data suggest that hPIV2 V protein inhibits caspase-1-mediated maturation of IL-1β via its interaction with caspase-1.

The genome RNA of human parainfluenza virus type 2 (hPIV2) that acts as the template for the polymerase complex is entirely encapsidated by the nucleoprotein (NP). Recently, the crystal structure of NP of PIV5, a virus closely related to hPIV2, was resolved in association with RNA. Ten amino acids that contact the bound RNA were identified and are strictly conserved between PIV5 and hPIV2 NP. Mutation of hPIV2 NP Q202 (which contacts a base rather than the RNA backbone) to various amino acids resulted in an over 30-fold increase of polymerase activity as evidenced by a minireplicon assay, even though the RNA-binding affinity was unaltered. Using various modified minireplicons, we found that the enhanced reporter gene expression could be accounted for by increased minigenome replication, whereas mRNA synthesis itself was not affected by Q202 mutation. Moreover, the enhanced activities were still observed in minigenomes partially lacking the leader sequence and which were not of hexamer genome length. Unexpectedly, recombinant hPIV2 possessing the NP Q202A mutation could not be recovered from cDNA. IMPORTANCE We examined the importance of amino acids in the putative RNA-binding domain of hPIV2 NP for polymerase activity using minireplicons. Abnormally enhanced genome replication was observed upon substitution mutation of the NP Q202 position to various amino acids. Surprisingly, this mutation enabled polymerase to use minigenomes that were partially lacking the leader sequence and not of hexamer genome length. This mutation does not affect fundamental properties of NP, e.g., recognition of gene junctional and editing signals. However, the strongly enhanced polymerase activity may not be viable for the infectious life cycle. This report highlights the potential of the polymerase complex with point mutations in NP and helps our detailed understanding of the molecular basis of gene expression.

A stable packaging cell line (Vero/BC-F) constitutively expressing fusion (F) protein of the human parainfluenza virus type 2 (hPIV2) was established for production of the F-defective and single round-infectious hPIV2 vector in a strategy for recombinant vaccine development. The F gene expression has not evoked cytostatic or cytotoxic effects on the Vero/BC-F cells and the F protein was physiologically active to induce syncytial formation with giant polykaryocytes when transfected with a plasmid expressing hPIV2 hemagglutinin-neuraminidase (HN). Transduction of the F-defective replicon RNA into the Vero/BC-F cells led to the release of the infectious particles that packaged the replicon RNA (named as hPIV2ΔF) without detectable mutations, limiting the infectivity to a single round. The maximal titer of the hPIV2ΔF was 6.0 × 10(8) median tissue culture infections dose per ml. The influenza A virus M2 gene was inserted into hPIV2ΔF, and the M2 protein was found to be highly expressed in a human lung cancer cell line after transduction. Furthermore, in vivo airway infection experiments revealed that the hPIV2ΔF was capable of delivering transgenes to hamster tracheal cells. Thus, non-transmissible or single round-infectious hPIV2 vector will be potentially applicable to human gene therapy or recombinant vaccine development.

For most parainfluenza viruses, a virus type-specific interaction between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins is a prerequisite for mediating virus-cell fusion and cell-cell fusion. The molecular basis of this functional interaction is still obscure partly because it is unknown which region of the F protein is responsible for the physical interaction with the HN protein. Our previous cell-cell fusion assay using the chimeric F proteins of parainfluenza virus 5 (PIV5) and simian virus 41 (SV41) indicated that replacement of two domains in the head region of the PIV5 F protein with the SV41 F counterparts bestowed on the PIV5 F protein the ability to induce cell-cell fusion on coexpression with the SV41 HN protein while retaining its ability to induce fusion with the PIV5 HN protein. In the study presented here, we furthered the chimeric analysis of the F proteins of PIV5 and SV41, finding that the PIV5 F protein could be converted to an SV41 HN-specific chimeric F protein by replacing five domains in the head region with the SV41 F counterparts. The five SV41 F-protein-derived domains of this chimera were then divided into 16 segments; 9 out of 16 proved to be not involved in determining its specificity for the SV41 HN protein. Finally, mutational analyses of a chimeric F protein, which harbored seven SV41 F-protein-derived segments, revealed that replacement of at most 21 amino acids of the PIV5 F protein with the SV41 F-protein counterparts was enough to convert its HN protein specificity.

Paramyxovirus V proteins block Toll-like receptor 7 (TLR7)- and TLR9-dependent signaling leading to alpha interferon production. Our recent study has provided evidence that interaction of the V proteins with IRF7 is important for the blockade. However, the detailed mechanisms still remain unclear. Here we reexamined the interaction of the human parainfluenza virus type 2 (HPIV2) V protein with signaling molecules involved in TLR7/9-dependent signaling. Immunoprecipitation experiments in HEK293T cells transfected with V protein and one of the signaling molecules revealed that the V protein interacted with not only IRF7 but also TRAF6, IKKα, and MyD88. Whereas overexpression of TRAF6 markedly enhanced the level of V protein associating with IRF7, IKKα, and MyD88 in HEK293T cells, the level of V protein associating with TRAF6 was little affected by overexpression of IRF7, IKKα, and MyD88. Moreover, knockdown or knockout of endogenous TRAF6 in HEK293T or mouse embryonic fibroblast cells resulted in dissociation of the V protein from IRF7, IKKα, and MyD88. These results demonstrate that binding of the V protein to IRF7, IKKα, and MyD88 is largely indirect and mediated by endogenous TRAF6. It was found that the V protein inhibited TRAF6-mediated lysine 63 (K63)-linked polyubiquitination of IRF7, which is prerequisite for IRF7 activation. Disruption of the tryptophan-rich motif of the V protein significantly affected its TRAF6-binding efficiency, which correlated well with the magnitude of inhibition of K63-linked polyubiquitination and the resultant activation of IRF7. Taken together, these results suggest that the HPIV2 V protein prevents TLR7/9-dependent interferon induction by inhibiting TRAF6-mediated K63-linked polyubiquitination of IRF7.

Rho GTPases are involved in a variety of cellular activities and are regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We found that the activation of Rho GTPases by lysophosphatidic acid promotes the growth of human parainfluenza virus type 2 (hPIV-2). Furthermore, hPIV-2 infection causes activation of RhoA, a Rho GTPase. We hypothesized that Graf1 (also known as ARHGAP26), a GAP, regulates hPIV-2 growth by controlling RhoA signaling. Immunofluorescence analysis showed that hPIV-2 infection altered Graf1 localization from a homogenous distribution within the cytoplasm to granules. Graf1 colocalized with hPIV-2 P, NP, and L proteins. Graf1 interacts with P and V proteins via their N-terminal common region, and the C-terminal Src homology 3 domain-containing region of Graf1 is important for these interactions. In HEK293 cells constitutively expressing Graf1, hPIV-2 growth was inhibited, and RhoA activation was not observed during hPIV-2 infection. In contrast, Graf1 knockdown restored hPIV-2 growth and RhoA activation. Overexpression of hPIV-2 P and V proteins enhanced hPIV-2-induced RhoA activation. These results collectively suggested that hPIV-2 P and V proteins enhanced hPIV-2 growth by binding to Graf1 and that Graf1 inhibits hPIV-2 growth through RhoA inactivation. IMPORTANCE Robust growth of hPIV-2 requires Rho activation. hPIV-2 infection causes RhoA activation, which is suppressed by Graf1. Graf1 colocalizes with viral RNP (vRNP) in hPIV-2-infected cells. We found that Graf1 interacts with hPIV-2 P and V proteins. We also identified regions in these proteins which are important for this interaction. hPIV-2 P and V proteins enhanced the hPIV-2 growth via binding to Graf1, while Graf1 inhibited hPIV-2 growth through RhoA inactivation.

Tetherin (BST-2/CD317/HM1.24) is an antiviral membrane protein that prevents the release of enveloped viruses from the cell surface. We found that the growth of human parainfluenza virus type 2 (hPIV-2), but not that of V protein-deficient recombinant hPIV-2, was inhibited by tetherin. V protein immunoprecipitates with tetherin, and this interaction requires its C-terminal Trp residues. The glycosyl phosphatidylinositol attachment signal of tetherin, but not its cytoplasmic tail, was necessary for its binding with V. The distribution of the V protein clearly changed when co-expressed with tetherin in plasmid-transfected cells. hPIV-2 infection of HeLa cells reduced cell surface tetherin without affecting total cellular tetherin. This reduction also occurred in HeLa cells constitutively expressing V, whereas mutated V protein did not affect the cell surface tetherin. Our results suggest that hPIV-2 V protein antagonizes tetherin by binding it and reducing its presence at the cell surface.

Plasmacytoid dendritic cells (pDCs) do not produce alpha interferon (IFN-α) unless viruses cause a systemic infection or overcome the first-line defense provided by conventional DCs and macrophages. We show here that even paramyxoviruses, whose infections are restricted to the respiratory tract, have a V protein able to prevent Toll-like receptor 7 (TLR7)- and TLR9-dependent IFN-α induction specific to pDCs. Mutational analysis of human parainfluenza virus type 2 demonstrates that the second Trp residue of the Trp-rich motif (Trp-X 3 -Trp-X 9 -Trp) in the C-terminal domain unique to V, a determinant for IRF7 binding, is critical for the blockade of TLR7/9-dependent signaling.

The large RNA polymerase (L) protein of human parainfluenza virus type 2 (hPIV2) binds the nucleocapsid, phosphoprotein, and V protein, as well as itself, and these interactions are essential for transcription and replication of the viral RNA genome. Although all of these interactions were found to be mediated through the domains within the N terminus of L, the C terminus of the L protein was also required for minigenome reporter gene expression. We have identified a highly conserved rubulavirus domain near the C terminus of the L protein that is required for mRNA synthesis but not for genome replication. Remarkably, this region of L shares homology with a conserved region of cellular capping enzymes that binds GTP and forms a lysyl-GMP enzyme intermediate, the first step in the cellular capping reaction. We propose that this conserved region of L also binds GTP (or GDP) to carry out the second step of the unconventional nonsegmented negative-strand virus capping reaction.

In wild-type human parainfluenza virus type 2 (WT HPIV2), one gene (the P/V gene) encodes both the polymerase-associated phosphoprotein (P) and the accessory V protein. We generated a HPIV2 virus (rHPIV2-V(ko)) in which the P/V gene encodes only the P protein to examine the role of V in replication in vivo and as a potential live attenuated virus vaccine. Preventing expression of V protein severely impaired virus recovery from cDNA and growth in vitro, particularly in IFN-competent cells. rHPIV2-V(ko), unlike WT HPIV2, strongly induced IFN-beta and permitted IFN signaling, leading to establishment of a robust antiviral state. rHPIV2-V(ko) infection induced extensive syncytia and cytopathicity that was due to both apoptosis and necrosis. Replication of rHPIV2-V(ko) was highly restricted in the respiratory tract of African green monkeys and in differentiated primary human airway epithelial (HAE) cultures, suggesting that V protein is essential for efficient replication of HPIV2 in organized epithelial cells and that rHPIV2-V(ko) is over-attenuated for use as a live attenuated vaccine.

The fusion (F) protein of parainfluenza virus 5 (PIV-5) strain W3A is able to induce cell fusion when it is expressed alone in baby hamster kidney cells, whilst the F protein of PIV-5 strain WR induces cell fusion only when co-expressed with the haemagglutinin–neuraminidase (HN) protein. It has been shown previously that when Leu-22 of the WR F protein is replaced with the W3A F counterpart (Pro-22), the resulting mutant L22P exhibits HN-independent fusion activity. Furthermore, previous chimeric analysis between L22P and the F protein of PIV-5 strain T1 has suggested that Glu-132 also contributes to the HN-independent fusion activity of L22P. It was shown here that substitution of Glu-132 of L22P with various amino acids including the T1 F protein counterpart (Lys-132) resulted in a reduction in fusion activity, whereas substitution with Asp was the exception in being tolerated. Interestingly, reduced fusion activity of an L22P mutant that harboured the E132K substitution could be restored by an additional D416K substitution but not by a D416E mutation, suggesting that the presence of the same charge at positions 132 and 416 is important for the HN-independent fusion activity. In contrast, substitution of Leu-22 of the WR F protein with various amino acids except those with aliphatic side chains resulted in acquisition of fusion activity, suggesting that the HN dependence of the WR F protein in the induction of cell fusion is attributable to the hydrophobicity of Leu-22. These results indicate that at least three amino acids are involved in the HN-independent fusion activity of the PIV-5 F protein.

The monoclonal antibody M1-1A, specific for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 2 virus (HPIV2), blocks virus-induced cell-cell fusion without affecting the hemagglutinating and neuraminidase activities. F13 is a neutralization escape variant selected with M1-1A and contains amino acid mutations N83Y and M186I in the HN protein, with no mutation in the fusion protein. Intriguingly, F13 exhibits reduced ability to induce cell-cell fusion despite its multistep replication. To investigate the potential role of HPIV2 HN protein in the regulation of cell-cell fusion, we introduced these mutations individually or in combination to the HN protein in the context of recombinant HPIV2. Following infection at a low multiplicity, Vero cells infected with the mutant virus H-83/186, which carried both the N83Y and M186I mutations, remained as nonfused single cells at least for 24 h, whereas most of the cells infected with wild-type virus mediated prominent cell-cell fusion within 24 h. On the other hand, the cells infected with the mutant virus, carrying either the H-83 or H-186 mutation, mediated cell-cell fusion but less efficiently than those infected with wild-type virus. Irrespective of the ability to cause cell-cell fusion, however, every virus could infect all the cells in the culture within 48 h after the initial infection. These results indicated that both the N83Y and M186I mutations in the HN protein are involved in the regulation of cell-cell fusion. Notably, the limited cell-cell fusion by H-83/186 virus was greatly promoted by lysophosphatidic acid, a stimulator of the Ras and Rho family GTPases.

The human parainfluenza virus type 2 (hPIV2) V protein plays important roles in inhibiting the host interferon response and promoting virus growth, but its role in hPIV2 replication and transcription is not clear. A green fluorescent protein (GFP)-expressing a negative-sense minigenomic construct of hPIV2 has been established by standard technology, with helper plasmids expressing the nucleocapsid protein (NP), phosphoprotein (P), and large RNA polymerase (L) protein, to examine the role of V protein. We found that the simultaneous expression of wild-type V protein in the minigenome system inhibited GFP expression, at least in part, by inhibiting minigenome replication. In contrast, expression of C terminally truncated or mutant hPIV2 V proteins had no effect. Moreover, the V protein of simian virus 41, the rubulavirus most closely related virus to hPIV2, also inhibited GFP expression, whereas that of PIV5, a more distantly related rubulavirus, did not. Using these other rubulavirus V proteins, as well as various mutant hPIV2 V proteins, we found that the ability of V protein to inhibit GFP expression correlated with its ability to bind to L protein via its C-terminal V protein-specific region, but there was no correlation with NP binding. A possible role for this inhibition of genome replication in promoting viral fitness is discussed.

When K562 cells were infected with Newcastle disease virus (NDV) or human parainfluenza type 2 virus (hPIV-2), polykaryocyte formation could not be detected. Failure of multinucleated giant cell formation in K562 cells infected with either NDV or hPIV-2 is due to disturbance of the viral envelope-cell fusion step or to defect in the cell-cell fusion step, respectively. Especially, NDV completely replicated in K562 cells, and the hemagglutinin-neuraminidase and fusion proteins expressed on the cell surface of NDV-infected K562 cell were fully functional for fusion inducing activity. Therefore, the cell membranes of K562 cells are considered to be resistant to virus-induced cell fusion. Membrane fusion is regulated by many host factors including membrane fluidity, cytoskeletal systems, and fusion regulatory proteins system. An unknown regulatory mechanism of virus-induced cell fusion may function on the cell surface of K562 cells.

Gene expression of nonsegmented negative-strand RNA viruses (nsNSVs) such as parainfluenza viruses requires the RNA synthesis activity of their polymerase L protein; however, the detailed mechanism of this process is poorly understood. In this study, a parainfluenza minireplicon assay expressing secretory Gaussia luciferase (Gluc) was established to analyze large protein (L) activity. Measurement of Gluc expression in the culture medium of cells transfected with the minigenome and viral polymerase components enabled quick and concise calculation of L activity. By comparing the amino acid sequences in conserved region III (CRIII), a putative polymerase-active domain of the L protein, two strictly conserved aspartates were identified in all families of nsNSV. A series of L mutants from human parainfluenza virus type 2 and parainfluenza virus type 5 showed that these aspartates are necessary for reporter gene expression. It was also confirmed that these aspartates are important for the production of viral mRNA and antigenome cRNA, but not for a polymerase-complex formation. These findings suggest that these two aspartates are key players in the nucleotidyl transfer reaction using two metal ions.

It is commonly accepted that the temperature-sensitive phenotype of Sendai virus (SeV) persistently infected cells is caused by the M and/or HN proteins. Expression level of the L, M, HN, and V proteins is extremely low in L929 cells persistently infected with SeVpi (L929/SeVpi cells) incubated at 38 degrees C. The HN protein quickly disappears in L929/SeVpi cells following a temperature shift up to 38 degrees C, and pulse-chase experiments show that the Lpi, HNpi, and Mpi proteins are unstable at 38 degrees C. Following a temperature shift either upward or downward, M protein is translocated into the nucleus and then localizes to the perinuclear region. None of virus-specific polypeptides are detected in the cells primarily infected with SeVpi and incubated at 38 degrees C and virus proteins are not pulse-labeled at 38 degrees C, indicating that temperature-sensitive step is at an early stage of infection. The Mpi protein is transiently located in the nucleus of the SeVpi primarily infected cells. Recombinant SeVs possessing the HNpi or/and Mpi proteins are not temperature-sensitive. The HN protein is expressed at very low levels and the F protein localizes to the perinuclear region in rSeV(Mpi)-infected cells incubated at 38 degrees C for 18 h. rSeVs having the Mpi protein exhibit lower cytotoxicity and are incapable of establishing persistent infection. Amino acid 116 of the Mpi protein is related to the nuclear translocation and lower cytopathogenesis, whereas aa183 is involved in the interaction between M protein and viral glycoproteins.

Human parainfluenza virus type 1 (HPIV1), a major cause of croup in infants and young children, accounts for 6% of hospitalizations for pediatric respiratory tract disease. The antigenically related Sendai virus, referred to here as murine PIV1 (MPIV1), is being considered for use as a live-attenuated vaccine to protect against HPIV1 (J. L. Hurwitz, K. F. Soike, M. Y., Sangster, A. Portner, R. E. Sealy, D. H. Dawson, and C. Coleclough, 1997, Vaccine 15(5), 533–540) and also as a recombinant vaccine vector expressing antigens to protect against viral disease in humans. However, in the 1950s MPIV1 was reported to have been isolated from humans, suggesting that zoonotic transmission might have occurred. It is therefore important to examine the ability of MPIV1 to replicate in nonhuman primates, i.e., surrogate hosts for humans. In the present study the level of replication of MPIV1 and HPIV1 was compared in African green monkeys and chimpanzees. Surprisingly, MPIV1 replicated as efficiently as HPIV1 in the upper and lower respiratory tract of African green monkeys at doses of 104 and 106 and replicated only slightly less efficiently at both sites in chimpanzees. African green monkeys immunized with MPIV1 were highly resistant to subsequent challenge with HPIV1 even though MPIV1 did not induce a detectable HPIV1-neutralizing antibody response. The high level of replication of MPIV1 observed in the upper and lower respiratory tract of these primates suggests that MPIV1 likely would require significant attenuation before it could be given to humans as a vaccine against HPIV1 or as a vaccine vector. Its ability to efficiently replicate in nonhuman primates suggests that MPIV1 lacks a significant host range restriction in primates and could theoretically cause zoonotic disease in humans.

Human parainfluenza type 2 virus (hPIV-2)-infected HeLa (HeLa-CA) cells and hPIV-2 V-expressing HeLa (HeLa-V) cells show high resistance to alpha/beta interferons (IFN-α/β) irrespective of whether vesicular stomatitis virus or Sindbis virus is used as a challenge virus. When Sindbis virus is used, these cells show high susceptibility to human IFN-γ. Furthermore, the multiplication of HeLa-V cells is not inhibited by IFN-α/β. HeLa cells expressing the N-terminally truncated V protein show resistance to IFN-α/β, showing that the IFN resistance determinant maps to the cysteine-rich V-specific domain. A complete defect of Stat2 is found in HeLa-CA and HeLa-V cells, whereas the levels of Stat1 expression are not significantly different among HeLa, HeLa-CA, HeLa-P, and HeLa-V cells, indicating that IFN-α/β resistance of HeLa-CA and HeLa-V cells is due to a defect of Stat2. HeLa-SV41V cells show high resistance to all IFNs, and no expression of Stat1 can be detected. Stat2 mRNA is fully detected in HeLa-V cells. Stat2 was scarcely pulse-labeled in the HeLa-V cells, indicating that synthesis of Stat2 is suppressed or Stat2 is very rapidly degraded in HeLa-V cells. The V protein suppresses the in vitro translation of Stat2 mRNA more extensively than that of Stat1 mRNA. An extremely small amount of Stat2 can be detected in HeLa-V cells treated with proteasome inhibitors. The half-life of Stat2 is approximately 3.5 and 2 h in uninfected and hPIV-2-infected HeLa cells, respectively. This study shows that synthesis of Stat2 may be suppressed and Stat2 degradation is also enhanced in hPIV-2-infected HeLa and HeLa-V cells.

Eleven monoclonal antibodies (MAbs) directed against the large (L) protein of human parainfluenza type 2 virus (hPIV-2) were prepared to examine the interactions of the L protein with other viral proteins. Coimmunoprecipitation assays using these MAbs revealed that the L protein directly interacted with the phospho- (P) and nucleocapsid (NP) proteins in vivo and in vitro. Mutational analysis of the P or NP protein was performed to identify the region(s) on these proteins interacting with L protein, indicating that amino acids 278–353 on the P protein and amino acids 403–494 on the NP protein are essential for the binding to the L protein.

Using a plasmid expression system in HeLa cells, we have previously shown that the fusion (F) protein of simian virus 41 (SV-41) induces cell fusion when coexpressed with the haemagglutinin-neuraminidase (HN) protein of human parainfluenza virus type 2 (PIV-2), while the PIV-2 F protein does not induce cell fusion with the SV-41 HN protein. In the present study, we found that the PIV-2 F protein induced extensive cell fusion with the HN protein of mumps virus (MuV), whereas the SV-41 F protein did not. Chimaeric analyses of the F proteins of PIV-2 and SV-41 identified two regions (designated M1 and M2) on the PIV-2 F protein, either of which was necessary for chimaeric F proteins to show fusogenic activity with the MuV HN protein. Subsequently, two additional regions (P1 and P2) were identified on the PIV-2 F protein, both of which were necessary for chimaeric F proteins to prevent induction of cell fusion with the SV-41 HN protein. Consequently, it was proved that a given chimaeric F protein, harbouring regions P1 and P2 together with either of region M1 or M2, induced cell fusion specifically with HN proteins of PIV-2 and MuV, the same as the PIV-2 F protein. Region M2 was located at the membrane proximal end of the PIV-2 F1 ectodomain, while regions P1, M1 and P2 clustered together in the middle of the ectodomain. These regions on the PIV-2 F protein may be involved in a putative functional interaction with HN proteins, which is considered to be a prerequisite for cell fusion.

Fusion regulatory protein-1 (FRP-1) regulates virus-mediated cell fusion and induces poly-karyocyte formation of monocytes without any fusogen. We have recently reported that FRP-1 and the 4F2/CD98 heavy chain are identical molecules. Cell fusion in Newcastle disease virus (NDV)-infected HeLa cells was enhanced when cells were incubated with anti-FRP-1 MAb. Anti-FRP-1 MAbs also induced human immunodeficiency virus gp160-mediated cell fusion. However, HBJ127, an anti-FRP-1/4F2/CD98 MAb that enhanced cell fusion in NDV-infected cells, delayed human parainfluenza virus type 2 (HPIV-2)-induced cell fusion in HeLa cells, although these viruses belong to the same genus Rubulavirus. No anti-FRP-1 MAbs enhanced cell fusion in HPIV-2-infected HeLa cells. Anti-FRP-1 MAbs including HBJ127 showed no effect on virus growth and expression levels of virus-specific poly-peptides in HPIV-2-infected HeLa cells, indicating that the delay in cell fusion by an anti-FRP-1 MAb is not due to suppression of virus replication. When HeLa cells were transfected with an expression vector harbouring HPIV-2 HN and F genes, cell fusion was also suppressed by HBJI27, but the effect was weak in comparison with virus-infected cells. These data indicate anti-FRP-1 antibodies not only induce/enhance, but also inhibit/delay virus-induced cell fusion and therefore FRP-1 molecules are multifunctional.

The paramyxovirus phospho- (P) and nucleocapsid (NP) proteins are involved in transcription and replication of the viral genome. To study the interaction between NP and P proteins, we established HeLa cell lines that constitutively expressed the NP and/or P proteins of human parainfluenza virus type 2 (hPIV-2). Co-immunoprecipitation assays revealed that the NP and P proteins can form complexes in HeLa cells expressing both proteins (HeLa-NP + P cells) and in mixed cell lysates of HeLa-NP and HeLa-P cells. Deletion mutant analysis of the P protein was performed to identify the regions of P protein that interact with NP protein. The results indicate that two independent NP-binding sites exist on P protein: one is located in the N-terminal part of the protein, aa 1–47, and the other in the C-terminal part, aa 357–395. In addition, cells co-expressing NP and P proteins with N-terminal deletions showed immunofluorescence staining patterns (granular pattern) similar to those found in hPIV-2-infected cells. However, cells co-expressing NP and P proteins with C-terminal deletions showed a different immunofluorescence staining pattern (diffuse pattern), indicating that the C-terminal region is required for granule formation.

cDNAs encoding human parainfluenza virus type 4A and type 4B (hPIV-4A and -4B) fusion (F) proteins were cloned and sequenced. The predicted amino acid sequences of the F proteins had similar characteristic traits to those reported for the F proteins of other paramyxoviruses. They were more closely related to the F proteins of simian virus 5 (SV5), mumps virus (MuV), hPIV-2 and Newcastle disease virus (NDV) than to the F proteins of hPIV-1, hPIV-3, Sendai virus (SV) and measles virus (MV). In addition, hPIV-4A, hPIV-4B, SV5 and MuV shared a common feature of genomic organization: there was a small ORF between the F and haemagglutinin-neuraminidase (HN)-coding sequences, implying a common ancestry.

Previously, we obtained a neutralizing monoclonal antibody directed against the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 2 virus (PIV2), which was able to prevent cell fusion without affecting the hemagglutinating and neuraminidase activities. In this study, four escape mutants of PIV2 have been obtained under pressure of the monoclonal antibody. Intriguingly, the HN protein of each mutant proved to have two amino acid substitutions, one of which is at 83Asn or 91Lys, and another one is at 150Leu, 160Ala, or 186Met. One mutant designated F13, which has substitutions at 83Asn and 186Met in the HN protein, could not cause cell fusion in HeLa cells despite its multiple replication, while the other mutants formed typical syncytial cells. The deduced amino acid sequence of F13 fusion (F) protein proved to be identical to that of wild-type F protein, and furthermore, protein expression analyses have revealed that the low-fusion phenotype of F13 was due to its mutated HN protein, whose antigenicity to the monoclonal antibody was abolished by the single mutation at 83Asn. These observations have suggested that the principal epitope for the monoclonal antibody resides in the presumed stalk domain of the HN protein, which may play an important role in promoting cell fusion.

For most paramyxoviruses, virus type-specific interaction between fusion (F) protein and attachment protein (hemagglutinin-neuraminidase [HN], hemagglutinin [H], or glycoprotein [G]) is a prerequisite for mediating virus-cell fusion and cell-cell fusion. Our previous cell-cell fusion assay using the chimeric F proteins of human parainfluenza virus 2 (HPIV2) and simian virus 41 (SV41) suggested that the middle region of the HPIV2 F protein contains the site(s) that determines its specificity for the HPIV2 HN protein. In the present study, we further investigated the sites of the F protein that could be critical for determining the HN protein specificity. By analyzing the reported structure of the F protein of parainfluenza virus 5 (PIV5), we found that four major domains (M1, M2, M3, and M4) and five minor domains (A to E) in the middle region of the PIV5 F protein were exposed on the trimer surface. We then replaced these domains with the SV41 F counterparts individually or in combination and examined whether the resulting chimeras could mediate cell-cell fusion when coexpressed with the SV41 HN protein. The results showed that a chimera designated M(1+2), which harbored SV41 F-derived domains M1 and M2, mediated cell-cell fusion with the coexpressed SV41 HN protein, suggesting that these domains are involved in determining the HN protein specificity. Intriguingly, another chimera which harbored the SV41 F-derived domain B in addition to domains M1 and M2 showed increased specificity for the SV41 HN protein compared to that of M(1+2), although it was capable of mediating cell-cell fusion by itself.

The complete nucleotide sequence of the simian virus 41 (SV41) large (L) protein gene was determined. The L gene spanned 6883 nucleotides including a putative trailer RNA, and the L mRNA contained a single large open reading frame encoding a polypeptide of 2269 amino acids. Dot-matrix comparisons under stringent conditions identified domains highly conserved among paramyxoviruses. Domain 3 is the most highly conserved, and has been hypothesized to be the RNA polymerase active site. A phylogenetic tree was constructed from the sequences of the L proteins of seven paramyxoviruses. SV41 was most closely related to human parainfluenza virus type 2 (HPIV-2), and SV41, HPIV-2 and SV5 form a subgroup. The intergenic sequences at the nucleocapsid protein-phosphoprotein and haemagglutinin-neuraminidase-L protein gene junctions, and the 5' trailer sequence of SV41 were also determined, and it was shown that the first 13 nucleotides of the 5' trailer sequence are complementary to those of the 3' leader sequence. The intergenic, and gene-start and -end sequences of SV41, HPIV-2 and SV5 are shown.

The nucleotide sequences of cDNAs of the simian virus 5 (SV5) nucleoprotein (NP) gene, and the 3' end of the genome and NP gene of SV41 were determined. The open reading frames of the SV5 and SV41 NP genes encode polypeptides with Mrs of 56,582 and 60,575, respectively, values which are consistent with those estimated by SDS-PAGE. The NP of human parainfluenza virus type 2 (hPIV-2) was more closely related to that of SV41 (amino acid sequence identity 70.5%) than that of SV5 (57.0%); the amino acid sequence identity between the NPs of SV41 and SV5 was 63.3%. The sequence of the 3' end of the genome of SV41 showed a high level of similarity to that of hPIV-2, the terminal 18 nucleotides being identical. It is concluded from these findings that SV41 is related most closely to hPIV-2, even though SV5 had been thought to be an animal type of hPIV-2.

Antigenic relationships of simian virus 41 (SV41) to other paramyxoviruses were examined by immunoprecipitation of isotope-labelled SV41-infected cell lysates with specific antisera. SV41 is closely related to the group comprising human parainfluenza virus 2 (HPIV-2), simian virus 5 (SV5), parainfluenza virus 4 and mumps virus. Slight cross-neutralization was detected between SV41, HPIV-2 and SV5. Anti-SV41 activities were detected in 21 of 1116 human serum specimens, indicating that a proportion of the human population is infected with SV41. The haemagglutinin-neuraminidase of SV41 was preferentially immunoprecipitated by anti-SV41 positive sera.

CD98 is a widely expressed cell surface heterodimeric protein of 125 kDa. Its expression is upregulated during lymphocyte activation induced by mitogen, superantigen, conventional antigen, and a combination of phorbol myristate acetate (PMA) and ionomycin. However, the role of CD98 in the immune system is not so well understood. The role of CD98 in murine T lymphocyte proliferation was investigated, especially in correlation with the interleukin 2 (IL-2)/interleukin 2 receptor (IL-2R) system. Monoclonal antibody (mAb) directed against murine CD98 heavy chain (mCD98HC) suppressed the proliferation of lymphocytes stimulated with concanavalin A (Con A). Anti-mCD98HC mAb did not suppress the expression of IL-2Ralpha. Anti-IL-2Ralpha mAb, which suppressed DNA synthesis, did not inhibit the expression of CD98HC. Murine IL-2 (recombinant), which induced considerable DNA synthesis by lymphocytes stimulated with a sub-optimal dose of Con A, did not induce CD98HC expression in lymphocytes. In addition, anti-mCD98HC mAb did not inhibit the production of IL-2 by lymphocyte stimulated with Con A. Taken together with these findings, it was speculated that the CD98 system is independent of the IL-2/IL-2R system in murine T lymphocyte activation.