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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 found that infection with human parainfluenza virus type 2 (hPIV-2), a member of the genus Orthorubulavirus, family Paramyxoviridae, causes filamentous actin (F-actin) formation to promote viral growth. In the present study, we investigated whether similar regulation of F-actin formation is observed in infections with other rubulaviruses, such as parainfluenza virus type 5 (PIV-5) and simian virus 41 (SV41). Infection with these viruses caused F-actin formation and RhoA activation, which promoted viral growth. These results indicate that RhoA-induced F-actin formation is important for efficient growth of these rubulaviruses. Only SV41 and hPIV-2 V and P proteins bound to Graf1, while the V and P proteins of PIV-5, mumps virus, and hPIV-4 did not bind to Graf1. In contrast, the V proteins of these rubulaviruses bound to both inactive RhoA and profilin 2. These results suggest that there are common and unique mechanisms involved in regulation of F-actin formation by members of the genus Orthorubulavirus.

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.

Hazara virus (HAZV) is closely related to Crimean-Congo hemorrhagic fever virus (CCHFV), but differs in that it is non-pathogenic to humans. Since HAZV was isolated for the first time in 1954, the biological characteristics of this virus, particularly its behavior within culture cells, have not been well-studied, despite its importance as a surrogate model for CCHFV. Nucleoprotein (N) is the main component of viral nucleocapsid and is the most abundant virion protein, it is believed to play a pivotal role in the viral lifecycle. Generation of a series of anti-HAZV N monoclonal antibodies has enabled us to directly examine the involvement of this protein on viral growth. Observation of HAZV-infected cells revealed that this infection caused apoptosis, which was further characterized by DNA ladder and elevated caspase-3/7 activity. HAZV titers initially increased in cell culture, but after reaching the peak titer began to rapidly decline. HAZV particles were found to be very unstable in culture medium at 37 °C, and virus particles tend to lose infectivity at that point. HAZV N appears to inhibit apoptosis, thus can potentially support efficient viral propagation.

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.

To prevent contamination when taking blood culture, there are various effective interventions. Whether there is greater efficacy by using a combination of these interventions has not been widely evaluated.Our six-element intervention bundle aimed to prevent contamination of blood culture in our emergency department (ED). Elements were: use of 1% chlorhexidine alcohol, alcohol wiping, hand hygiene, using sterile gloves, using holed sterile cover, and selection of upper extremities as the site of venipuncture. We compared the contamination rate of blood culture between the pre- and the post-intervention periods among all cases with two or more blood cultures taken in our ED. We also evaluated the rate of patients receiving vancomycin among all those transferred to the hospital from the ED.During the pre- and post-intervention periods, 460 and 450 cases were included in analysis, respectively. Contamination of blood culture occurred in 29 pre-intervention cases (6.3%) and five post-interventional cases (1.1%) (relative risk 0.18, 95% confidence interval 0.07 to 0.45; P 0.001). After bundle implementation, there was significant increase in adherence to using 1% chlorhexidine alcohol, alcohol wiping, hand hygiene, and using holed sterile covers. Among patients admitted to hospital, fewer patients received vancomycin during the post-intervention period than in the pre-intervention period (5.4% vs. 3.2%, P = 0.03).Our intervention bundle dramatically reduced the contamination rate when drawing blood culture in our ED.

Human parainfluenza virus type 2 (hPIV-2) proteins and genomes newly synthesized in the cytoplasm need to be transported to the plasma membrane where budding occurs. This mechanism, where Rab proteins regulate intracellular traffic by switching between GTP-bound active form and GDP-bound inactive form, is not fully understood. mRNA and protein expression levels of Rab8a, Rab11a, and Rab27a are not altered by hPIV-2 infection. hPIV-2 growth is affected by depletion of Rab27a but not Rab8a and Rab11a. Overexpression of a constitutively active mutant of Rab27a Q78L promotes the cell surface levels of fusion (F) and hemagglutinin-neuraminidase (HN) proteins in hPIV-2-infected cells without affecting viral mRNA levels. Increase in the cell surface level of F and HN proteins by Rab27a Q78L is noticeable when these proteins are coexpressed independent of hPIV-2 infection. Our results collectively suggest that the active form of Rab27a enhances hPIV-2 growth by promoting transport of F and HN proteins to the plasma membrane.

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.

Tetherin (BST-2/CD317/HM1.24) is an anti-viral factor that restricts the budding of several enveloped viruses. Most of these viruses have evolved to encode tetherin antagonists. Our previous study demonstrated that the growth of human parainfluenza virus type 2 (hPIV-2), a member of the genus Rubulavirus in the family Paramyxoviridae, was inhibited by tetherin, and its V protein decreases the amount of cell surface tetherin by the interaction. In the present study, we investigated whether tetherin inhibits the growth of other rubulaviruses including PIV-5, mumps virus (MuV), simian virus 41, and hPIV-4, and whether their V proteins act as tetherin antagonists. Plaque assay demonstrated that the growth of PIV-5 and MuV was inhibited by tetherin. Flow cytometry and immunoblot analyses revealed that the infection of PIV-5 and MuV caused reduction of cell surface tetherin without affecting total amount of tetherin. Immunoprecipitation analysis showed that all V proteins of rubulaviruses tested bound to tetherin. These results suggest that tetherin antagonism by V proteins is common among the genus Rubulavirus.

Tetherin is an anti-viral factor that restricts viral budding through tethering virions to the cell surface. The human parainfluenza virus type 2 (hPIV-2) V protein decreases cell surface tetherin in HeLa cells, which constitutively express endogenous tetherin. However, the role of the hPIV-2 V protein in tetherin induction remains unclear. Here, we examined whether hPIV-2 infection itself induces tetherin in HEK293 cells that have no basal expression of tetherin. Unlike influenza A virus (IAV) infection, hPIV-2 infection induced neither tetherin mRNA nor protein expression. In contrast, robust tetherin induction was observed by infection of rPIV-2s carrying V mutants, in which either three Trp residues (W178H/W182E/W192A) or Cys residues (C209/211/214A) that are important for decreasing cell surface tetherin are mutated. hPIV-2 infection also inhibited the induction of tetherin expression by IFN-α and IAV infection. Furthermore, hPIV-2 V protein but not P and VW178H/W182E/W192A suppressed tetherin induction. Our data collectively suggest that the hPIV-2 V protein inhibits tetherin expression induced by several external stimuli.

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.

The RNA genome of human parainfluenza virus type 2 (hPIV2) is encapsidated by nucleoprotein (NP) to act as a template for RNA synthesis. We examined the importance of individual amino acids in the RNA-binding domain of hPIV2 NP for polymerase activity using a mini-replicon assay. We showed that substitution of tyrosine at amino acid position 260, located in the RNA-binding pocket of NP, severely reduced polymerase activity. The aromatic side-chain of Y260 may be required for the formation of stable contacts between nucleotides and basic amino acids, thereby affecting promoter recognition by the viral polymerase.

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.

Leader sequence, located at the 3′ terminus of paramyxovirus genomes, determines the degree of viral transcription and replication. The essential nucleotides in the leader sequence that influence viral propagation, however, have not been investigated in detail. In the present study, we show that polymerase complex of human parainfluenza virus type 2 (hPIV2) uses a luciferase-encoding hPIV2 mini-genome possessing the leader sequence from other closely related viruses as a template. Furthermore, we demonstrate that although hPIV2 polymerase complex can recognize the leader sequence of hPIV4B, mumps virus (MuV) and PIV5 as well as Newcastle disease virus (NDV), it cannot recognize measles virus, hPIV1, Sendai virus (SeV) or hPIV3. We could obtain the chimeric hPIV2 possessing the leader sequence from hPIV4B, MuV and PIV5, but not from other species, including NDV and SeV. These results reveal that although hPIV2 polymerase complex can recognize the leader sequence from rubulaviruses to achieve efficient viral infection, this does not apply to viruses belonging to other genus. A comparison of leader sequence nucleotides among paramyxoviruses highlights the importance of the conservation in the first 13 nucleotides for infectious hPIV2 growth.

Hazara virus (HAZV) is a member of the genus Orthonairovirus of the family Nairoviridae. HAZV is closely related to Crimean-Congo hemorrhagic fever virus but differs in that it is non-pathogenic to humans. To establish an infection model system, we tested whether embryonated chicken eggs, which are classically used for evaluating viral pathogenicity, are susceptible to HAZV infection. We demonstrated that HAZV replicates well in embryonated chicken eggs and kills 100% of the embryos. This can be a valuable tool to evaluate the lethality of nairoviruses in a biosafety level 2 laboratory.

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.

Leptin receptor belongs to the class I cytokine receptor superfamily, which mediates multiple physiological roles in mammals. However, the leptin system is poorly understood in birds, as the evidence for the existence of a natural ligand of the receptor in birds is controversial. As part of a strategy to reveal the physiological significance of leptin in birds, we isolated a monoclonal antibody (mAb) against a chicken leptin receptor (chLEPR). Based on the cDNA sequence for chLEPR, a peptide coding for the cytoplasmic domain of chLEPR was expressed in Escherichia coli and this was used to immunize mice to obtain the mAb. The anti-chLEPR mAb recognized proteins migrated at approximately 180 kDa by Western blot analysis using cellular extracts prepared from COS-7 cells transfected with chLEPR expression vector. By Western blot analysis using the same mAb, an immunoreactive band migrated at 180 kDa was detected in the chicken brain and Leghorn male hepatoma (LMH) cells, and which was similar to the size observed in the in vitro transfection study. Taken together, the chLEPR mAb obtained in the present study cross-reacted, at least, with long isoform chLEPR, suggesting that LEPR mRNA expressed in chicken tissues is likely to be translated. The chLEPR mAb, which has not been described elsewhere, enables us to explore the expression and localization of the receptor in the chicken tissues at the protein level. Therefore, this antibody would be a powerful tool in studying and understanding the regulation and function of leptin and its receptors in birds.

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.

Membrane fusion is an important event in the functioning of a living organism. Life starts as a sperm fuses with the membrane of an egg, leading to its fertilization. Membrane fusion is also required for myogenesis, osteogenesis and placenta formation. Multinucleated giant cells originating from monocytes-macrophages are associated with granulomatous lesions formed in response to foreign bodies, viruses, and bacteria. The CD4 molecule acts as a receptor for HIV. The major virus envelope glycoprotein, gp120, attaches to CD4 molecules expressed on the host cell surface. After binding to CD4 on the target cells, HIV is internalized via direct, pH-independent fusion of the viral and cell membranes. However, attachment of HIV to CD4 on the target cells is not sufficient for fusion. Interaction of gp160-expressing cells with neighboring cells bearing surface CD4 molecules is also required for syncytium formation. Syncytium formation and subsequent generalized cell fusion have been reported as potentially important mechanisms of virus-induced cytotoxic effects. Some antibodies against CD98/FRP-1 suppressed virus-induced cell fusion and CD98-mediated cell fusion of monocytes, indicating that CD98/FRP-1 molecules are able to regulate cell fusion. In this study, the functional interaction between CD98 and CD147 was investigated. Three kinds (Ab1, 2, and 3) of anti-CD147 and three kinds of anti-CD98 antibodies were used. Ab1 suppressed CD98-mediated cell fusion, but showed no effect on cell aggregation of Cd(+)U2ME-7 cells, U937-2 cells expressing HIV gp160. On the other hand, Ab2 enhanced the CD98-mediated cell fusion. Ab1 showed suppressive effect at early stage and Ab2 showed enhancing effect at later stage. Ab2 and 3 suppressed the spontaneous cell agglutination and cell fusion of Cd(+)JME-2 cells, Jurkat cells expressing HIV gp160. Ab2 suppressed CD98-mediated cell fusion, but showed no effect on cell aggregation of Cd(+)JME-2 cells. Ab2 cancelled suppression of cell fusion induced by suppressive antibody against CD98. Ab2 and 3 also suppressed CD98-mediated cell fusion of monocytes. This study indicates the functional interaction between CD98 and CD147 in the regulation of cell fusion.

Introduction and aims: The human system l -amino acid transporter1 (hLAT1) is one of the CD98 light chains and its gene has been mapped to chromosome 16q24. Our preliminary findings have indicated that in HeLa S3 cells there are transcripts whose nucleotide sequences are very similar but not identical to that of the amino acid transporter. This study intends to examine whether these novel transcripts have biological significance through elucidating their genetic aspects and expression profiles in human cells. Methods: The expression levels of the transcripts were quantified by real-time PCR analysis. Chromosomal mapping of the gene was performed by fluorescence in situ hybridization (FISH). Results: Three types of transcripts were identified and their nucleotide sequences were aligned with the chromosome 16p12 clone with high identity. They encoded 180- or 190-amino acid proteins, showing 92–94% of amino acid identity to the amino-terminal region of the hLAT1 (507 amino acids). However, their 3′ non-coding sequences did not show homology to the nucleotide sequence of the amino acid transporter. Their genes were mapped to chromosome 16p11.2–p13.1 as low-copy repeats (LCRs). The transcription of one of these genes in peripheral blood mononuclear cells was significantly up-regulated when the cells were stimulated with concanavalin A. Conclusion: We have characterized the three truncated paralogs of the hLAT1 gene. It is suggested that the expression of one of these paralogs may play an important role in the activation of peripheral blood mononuclear cells.