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Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus responsible for adult T-cell leukemia/lymphoma, a severe and fatal CD4+ T-cell malignancy. Additionally, HTLV-1 can lead to a chronic progressive neurodegenerative disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis. Unfortunately, the prognosis for HTLV-1-related diseases is generally poor, and effective treatment options are limited. In this study, we designed and synthesized a codon optimized HTLV-1 envelope (Env) mRNA encapsulated in a lipid nanoparticle (LNP) and evaluated its efficacy as a vaccine candidate in an established rabbit model of HTLV-1 infection and persistence. Immunization regimens included a prime/boost protocol using Env mRNA-LNP or control green fluorescent protein (GFP) mRNA-LNP. After immunization, rabbits were challenged by intravenous injection with irradiated HTLV-1 producing cells. Three rabbits were partially protected and three rabbits were completely protected against HTLV-1 challenge. These rabbits were then rechallenged 15 weeks later, and two rabbits maintained sterilizing immunity. In Env mRNA-LNP immunized rabbits, proviral load and viral gene expression were significantly lower. After viral challenge in the Env mRNA-LNP vaccinated rabbits, an increase in both CD4+/IFN-γ+ and CD8+/IFN-γ+ T-cells was detected when stimulating with overlapping Env peptides. Env mRNA-LNP elicited a detectable anti-Env antibody response after prime/boost vaccination in all animals and significantly higher levels of neutralizing antibody activity. Neutralizing antibody activity was correlated with a reduction in proviral load. These findings hold promise for the development of preventive strategies and therapeutic interventions against HTLV-1 infection and its associated diseases.IMPORTANCEmRNA vaccine technology has proven to be a viable approach for effectively triggering immune responses that protect against or limit viral infections and disease. In our study, we synthesized a codon optimized human T-cell leukemia virus type 1 (HTLV-1) envelope (Env) mRNA that can be delivered in a lipid nanoparticle (LNP) vaccine approach. The HTLV-1 Env mRNA-LNP produced protective immune responses against viral challenge in a preclinical rabbit model. HTLV-1 is primarily transmitted through direct cell-to-cell contact, and the protection offered by mRNA vaccines in our rabbit model could have significant implications for optimizing the development of other viral vaccine candidates. This is particularly important in addressing the challenge of enhancing protection against infections that rely on cell-to-cell transmission., Competing Interests: The authors declare no conflict of interest.

Respiratory syncytial virus (RSV) has been reported to use CX3CR1 in vitro as a receptor on cultured primary human airway epithelial cultures. To evaluate CX3CR1 as the receptor for RSV in vivo , we used the cotton rat animal model because of its high permissiveness for RSV infection. Sequencing the cotton rat CX3CR1 gene revealed 91% amino acid similarity to human CX3CR1. Previous work found that RSV binds to CX3CR1 via its attachment glycoprotein (G protein) to infect primary human airway cultures. To determine whether CX3CR1-G protein interaction is necessary for RSV infection, recombinant RSVs containing mutations in the CX3CR1 binding site of the G protein were tested in cotton rats. In contrast to wild-type virus, viral mutants did not grow in the lungs of cotton rats. When RSV was incubated with an antibody blocking the CX3CR1 binding site of G protein and subsequently inoculated intranasally into cotton rats, no virus was found in the lungs 4 days postinfection. In contrast, growth of RSV was not affected after preincubation with heparan sulfate (the receptor for RSV on immortalized cell lines). A reduction in CX3CR1 expression in the cotton rat lung through the use of peptide-conjugated morpholino oligomers led to a 10-fold reduction in RSV titers at day 4 postinfection. In summary, these results indicate that CX3CR1 functions as a receptor for RSV in cotton rats and, in combination with data from human airway epithelial cell cultures, strongly suggest that CX3CR1 is a primary receptor for naturally acquired RSV infection. IMPORTANCE The knowledge about a virus receptor is useful to better understand the uptake of a virus into a cell and potentially develop antivirals directed against either the receptor molecule on the cell or the receptor-binding protein of the virus. Among a number of potential receptor proteins, human CX3CR1 has been demonstrated to act as a receptor for respiratory syncytial virus (RSV) on human epithelial cells in tissue culture. Here, we report that the cotton rat CX3CR1, which is similar to the human molecule, acts as a receptor in vivo . This study strengthens the argument that CX3CR1 is a receptor molecule for RSV.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract (LRT) infections, with increased severity in high-risk human populations, such as infants, the immunocompromised, and the elderly. Although the virus was identified more than 60 years ago, there is still no licensed vaccine available. Over the years, several vaccine delivery strategies have been evaluated. In this study, we developed two recombinant vesicular stomatitis virus (rVSV) vector-based vaccine candidates expressing the RSV-G (attachment) protein (rVSV-G) or F (fusion) protein (rVSV-F). All vectors were evaluated in the cotton rat animal model for their in vivo immunogenicity and protective efficacy against an RSV-A2 virus challenge. Intranasal (i.n.) delivery of rVSV-G and rVSV-F together completely protected the lower respiratory tract (lungs) at doses as low as 10 3 PFU. In contrast, doses greater than 10 6 PFU were required to protect the upper respiratory tract (URT) completely. Reimmunization of RSV-immune cotton rats was most effective with rVSV-F. In immunized animals, overall antibody responses were sufficient for protection, whereas CD4 and CD8 T cells were not necessary. A prime-boost immunization regimen increased both protection and neutralizing antibody titers. Overall, mucosally delivered rVSV-vector-based RSV vaccine candidates induce protective immunity and therefore represent a promising immunization regimen against RSV infection. IMPORTANCE Even after decades of intensive research efforts, a safe and efficacious RSV vaccine remains elusive. Expression of heterologous antigens from rVSV vectors has demonstrated several practical and safety advantages over other virus vector systems and live attenuated vaccines. In this study, we developed safe and efficacious vaccine candidates by expressing the two major immunogenic RSV surface proteins in rVSV vectors and delivering them mucosally in a prime-boost regimen. The main immune parameter responsible for protection was the antibody response. These vaccine candidates induced complete protection of both the upper and lower respiratory tracts., (Copyright © 2021 American Society for Microbiology.)

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in children of <5 years of age worldwide, infecting the majority of infants in their first year of life. Despite the widespread impact of this virus, no vaccine is currently available. For more than 50 years, live attenuated vaccines (LAVs) have been shown to protect against other childhood viral infections, offering the advantage of presenting all viral proteins to the immune system for stimulation of both B and T cell responses and memory. The RSV LAV candidate described here, rgRSV-L(G1857A)-G(L208A), contains two modifications: an attenuating mutation in the S -adenosylmethionine (SAM) binding site of the viral mRNA cap methyltransferase (MTase) within the large (L) polymerase protein and a mutation in the attachment (G) glycoprotein that inhibits its cleavage during production in Vero cells, resulting in virus with a "noncleaved G" (ncG). RSV virions containing the ncG have an increased ability to infect primary well-differentiated human bronchial epithelial (HBE) cultures which model the in vivo site of immunization, the ciliated airway epithelium. This RSV LAV candidate is produced efficiently in Vero cells, is highly attenuated in HBE cultures, efficiently induces neutralizing antibodies that are long lasting, and provides protection against an RSV challenge in the cotton rat, without causing enhanced disease. Similar results were obtained in a rhesus macaque. IMPORTANCE Globally, respiratory syncytial virus (RSV) is a major cause of death in children under 1 year of age, yet no vaccine is available. We have generated a novel RSV live attenuated vaccine candidate containing mutations in the L and G proteins. The L polymerase mutation does not inhibit virus yield in Vero cells, the cell type required for vaccine production, but greatly reduces virus spread in human bronchial epithelial (HBE) cultures, a logical in vitro predictor of in vivo attenuation. The G attachment protein mutation reduces its cleavage in Vero cells, thereby increasing vaccine virus yield, making vaccine production more economical. In cotton rats, this RSV vaccine candidate is highly attenuated at a dose of 10 5 PFU and completely protective following immunization with 500 PFU, 200-fold less than the dose usually used in such studies. It also induced long-lasting antibodies in cotton rats and protected a rhesus macaque from RSV challenge. This mutant virus is an excellent RSV live attenuated vaccine candidate., (Copyright © 2021 American Society for Microbiology.)

Human respiratory syncytial virus (RSV) is the leading viral cause of lower respiratory tract disease in infants and children worldwide. Currently, there are no FDA-approved vaccines to combat this virus. The large (L) polymerase protein of RSV replicates the viral genome and transcribes viral mRNAs. The L protein is organized as a core ring-like domain containing the RNA-dependent RNA polymerase and an appendage of globular domains containing an mRNA capping region and a cap methyltransferase region, which are linked by a flexible hinge region. Here, we found that the flexible hinge region of RSV L protein is tolerant to amino acid deletion or insertion. Recombinant RSVs carrying a single or double deletion or a single alanine insertion were genetically stable, highly attenuated in immortalized cells, had defects in replication and spread, and had a delay in innate immune cytokine responses in primary, well-differentiated, human bronchial epithelial (HBE) cultures. The replication of these recombinant viruses was highly attenuated in the upper and lower respiratory tracts of cotton rats. Importantly, these recombinant viruses elicited high levels of neutralizing antibody and provided complete protection against RSV replication. Taken together, amino acid deletions or insertions in the hinge region of the L protein can serve as a novel approach to rationally design genetically stable, highly attenuated, and immunogenic live virus vaccine candidates for RSV. IMPORTANCE Despite tremendous efforts, there are no FDA-approved vaccines for human respiratory syncytial virus (RSV). A live attenuated RSV vaccine is one of the most promising vaccine strategies for RSV. However, it has been a challenge to identify an RSV vaccine strain that has an optimal balance between attenuation and immunogenicity. In this study, we generated a panel of recombinant RSVs carrying a single and double deletion or a single alanine insertion in the large (L) polymerase protein that are genetically stable, sufficiently attenuated, and grow to high titer in cultured cells, while retaining high immunogenicity. Thus, these recombinant viruses may be promising vaccine candidates for RSV., (Copyright © 2020 American Society for Microbiology.)

A clinical isolate of measles virus (MeV) bearing a single amino acid alteration in the viral fusion protein (F; L454W) was previously identified in two patients with lethal sequelae of MeV central nervous system (CNS) infection. The mutation dysregulated the viral fusion machinery so that the mutated F protein mediated cell fusion in the absence of known MeV cellular receptors. While this virus could feasibly have arisen via intrahost evolution of the wild-type (wt) virus, it was recently shown that the same mutation emerged under the selective pressure of small-molecule antiviral treatment. Under these conditions, a potentially neuropathogenic variant emerged outside the CNS. While CNS adaptation of MeV was thought to generate viruses that are less fit for interhost spread, we show that two animal models can be readily infected with CNS-adapted MeV via the respiratory route. Despite bearing a fusion protein that is less stable at 37°C than the wt MeV F, this virus infects and replicates in cotton rat lung tissue more efficiently than the wt virus and is lethal in a suckling mouse model of MeV encephalitis even with a lower inoculum. Thus, either during lethal MeV CNS infection or during antiviral treatment in vitro , neuropathogenic MeV can emerge, can infect new hosts via the respiratory route, and is more pathogenic (at least in these animal models) than wt MeV. IMPORTANCE Measles virus (MeV) infection can be severe in immunocompromised individuals and lead to complications, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE) occur even in the face of an intact immune response. While they are relatively rare complications of MeV infection, MIBE and SSPE are lethal. This work addresses the hypothesis that despite a dysregulated viral fusion complex, central nervous system (CNS)-adapted measles virus can spread outside the CNS within an infected host., (Copyright © 2019 American Society for Microbiology.)

Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F-derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The self-assembly feature enhances biodistribution and the half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides., Importance: Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. We show here that specific biophysical properties regulate the in vivo efficacy of MV F-derived peptides., (Copyright © 2016 American Society for Microbiology.)

Unlabelled: Human metapneumovirus (hMPV) is a major causative agent of upper- and lower-respiratory-tract infections in infants, the elderly, and immunocompromised individuals worldwide. Like all pneumoviruses, hMPV encodes the zinc binding protein M2-1, which plays important regulatory roles in RNA synthesis. The M2-1 protein is phosphorylated, but the specific role(s) of the phosphorylation in viral replication and pathogenesis remains unknown. In this study, we found that hMPV M2-1 is phosphorylated at amino acid residues S57 and S60. Subsequent mutagenesis found that phosphorylation is not essential for zinc binding activity and oligomerization, whereas inhibition of zinc binding activity abolished the phosphorylation and oligomerization of the M2-1 protein. Using a reverse genetics system, recombinant hMPVs (rhMPVs) lacking either one or both phosphorylation sites in the M2-1 protein were recovered. These recombinant viruses had a significant decrease in both genomic RNA replication and mRNA transcription. In addition, these recombinant viruses were highly attenuated in cell culture and cotton rats. Importantly, rhMPVs lacking phosphorylation in the M2-1 protein triggered high levels of neutralizing antibody and provided complete protection against challenge with wild-type hMPV. Collectively, these data demonstrated that phosphorylation of the M2-1 protein upregulates hMPV RNA synthesis, replication, and pathogenesis in vivo, Importance: The pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Currently, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional antitermination factor. In this work, we found that phosphorylation of M2-1 is essential for virus replication and pathogenesis in vivo Recombinant hMPVs lacking phosphorylation in M2-1 exhibited limited replication in the upper and lower respiratory tract and triggered strong protective immunity in cotton rats. This work highlights the important role of M2-1 phosphorylation in viral replication and that inhibition of M2-1 phosphorylation may serve as a novel approach to develop live attenuated vaccines as well as antiviral drugs for pneumoviruses., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Human parainfluenza viruses (HPIVs) cause widespread respiratory infections, with no vaccines or effective treatments. We show that the molecular determinants for HPIV3 growth in vitro are fundamentally different from those required in vivo and that these differences impact inhibitor susceptibility. HPIV infects its target cells by coordinated action of the hemagglutinin-neuraminidase receptor-binding protein (HN) and the fusion envelope glycoprotein (F), which together comprise the molecular fusion machinery; upon receptor engagement by HN, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. Peptides derived from key regions of F can potently inhibit HPIV infection at the entry stage, by interfering with the structural transition of F. We show that clinically circulating viruses have fusion machinery that is more stable and less readily activated than viruses adapted to growth in culture. Fusion machinery that is advantageous for growth in human airway epithelia and in vivo confers susceptibility to peptide fusion inhibitors in the host lung tissue or animal, but the same fusion inhibitors have no effect on viruses whose fusion glycoproteins are suited for growth in vitro. We propose that for potential clinical efficacy, antivirals should be evaluated using clinical isolates in natural host tissue rather than lab strains of virus in cultured cells. The unique susceptibility of clinical strains in human tissues reflects viral inhibition in vivo., Importance: Acute respiratory infection is the leading cause of mortality in young children under 5 years of age, causing nearly 20% of childhood deaths worldwide each year. The paramyxoviruses, including human parainfluenza viruses (HPIVs), cause a large share of these illnesses. There are no vaccines or drugs for the HPIVs. Inhibiting entry of viruses into the human cell is a promising drug strategy that blocks the first step in infection. To develop antivirals that inhibit entry, it is critical to understand the first steps of infection. We found that clinical viruses isolated from patients have very different entry properties from those of the viruses generally studied in laboratories. The viral entry mechanism is less active and more sensitive to fusion inhibitory molecules. We propose that to interfere with viral infection, we test clinically circulating viruses in natural tissues, to develop antivirals against respiratory disease caused by HPIVs., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Unlabelled: The paramyxoviruses human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (hPIV3) are responsible for the majority of pediatric respiratory diseases and inflict significant economic loss, health care costs, and emotional burdens. Despite major efforts, there are no vaccines available for these viruses. The conserved region VI (CR VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at positions guanine N-7 (G-N-7) and ribose 2'-O. In this study, we generated a panel of recombinant hMPVs carrying mutations in the S-adenosylmethionine (SAM) binding site in CR VI of L protein. These recombinant viruses were specifically defective in ribose 2'-O methylation but not G-N-7 methylation and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of cotton rats. Importantly, vaccination of cotton rats with these recombinant hMPVs (rhMPVs) with defective MTases triggered a high level of neutralizing antibody, and the rats were completely protected from challenge with wild-type rhMPV. Collectively, our results indicate that (i) amino acid residues in the SAM binding site in the hMPV L protein are essential for 2'-O methylation and (ii) inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for hMPV and perhaps other paramyxoviruses, such as hRSV and hPIV3., Importance: Human paramyxoviruses, including hRSV, hMPV, and hPIV3, cause the majority of acute upper and lower respiratory tract infections in humans, particularly in infants, children, the elderly, and immunocompromised individuals. Currently, there is no licensed vaccine available. A formalin-inactivated vaccine is not suitable for these viruses because it causes enhanced lung damage upon reinfection with the same virus. A live attenuated vaccine is the most promising vaccine strategy for human paramyxoviruses. However, it remains a challenge to identify an attenuated virus strain that has an optimal balance between attenuation and immunogenicity. Using reverse genetics, we generated a panel of recombinant hMPVs that were specifically defective in ribose 2'-O methyltransferase (MTase) but not G-N-7 MTase. These MTase-defective hMPVs were genetically stable and sufficiently attenuated but retained high immunogenicity. This work highlights a critical role of 2'-O MTase in paramyxovirus replication and pathogenesis and a new avenue for the development of safe and efficacious live attenuated vaccines for hMPV and other human paramyxoviruses., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Human norovirus (NoV) accounts for 95% of nonbacterial gastroenteritis worldwide. Currently, there is no vaccine available to combat human NoV as it is not cultivable and lacks a small-animal model. Recently, we demonstrated that recombinant vesicular stomatitis virus (rVSV) expressing human NoV capsid protein (rVSV-VP1) induced strong immunities in mice (Y. Ma and J. Li, J. Virol. 85:2942-2952, 2011). To further improve the safety and efficacy of the vaccine candidate, heat shock protein 70 (HSP70) was inserted into the rVSV-VP1 backbone vector. A second construct was generated in which the firefly luciferase (Luc) gene was inserted in place of HSP70 as a control for the double insertion. The resultant recombinant viruses (rVSV-HSP70-VP1 and rVSV-Luc-VP1) were significantly more attenuated in cell culture and viral spread in mice than rVSV-VP1. At the inoculation dose of 1.0 × 10(6) PFU, rVSV-HSP70-VP1 triggered significantly higher vaginal IgA than rVSV-VP1 and significantly higher fecal and vaginal IgA responses than rVSV-Luc-VP1, although serum IgG and T cell responses were similar. At the inoculation dose of 5.0 × 10(6) PFU, rVSV-HSP70-VP1 stimulated significantly higher T cell, fecal, and vaginal IgA responses than rVSV-VP1. Fecal and vaginal IgA responses were also significantly increased when combined vaccination of rVSV-VP1 and rVSV-HSP70 was used. Collectively, these data indicate that (i) insertion of an additional gene (HSP70 or Luc) into the rVSV-VP1 backbone further attenuates the VSV-based vaccine in vitro and in vivo, thus improving the safety of the vaccine candidate, and (ii) HSP70 enhances the human NoV-specific mucosal and T cell immunities triggered by a VSV-based human NoV vaccine., Importance: Human norovirus (NoV) is responsible for more than 95% of acute nonbacterial gastroenteritis worldwide. Currently, there is no vaccine for this virus. Development of a live attenuated vaccine for human NoV has not been possible because it is uncultivable. Thus, a live vector-based vaccine may provide an alternative vaccine strategy. In this study, we developed a vesicular stomatitis virus (VSV)-based human NoV vaccine candidate. We constructed rVSV-HSP70-VP1, coexpressing heat shock protein (HSP70) and capsid (VP1) genes of human NoV, and rVSV-Luc-VP1, coexpressing firefly luciferase (Luc) and VP1 genes. We found that VSVs with a double gene insertion were significantly more attenuated than VSV with a single VP1 insertion (rVSV-VP1). Furthermore, we found that coexpression or coadministration of HSP70 from VSV vector significantly enhanced human NoV-specific mucosal immunity. Collectively, we developed an improved live vectored vaccine candidate for human NoV which will be useful for future clinical studies.

Unlabelled: Human metapneumovirus (hMPV) is a relatively recently identified paramyxovirus that causes acute upper and lower respiratory tract infection. Entry of hMPV is unusual among the paramyxoviruses, in that fusion is accomplished by the fusion (F) protein without the attachment glycoprotein (G protein). It has been suggested that hMPV F protein utilizes integrin αvβ1 as a cellular receptor. Consistent with this, the F proteins of all known hMPV strains possess an integrin-binding motif ((329)RGD(331)). The role of this motif in viral entry, infectivity, and pathogenesis is poorly understood. Here, we show that α5β1 and αv integrins are essential for cell-cell fusion and hMPV infection. Mutational analysis found that residues R329 and G330 in the (329)RGD(331) motif are essential for cell-cell fusion, whereas mutations at D331 did not significantly impact fusion activity. Furthermore, fusion-defective RGD mutations were either lethal to the virus or resulted in recombinant hMPVs that had defects in viral replication in cell culture. In cotton rats, recombinant hMPV with the R329K mutation in the F protein (rhMPV-R329K) and rhMPV-D331A exhibited significant defects in viral replication in nasal turbinates and lungs. Importantly, inoculation of cotton rats with these mutants triggered a high level of neutralizing antibodies and protected against hMPV challenge. Taken together, our data indicate that (i) α5β1 and αv integrins are essential for cell-cell fusion and viral replication, (ii) the first two residues in the RGD motif are essential for fusion activity, and (iii) inhibition of the interaction of the integrin-RGD motif may serve as a new target to rationally attenuate hMPV for the development of live attenuated vaccines., Importance: Human metapneumovirus (hMPV) is one of the major causative agents of acute respiratory disease in humans. Currently, there is no vaccine or antiviral drug for hMPV. hMPV enters host cells via a unique mechanism, in that viral fusion (F) protein mediates both attachment and fusion activity. Recently, it was suggested that hMPV F protein utilizes integrins as receptors for entry via a poorly understood mechanism. Here, we show that α5β1 and αv integrins are essential for hMPV infectivity and F protein-mediated cell-cell fusion and that the integrin-binding motif in the F protein plays a crucial role in these functions. Our results also identify the integrin-binding motif to be a new, attenuating target for the development of a live vaccine for hMPV. These findings not only will facilitate the development of antiviral drugs targeting viral entry steps but also will lead to the development new live attenuated vaccine candidates for hMPV.

The major inducible 70-kDa heat shock protein (hsp70) is host protective in a mouse model of measles virus (MeV) brain infection. Transgenic constitutive expression of hsp70 in neurons, the primary target of MeV infection, abrogates neurovirulence in neonatal H-2(d) congenic C57BL/6 mice. A significant level of protection is retained after depletion of T lymphocytes, implicating innate immune mechanisms. The focus of the present work was to elucidate the basis for hsp70-dependent innate immunity using this model. Transcriptome analysis of brains from transgenic (TG) and nontransgenic (NT) mice 5 days after infection identified type I interferon (IFN) signaling, macrophage activation, and antigen presentation as the main differences linked to survival. The pivotal role of type I IFN in hsp70-mediated protection was demonstrated in mice with a genetically disrupted type I IFN receptor (IFNAR(-/-)), where IFNAR(-/-) eliminated the difference in survival between TG and NT mice. Brain macrophages, not neurons, are the predominant source of type I IFN in the virus-infected brain, and in vitro studies provided a mechanistic basis by which MeV-infected neurons can induce IFN-β in uninfected microglia in an hsp70-dependent manner. MeV infection induced extracellular release of hsp70 from mouse neuronal cells that constitutively express hsp70, and extracellular hsp70 induced IFN-β transcription in mouse microglial cells through Toll-like receptors 2 and 4. Collectively, our results support a novel axis of type I IFN-dependent antiviral immunity in the virus-infected brain that is driven by hsp70.

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are closely related but pathogenically distinct human retroviruses. The antisense strand of the HTLV-1 genome encodes HTLV-1 basic leucine zipper (b-ZIP) protein (HBZ), a protein that inhibits Tax-mediated viral transcription, enhances T-cell proliferation, and promotes viral persistence. Recently, an HTLV-2 antisense viral protein (APH-2) was identified. Despite its lack of a typical b-ZIP domain, APH-2, like HBZ, interacts with cyclic AMP response element binding protein (CREB) and downregulates Tax-mediated viral transcription. Here, we provide evidence that the APH-2 C-terminal LXXLL motif is important for CREB binding and Tax repression. In order to investigate the functional role of APH-2 in the HTLV-2-mediated immortalization of primary T lymphocytes in vitro and in HTLV-2 infection in vivo, we generated APH-2 mutant viruses. In cell cultures, the immortalization capacities of APH-2 mutant viruses were indistinguishable from that of wild-type HTLV-2 (wtHTLV-2), indicating that, like HBZ, APH-2 is dispensable for viral infection and cellular transformation. In vivo, rabbits inoculated with either wtHTLV-2 or APH-2 mutant viruses established a persistent infection. However, the APH-2 knockout virus displayed an increased replication rate, as measured by an increased viral antibody response and a higher proviral load. In contrast to HTLV-1 HBZ, we show that APH-2 is dispensable for the establishment of an efficient infection and persistence in a rabbit animal model. Therefore, antisense proteins of HTLV-1 and HTLV-2 have evolved different functions in vivo, and further comparative studies will provide fundamental insights into the distinct pathobiologies of these two viruses.

Measles virus (MV) vaccine effectively protects seronegative individuals against infection. However, inhibition of vaccine-induced seroconversion by maternal antibodies after vaccination remains a problem, as it leaves infants susceptible to MV infection. In cotton rats, passive transfer of MV-specific IgG mimics maternal antibodies and inhibits vaccine-induced seroconversion. Here, we report that immunization in the presence of passively transferred IgG inhibits the secretion of neutralizing antibodies but not the generation of MV-specific B cells. This finding suggested that MV-specific B cells require an additional stimulus to mature into antibody-secreting plasma cells. In order to provide such a stimulus, we generated a recombinant Newcastle disease virus (NDV) expressing the MV hemagglutinin (NDV-H). In contrast to MV, NDV-H induced high levels of type I interferon in plasmacytoid dendritic cells and in lung tissue. In cotton rats immunized with NDV-H, neutralizing antibodies were also generated in the presence of passively transferred antibodies. In the latter case, however, the level and kinetics of antibody generation were reduced. In vitro, alpha interferon stimulated the activation of MV-specific B cells from MV-immune spleen cells. NDV infection (which induces alpha interferon) had the same effect, and stimulation could be abrogated by antibodies neutralizing alpha interferon, but not interleukin 6 (IL-6). In vivo, coapplication of UV-inactivated MV with NDV led to increased MV-specific antibody production in the presence and absence of passively transferred antibodies. These data indicate that MV-specific B cells are being generated after immunization in the presence of maternal antibodies and that the provision of alpha interferon as an additional signal leads to antibody secretion.

Many RNA and DNA viruses activate serine-threonine kinase AKT to increase virus replication. In contrast, measles virus (MV) infection leads to downregulation of AKT. This is thought to be beneficial for the virus because it correlates with immune suppression. To determine whether this is a sacrifice for the virus, we used a recombinant virus and transfected cells expressing constitutively active AKT and evaluated its effect on virus replication. In vitro, overexpression of AKT did not influence virus replication but did affect (cell-type dependent) virus release. In vivo, the recombinant virus did not abrogate inhibition of proliferation of spleen cells from MV-infected cotton rats.

Three discrete activities of the paramyxovirus hemagglutinin-neuraminidase (HN) protein, receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein, each affect the promotion of viral fusion and entry. For human parainfluenza virus type 3 (HPIV3), the effects of specific mutations that alter these functions of the receptor-binding protein have been well characterized using cultured monolayer cells, which have identified steps that are potentially relevant to pathogenesis. In the present study, proposed mechanisms that are relevant to pathogenesis were tested in natural host cell cultures, a model of the human airway epithelium (HAE) in which primary HAE cells are cultured at an air-liquid interface and retain functional properties. Infection of HAE cells with wild-type HPIV3 and variant viruses closely reflects that seen in an animal model, the cotton rat, suggesting that HAE cells provide an ideal system for assessing the interplay of host cell and viral factors in pathogenesis and for screening for inhibitory molecules that would be effective in vivo. Both HN's receptor avidity and the function and timing of F activation by HN require a critical balance for the establishment of ongoing infection in the HAE, and these HN functions independently modulate the production of active virions. Alterations in HN's F-triggering function lead to the release of noninfectious viral particles and a failure of the virus to spread. The finding that the dysregulation of F triggering prohibits successful infection in HAE cells suggests that antiviral strategies targeted to HN's F-triggering activity may have promise in vivo.

Measles virus infection leads to immune suppression. A potential mechanism is the reduction of interleukin 12 (IL-12) secretion during acute measles, resulting in a TH2 response. Studies in humans have reported conflicting results, detecting either a TH2 or a TH1 response. We have investigated the correlation between a TH2 response and immune suppression in specific-pathogen-free inbred cotton rats which were infected with measles vaccine and wild-type viruses. After infection of bone marrow-derived macrophages with wild-type virus, IL-12 secretion was reduced in contrast to the level for vaccine virus infection. In bronchoalveolar lavage cells, IL-12 secretion was suppressed after infection with both wild-type and vaccine virus on days 2, 4, and 6 and was detectable on days 8 and 10. After stimulation of mediastinal lymph node and spleen cells with UV-inactivated measles virus at various time points after infection, gamma interferon but no IL-4 was found. After stimulation with phorbol myristate acetate-ionomycin, high gamma interferon and low IL-4 levels were detected. To investigate whether the secretion of IL-4 contributes to immune suppression, a recombinant vaccine virus was created which secretes cotton rat IL-4. After infection with this recombinant virus, IL-4 secretion was enhanced. However, neither inhibition of concanavalin A-stimulated spleen cells nor keyhole limpet hemocyanin-specific proliferation of spleen cells was altered after infection with the recombinant virus in comparison to the levels with the parental virus. Our data indicate that measles virus infection leads to a decrease in IL-12 secretion and an increase in IL-4 secretion, but this does not seem to correlate with immune suppression.

In vitro studies show that hsp70 promotes gene expression for multiple viral families, although there are few reports on the in vivo significance of virus-hsp70 interaction. Previously we showed that hsp70-dependent stimulation of Edmonston measles virus (Ed MeV) transcription caused an increased cytopathic effect and mortality in transgenic hsp70-overexpressing C57BL/6 mice (H-2(b)). The response to MeV infection is influenced by the major histocompatibility complex haplotype; H-2(d) mice are resistant to brain infection due to robust antiviral immune responses, whereas H-2(b) mice are susceptible due to deficiencies in this response. We therefore tested the hypothesis that the outcome of MeV-hsp70 interaction may be dependent upon the host H-2 haplotype. The impact of selective neuronal hsp70 overexpression on Ed MeV brain infection was tested with congenic C57BL/10 H-2(d) neonatal mice. In this context, hsp70 overexpression conferred complete protection against virus-induced mortality, compared to >30% mortality in nontransgenic mice. Selective depletion of T-cell populations showed that transgenic mice exhibit a diminished reliance on T cells for protection. Brain transcript analysis indicated enhanced innate immune activation and signaling through Toll-like receptors 2 and 4 at early times postinfection for transgenic infected mice relative to those for nontransgenic infected mice. Collectively, results suggest that hsp70 can enhance innate antiviral immunity through Toll-like receptor signaling, supporting a protective role for physiological responses that enhance tissue levels of hsp70 (e.g., fever), and that the H-2 haplotype determines the effectiveness of this response.

Acute measles in children can be prevented by immunization with the live attenuated measles vaccine virus. Although immunization is able to induce CD4 and CD8 T cells as well as neutralizing antibodies, only the latter have been correlated with protective immunity. CD8 T cells, however, have been documented to be important in viral clearance in the respiratory tract, whereas CD4 T cells have been shown to be protective in a mouse encephalitis model. In order to investigate the CD4 T-cell response in infection of the respiratory tract, we have defined a T-cell epitope in the hemagglutinin (H) protein for immunization and developed a monoclonal antibody for depletion of CD4 T cells in the cotton rat model. Although the kinetics of CD4 T-cell development correlated with clearance of virus, the depletion of CD4 T cells during the primary infection did not influence viral titers in lung tissue. Immunization with the H epitope induced a CD4 T-cell response but did not protect against infection. Immunization in the presence of maternal antibodies resulted in the development of a CD4 T-cell response which (in the absence of neutralizing antibodies) did not protect against infection. In summary, CD4 T cells do not seem to protect against infection after immunization and do not participate in clearance of virus infection from lung tissue during measles virus infection. We speculate that the major role of CD4 T cells is to control and clear virus infection from other affected organs like the brain.

Transient hyperthermia such as that experienced during febrile episodes increases expression of the major inducible 70-kDa heat shock protein (hsp72). Despite the relevance of febrile episodes to viral pathogenesis and the multiple in vitro roles of heat shock proteins in viral replication and gene expression, the in vivo significance of virus-heat shock protein interactions is unknown. The present work determined the in vivo relationship between hsp72 levels and neurovirulence of an hsp72-responsive virus using the mouse model of measles virus (MV) encephalitis. Transgenic C57BL/6 mice were created to constitutively overexpress hsp72 in neurons, and these mice were inoculated intracranially with Edmonston MV (Ed MV) at 42 h of age. The mean viral RNA burden in brain was approximately 2 orders of magnitude higher in transgenic animals than in nontransgenic animals 2 to 4 weeks postinfection, and this increased burden was associated with a fivefold increase in mortality. Mice were also challenged with an Ed MV variant exhibiting an attenuated in vitro response to hsp72-dependent stimulation of viral transcription (Ed N-522D). This virus exhibited an attenuated neuropathogenicity in transgenic mice, where mortality and viral RNA burdens were not significantly different from nontransgenic mice infected with either Ed N-522D or parent Ed MV. Collectively, these results indicate that hsp72 levels can serve as a host determinant of viral neurovirulence in C57BL/6 mice, reflecting the direct influence of hsp72 on viral gene expression.

The major inducible 70-kDa heat shock protein (hsp72) increases measles virus (MV) transcription and genome replication. This stimulatory effect is attributed to hsp72 interaction with two highly conserved hydrophobic domains in the nucleocapsid protein (N) C terminus of Edmonston MV. These domains are known as Box-2 and Box-3. A single amino acid substitution in Box-3 of Edmonston MV (i.e., N522D) disrupts hsp72 binding. The prevalence of the N522D substitution in contemporary wild-type MV isolates suggests that this sequence has been positively selected. The present work determined if the N522D substitution enhances viral fitness and the degree to which any fitness advantage is influenced by hsp72 levels. Both parent Edmonston MV (Ed N) and an N522D substitution mutant (Ed N-522D) exhibited similar growth on Vero and murine neuroblastoma cells and in cotton rat lung, although Ed N-522D virus exhibited an attenuated in vitro response to hsp72 overexpression. In contrast, mixed infections showed a significantly reduced in vitro and in vivo fitness of Ed N-522D virus. Results support the involvement of additional selectional pressures that maintain the circulation of virus containing N-522D despite the cost to viral fitness.

Infection of humans with wild-type measles virus leads to strong immune suppression and secondary infections, whereas immunization with an attenuated vaccine strain does not. Using the cotton rat model (Sigmodon hispidus), we investigated whether vaccine and wild-type viruses differ in viral spread and whether this is correlated with inhibition of of proliferation of spleen cells ex vivo after mitogen stimulation. After intranasal infection of cotton rats with wild-type and vaccine strains, it was found that wild-type virus replicates better in lung tissue, spreads to the mediastinal lymph nodes, and induces a more pronounced and longer-lasting inhibition of proliferation of spleen cells ex vivo after mitogen stimulation than does vaccine virus. To induce the same degree of proliferation inhibition, 1,000-fold less wild-type virus was required than vaccine virus. With this system, the virulence of various measles virus isolates and recombinant viruses was tested. Four (in humans and/or monkeys) highly pathogenic virus strains were immunosuppressive, whereas viruses of vaccine virus genotype A were not. Using virus pairs which, due to passage on fibroblasts versus lymphoid cells or due to a point mutation in the hemagglutinin (N481 --> Y), differed in their usage of the two receptor molecules CD46 and CD150 on human cells, it was found that viruses using exclusively CD150 in vitro spread to mediastinal lymph nodes and induced strong immune suppression. These data demonstrate that important parameters of virulence seen in humans, such as viral spread and immune suppression, are reflected in the cotton rat model.

Measles virus (MV) strain CAM/RB, which was adapted to growth in the brain of newborn rodents, is highly neurovirulent. It has been reported earlier that experimentally selected virus variants escaping from the monoclonal antibodies (MAbs) Nc32 and L77 to hemagglutinin (H) preserved their neurovirulence, whereas mutants escaping MAbs K71 and K29 were found to be strongly attenuated (U. G. Liebert et al., J. Virol. 68:1486-1493, 1994). To investigate the molecular basis of these findings, we have generated a panel of recombinant MVs expressing the H protein from CAM/RB and introduced the amino acid substitutions thought to be responsible for antibody escape and/or neurovirulence. Using these recombinant viruses, we identified the amino acid changes conferring escape from the MAbs L77 (377R-->Q and 378M-->K), Nc32 (388G-->S), K71 (492E-->K and 550S-->P), and K29 (535E-->G). When the corresponding recombinant viruses were tested in brains of newborn rodents, we found that the mutations mediating antibody escape did not confer differential neurovirulence. In contrast, however, replacement of two different amino acids, at positions 195G-->R and 200S-->N, which had been described for the escape mutant set, caused the change in neurovirulence. Thus, antibody escape and neurovirulence appear not to be associated with the same structural alterations of the MV H protein.

BALB/c mice are resistant to measles virus (MV)-induced encephalitis due to their strong MV-specific CD4(+) T-cell response. Resistance is broken by neutralization of gamma interferon with monoclonal antibodies, indicating an important role for this pleiotropic cytokine. Here, we demonstrate that mouse gamma interferon has no direct antiviral effect in vitro and in vivo. The breakdown of resistance is due neither to a switch in the T-helper response nor to an impaired migration of CD4(+) T cells. Neutralization of gamma interferon interferes with the major histocompatibility complex class II-dependent antigen presentation and subsequent proliferation of CD4(+) T cells in vitro and in vivo. In consequence, the reduction in numbers of CD4(+) T cells below a protective threshold leads to susceptibility to MV-induced encephalitis.

In humans, maternal antibodies inhibit successful immunization against measles, because they interfere with vaccine-induced seroconversion. We have investigated this problem using the cotton rat model (Sigmodon hispidus). As in humans, passively transferred antibodies inhibit the induction of measles virus (MV)-neutralizing antibodies and protection after immunization with MV. In contrast, a recombinant vesicular stomatitis virus (VSV) expressing the MV hemagglutinin (VSV-H) induces high titers of neutralizing antibodies to MV in the presence of MV-specific antibodies. The induction of neutralizing antibodies increased with increasing virus dose, and all doses gave good protection from subsequent challenge with MV. Induction of antibodies by VSV-H was observed in the presence of passively transferred human or cotton rat antibodies, which were used as the models of maternal antibodies. Because MV hemagglutinin is not a functional part of the VSV-H envelope, MV-specific antibodies only slightly inhibit VSV-H replication in vitro. This dissociation of function and antigenicity is probably key to the induction of a neutralizing antibody in the presence of a maternal antibody.

Nonstructural proteins encoded by measles virus (MV) include the V protein which is translated from an edited P mRNA. V protein is not associated with intracellular or released viral particles and has recently been found to be dispensable for MV propagation in cell culture (H. Schneider, K. Kaelin, and M. A. Billeter, Virology 227:314-322, 1997). Using recombinant MVs (strain Edmonston [ED]) genetically engineered to overexpress V protein (ED-V+) or to be deficient for V protein (ED-V-), we found that in the absence of V both MV-specific proteins and RNAs accumulated to levels higher than those in the parental MV molecular clone (ED-tag), whereas MV-specific gene expression was strongly attenuated in human U-87 glioblastomas cells after infection with ED-V+. The titers of virus released from these cells 48 h after infection with either V mutant virus were lower than those from cells infected with ED-tag. Similarly, significantly reduced titers of infectious virus were reisolated from lung tissue of cotton rats (Sigmodon hispidus) after intranasal infection with both editing mutants compared to titers isolated from ED-tag-infected animals. In cell culture, expression of V protein led to a redistribution of MV N protein in doubly transfected Cos-7 cells, indicating that these proteins form heterologous complexes. This interaction was further confirmed by using a two-hybrid approach with both proteins expressed as Gal4 or VP16 fusion products. Moreover, V protein efficiently competed complexes formed between MV N and P proteins. These findings indicate that V protein acts to balance accumulation of viral gene products in cell culture, and this may be dependent on its interaction with MV N protein. Furthermore, expression of V protein may contribute to viral pathogenicity in vivo.