Evidence for an Underlying CD4 Helper and CD8 T-Cell Defect in B-Cell-Deficient Mice: Failure To Clear Persistent Virus Infection after Adoptive Immunotherapy with Virus-Specific Memory Cells from μMT/μMT Mice

Adoptive transfer of virus-specific memory lymphocytes can be used to identify factors and mechanisms involved in the clearance of persistent virus infections. To analyze the role of B cells in clearing persistent infection with lymphocytic choriomeningitis virus (LCMV), we used B-cell-deficient μMT/μMT (B−/−) mice. B−/− mice controlled an acute LCMV infection with the same kinetics and efficiency as B-cell-competent (B+/+) mice via virus-specific, major histocompatibility complex (MHC) class I-restricted CD8⁺ cytotoxic T lymphocytes (CTL). CTL from B−/− and B+/+ mice were equivalent in affinity to known LCMV CTL epitopes and had similar CTL precursor frequencies (pCTL). Adoptive transfer of memory cells from B+/+ mice led to virus clearance from persistently infected B+/+ recipients even after in vitro depletion of B cells, indicating that B cells or immunoglobulins are not required in the transfer population. In contrast, transfer of memory splenocytes from B−/− mice failed to clear virus. Control of virus was restored neither by transferring higher numbers of pCTL nor by supplementing B−/− memory splenocytes with LCMV-immune B cells or immune sera. Instead, B−/− mice were found to have a profound CD4 helper defect. Furthermore, compared to cultured splenocytes from B+/+ mice, those from B−/− mice secreted less gamma interferon (IFN-γ) and interleukin 2, with differences most pronounced for CD8 T cells. While emphasizing the importance of CD4 T-cell help and IFN-γ in the control of persistent infections, the CD4 T-helper and CD8 T-cell defects in B−/− mice suggest that B cells contribute to the induction of competent T effector cells.Cytotoxic T lymphocytes (CTL) have in general been associated with the resolution of both acute and chronic viral infections. As first shown by studies of lymphocytic choriomeningitis virus (LCMV) in mice, its natural host, a critical component of immune responses to virus infection is the induction of virus-specific major histocompatibility complex (MHC) class I-restricted CTL (reviewed in reference 14). Evidence that these cells can curtail acute viral infections and clear virus and viral genetic material from sera, peripheral blood leukocytes, and infected tissues came from adoptive transfer of LCMV memory CTL into mice persistently infected with LCMV (1, 25, 33, 47, 53).Studies with humans have correlated the presence of CTL with the control of acute infection and clearance of virus and the absence of CTL activity with persistent viral infections. Hence, humans with genetic deficiencies in the humoral compartment of the immune system but with an intact T-cell compartment overcome most viral infections and display immunological memory when challenged or reinfected with the same virus. For example, agammaglobulinemic children recover from acute measles infection as well as do fully immunocompetent individuals and resist reinfection (23). In contrast, individuals with genetic or acquired defects in the T-cell compartment generally cannot control viral infections. Similarly, activity of CTL specific for hepatitis B virus (HBV) is associated with control of acute HBV infection; in the absence of CTL, HBV persists (39). Additionally, anti-HIV CTL dramatically decrease the load of human immunodeficiency virus (HIV) in infected patients, whereas loss of CTL function is accompanied by regress from a relatively healthy clinical stage to AIDS or rapid development of disease after HIV infection (9, 32). Finally, diminished or missing CTL responses to human cytomegalovirus (HCMV) facilitate HCMV disease in individuals undergoing bone marrow transplantation (40). Adoptive transfer of HCMV MHC-restricted CTL into such patients prevented CMV viremia or CMV disease (55). Thus, understanding the requirements for initiation and maintenance of CTL activity is essential.Earlier, we and others documented the requirement for CD4 T-cell help (5, 16, 29, 48) and gamma interferon (IFN-γ) (48) in maintaining sufficient CTL activity in vivo and resolution of a chronic LCMV infection. Here, we evaluate the role of B lymphocytes in this process. Under the appropriate signals, B lymphocytes can differentiate into plasma cells to function as antibody-secreting cells. Trapping of antibody-antigen complexes as well as processing of antigen and peptide presentation within the MHC complex allows B cells to also function as antigen-presenting cells (APC) to T cells (22). Furthermore, B cells release numerous growth factors and cytokines that regulate immune responses (44).To ascertain the role of B lymphocytes in the clearance of both acute and persistent LCMV infections, we used μMT/μMT B-cell-deficient (B−/−) mice which lack functional B cells and antibody. Earlier studies showed that CD8 T cells from these mice were capable of controlling an acute LCMV infection and that there was no defect in generating CTL precursors (3). Our results confirm and expand these findings. We demonstrate that while adoptive transfer of memory cells from B+/+ mice easily clears infectious virus and viral material in an MHC-matched persistently infected recipient, transfer of similar cells from B−/− mice does not. However, failure to terminate the persistent infection does not result from absence of B cells in the transfer population. Apparently, B−/− mice have a fundamental defect in CD4 helper function as well as a quantitative deficiency in IFN-γ and interleukin 2 (IL-2) preferentially produced by CD8 T cells after LCMV infection. These results emphasize the essential role for CD4 T-lymphocyte help and IFN-γ in achieving CTL activity necessary for clearing a persistent LCMV infection and point to an expanded role for B cells in the development and maintenance of CD4 and CD8 T-cell functions.     

Cytotoxic T-Lymphocyte Precursor Frequencies in BALB/c Mice after Acute Respiratory Syncytial Virus (RSV) Infection or Immunization with a Formalin-Inactivated RSV Vaccine

A better understanding of the immune response to live and formalin-inactivated respiratory syncytial virus (RSV) is important for developing nonlive vaccines. In this study, major histocompatibility complex (MHC) class I- and II-restricted, RSV-specific cytotoxic T-lymphocyte precursor (CTLp) frequencies were determined in bronchoalveolar lavage (BAL) samples and spleen lymphocytes of BALB/c mice intranasally infected with live RSV or intramuscularly inoculated with formalin-inactivated RSV (FI-RSV). After RSV infection, both class I- and class II-restricted CTLps were detected by day 4 or 5 postinfection (p.i.). Peak CTLp frequencies were detected by day 7 p.i. The class II-restricted CTLp frequencies in the BAL following RSV infection were less than class I-restricted CTLp frequencies through day 14 p.i., during which class I-restricted CTLp frequencies remained elevated, but then declined by 48 days p.i. The frequencies of class II-restricted CTLps in the BAL were 2- to 10-fold less than those of class I-restricted CTLps. For spleen cells, frequencies of both MHC class I- and II-restricted CTLps to live RSV were similar. In contrast, class II-restricted CTLps predominated in FI-RSV-vaccinated mice. RSV challenge of vaccinated mice resulted in an increase in the frequency of class I-restricted CTLps at day 3 p.i. but did not enhance class II-restricted CTLp frequencies. These studies demonstrate differences in the CTLp response to live RSV infection compared with FI-RSV immunization and help define possible mechanisms of enhanced disease after FI-RSV immunization. In addition, these studies provide a quantitative means to address potential vaccine candidates by examining both MHC class I- and II-restricted CTLp frequencies.Respiratory syncytial virus (RSV) infection in infants and young children often results in lower respiratory tract disease and is a high priority for vaccine development (1, 2). Attempts to develop an effective live, inactivated, or subunit vaccine have been unsuccessful (24, 25, 28). Early efforts at vaccinating young children with a formalin-inactivated RSV (FI-RSV) vaccine failed to protect the children from naturally acquired infection and actually enhanced lower respiratory tract disease upon later virus infection (2, 15, 24, 25). This enhanced disease has created concern about the safety of any nonlive RSV vaccine and, consequently, understanding the pathogenesis of FI-RSV-induced enhanced disease is critically important to vaccine development. Studies with BALB/c mice suggest that induction of memory T cells producing Th2-like cytokines, as a result of FI-RSV vaccination, may be key to the pathogenesis of enhanced disease (6, 16, 28, 32, 40). Th2-like cytokine mRNA has been demonstrated in cells from lung tissue or bronchoalveolar lavage (BAL) specimens after RSV challenge of FI-RSV-immunized mice (17, 32, 40). In addition, in vivo studies using antibody (Ab) blockade showed that the enhanced histopathology in FI-RSV-immune mice challenged with live virus could be eliminated by using anti-interleukin-4 (IL-4) and anti-IL-10 Abs but not anti-IL-12 Abs (6). Recent evidence suggests that CD8⁺ T lymphocytes may be important in directing the type of inflammatory response to RSV in challenge of G glycoprotein-sensitized mice (21, 31).One aspect of the FI-RSV immune response that has not been well characterized is the cytotoxic T-lymphocyte (CTL) response. There is limited information on major histocompatibility complex (MHC) class I-restricted CTLs after FI-RSV immunization (29), while the information about the CTL response after live-RSV infection has been well documented. Several studies have shown class I-restricted CTLs to kill predominantly target cells expressing the M, N, or F RSV protein (5, 7, 9, 26, 29, 41). The role of CTLs in the immune response to RSV is well illustrated by in vivo depletion studies with BALB/c mice (8, 18, 30). These studies suggest that both CD4⁺ (class II) and CD8⁺ lymphocytes are important for clearing RSV and that both contribute to the inflammatory response associated with infection. A vaccinia virus construct expressing RSV membrane-associated, nonglycosylated protein M2 has been affiliated with short-term protection in the BALB/c mouse (7). This protein does not induce neutralizing Abs, and therefore, protection likely is mediated by CTLs. Passive transfer of CD8⁺ T lymphocytes has been associated with both clearance of the virus and enhanced histopathology (1).In this report, we describe studies of CTL precursor (CTLp) frequencies in both live-RSV-infected and FI-RSV-immunized mice for MHC class I- and class II-restricted target cells. These studies demonstrate clear differences in the CTLp response between RSV and FI-RSV immunizations and provide additional approaches to identifying potential FI-RSV-induced enhanced disease mechanisms.     

An Important Role for Major Histocompatibility Complex Class I-Restricted T Cells,and a Limited Role for Gamma Interferon,in Protection of Mice against Lethal Herpes Simplex Virus Infection

Herpes simplex virus (HSV) inhibits major histocompatibility complex (MHC) class I expression in infected cells and does so much more efficiently in human cells than in murine cells. Given this difference, if MHC class I-restricted T cells do not play an important role in protection of mice from HSV, an important role for these cells in humans would be unlikely. However, the contribution of MHC class I-restricted T cells to the control of HSV infection in mice remains unclear. Further, the mechanisms by which these cells may act to control infection, particularly in the nervous system, are not well understood, though a role for gamma interferon (IFN-γ) has been proposed. To address the roles of MHC class I and of IFN-γ, C57BL/6 mice deficient in MHC class I expression (β2 microglobulin knockout [β2KO] mice), in IFN-γ expression (IFN-γKO mice), or in both (IFN-γKO/β2KO mice) were infected with HSV by footpad inoculation. β2KO mice were markedly compromised in their ability to control infection, as indicated by increased lethality and higher concentrations of virus in the feet and spinal ganglia. In contrast, IFN-γ appeared to play at most a limited role in viral clearance. The results suggest that MHC class I-restricted T cells play an important role in protection of mice against neuroinvasive HSV infection and do so largely by mechanisms other than the production of IFN-γ.

Two gene products of herpes simplex virus (HSV) block presentation of viral proteins by class I major histocompatibility complex (MHC) molecules: the viral host shutoff protein (vhs), which is present in the viral particle, and the immediate-early protein ICP47 (1, 14, 41, 42). Through the sequential action of these proteins, antigen presentation by MHC class I is inhibited early in the viral replication cycle. ICP47 binds to human transporter associated with antigen-processing proteins (TAP), thereby inhibiting peptide loading on MHC class I and recognition by HSV-specific, MHC class I-restricted, CD8⁺ T cells (1, 14, 42, 43). This effect is greatest in nonhematopoietic cells in which the abundance of MHC class I and TAP are lower than in antigen-presenting cells (41). As a consequence, HSV is more likely to impair recognition of infected target cells in the tissues than to block the generation of antigen-specific CD8⁺ T cells. Consistent with this, recent studies indicate that HSV antigen-specific CD8⁺ cytotoxic-T-lymphocyte (CTL) precursors can be readily detected in the blood and cutaneous lesions of HSV-infected individuals (16, 31, 32). However, NK cells and HSV antigen-specific CD4⁺ T cells are detected earlier than antigen-specific CD8⁺ T cells in lesions of humans with recurrent HSV-2 disease (16). This finding has led to the proposal that gamma interferon (IFN-γ) produced by infiltrating NK and CD4⁺ T cells overrides the inhibitory effects of HSV on TAP function and MHC class I expression (22, 41), thereby allowing the eradication of virus by CD8⁺ T cells, whose numbers increase in lesions around the time of viral clearance (16, 31). In patients with AIDS, a lower frequency in the blood of HSV antigen-specific CD8⁺ CTL precursors is associated with more frequent and severe recurrences of genital disease (32). These correlative data suggest that CD8⁺ T cells may play an important role in the clearance of HSV in humans, at least from mucocutaneous lesions.ICP47 inhibits murine TAP poorly (1, 42), which may explain the greater ease with which anti-HSV CD8⁺ CTLs have been detected in mice than in humans (3, 8, 28, 34, 35). Despite the weak interaction of ICP47 with murine TAP, results of a recent study (12) suggested that ICP47 impairs CD8⁺ T-cell-dependent viral clearance from the nervous system: CD8⁺ T cells protected susceptible BALB/c or A/J mice from lethal, nervous system infection with an HSV mutant lacking ICP47 but did not appear to protect against infection with wild-type HSV or to contribute to clearance of either virus from the eye. These findings are consistent with data suggesting that CD8⁺ T cells limit persistence of HSV in the spinal ganglia and decrease spread to the central nervous system (35, 36). However, other studies have concluded that CD4⁺ T cells but not CD8⁺ T cells play the critical role in viral clearance and protection from lethal primary infection with wild-type HSV (20, 23, 24) or that either CD4⁺ or CD8⁺ T cells are sufficient for protection (26, 37). Since the effects of ICP47 are likely to be greater in humans than in mice, if MHC class I-restricted CD8⁺ T cells do not play an important role in protection of mice from lethal, neuroinvasive infection due to wild-type HSV, an important role in humans would be unlikely.The mechanisms by which T cells may limit the spread of infection in the nervous system are not clearly understood. Studies by Simmons and colleagues suggested that CD8⁺ T cells may lyse infected Schwann cells or satellite cells but that they probably do not lyse infected neurons (31, 32). They and others have proposed that CD8⁺ T cells protect neurons through the production of cytokines, in particular IFN-γ (35, 36). IFN-γ contributes to the clearance of HSV from mucocutaneous sites (4, 24, 25, 37, 44). However, the role of IFN-γ in protection from lethal, neuroinvasive infection is uncertain and may vary with the strain of mice, method used to inhibit IFN-γ function, and route of inoculation (4, 5, 24, 37, 44). IFN-γ is produced in the ganglia of mice with acute or latent HSV infection (5, 13, 19). Both CD4⁺ and CD8⁺ T cells (and NK cells) produce IFN-γ, but CD4⁺ T cells appear to be the predominant source of IFN-γ following intravaginal infection with HSV (24, 25). Thus, it is possible that the disparity in results regarding the relative importance of CD4⁺ and CD8⁺ T cells in protection from lethal, neuroinvasive HSV infection reflects their redundant roles in production of this cytokine or that IFN-γ and CD8⁺ T cells contribute independently to control of infection in the nervous system.To address in parallel the contributions of MHC class I-restricted T cells and of IFN-γ to protection of mice from HSV, MHC class I and CD8⁺ T-cell-deficient β2 microglobulin knockout (β2KO) mice, IFN-γ knockout (IFN-γKO) mice, and mice deficient in both MHC class I and IFN-γ expression (IFN-γKO/β2KO) were studied. The results indicated that loss of MHC class I expression in β2KO mice substantially increased their susceptibility to HSV, whereas the loss of IFN-γ expression had a much more limited effect. These findings indicate that MHC class I-restricted T cells play an important role in protection against neuroinvasive HSV infection in mice and that they do so largely by mechanisms other than the production of IFN-γ. Though MHC class I expression is more severely impaired in β2KO mice than in human cells infected with wild-type HSV, these findings support the notion that inhibition of MHC class I expression is an important factor in the virulence of this virus.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Protection against Lethal Encephalomyocarditis Virus Infection in the Absence of Serum-Neutralizing Antibodies

Although the ability of serum-neutralizing antibodies to protect against picornavirus infection is well established, the contribution of cell-mediated immunity to protection is uncertain. Using major histocompatibility complex class II-deficient (RHAβ^−/−) mice, which are unable to mediate CD4⁺ T-lymphocyte-dependent humoral responses, we demonstrated antibody-independent protection against lethal encephalomyocarditis virus (EMCV) infection in the natural host. The majority of RHAβ^−/− mice inoculated with 10⁴ PFU of attenuated Mengo virus (vMC₂₄) resolved infection and were resistant to lethal challenge with the highly virulent, serotypically identical cardiovirus, EMCV. Protection in these mice was in the absence of detectable serum-neutralizing antibodies. Depletion of CD8⁺ T lymphocytes prior to lethal EMCV challenge ablated protection in vMC₂₄-immunized RHAβ^−/− mice. The CD8⁺ T-lymphocyte-dependent protection observed in vivo may, in part, be the result of cytotoxic T-lymphocyte (CTL) activity, as CD8⁺ T splenocytes exhibited in vitro cytolysis of EMCV-infected targets. The existence of virus-specific CD8⁺ T-lymphocyte memory in these mice was demonstrated by increased expression of cell surface activation markers CD25, CD69, CD71, and CTLA-4 following antigen-specific reactivation in vitro. Although recall response in vMC₂₄-immunized RHAβ^−/− mice was intact and effectual shortly after immunization, protection abated over time, as only 3 of 10 vMC₂₄-immunized RHAβ^−/− mice survived when rechallenged 90 days later. The present study demonstrating CD8⁺ T-lymphocyte-dependent protection in the absence of serum-neutralizing antibodies, coupled with our previous results indicating that vMC₂₄-specific CD4⁺ T lymphocytes confer protection against lethal EMCV in the absence of prophylactic antibodies, suggests the existence of nonhumoral protective mechanisms against picornavirus infections.Picornaviruses are a family of positive-strand RNA viruses that are responsible for a variety of devastating human and animal diseases. The family is divided into six genera, enteroviruses, hepatoviruses, parechoviruses, rhinoviruses, aphthoviruses, and cardioviruses, that include such members as poliovirus, human rhinovirus, foot-and-mouth disease virus, and encephalomyocarditis virus (EMCV) (42). Mice are highly susceptible and considered the natural host for cardioviruses such as Mengo virus and EMCV, (7, 35), infections with which result in acute neurotropic disease producing rapid and lethal meningoencephalomyelitis (16, 47). The ability to protect mice against cardiovirus-induced disease by the elicitation or passive transfer of neutralizing antibodies is well documented (2, 13, 26, 41). Current dogma asserts that prophylaxis against picornavirus infection is afforded by serum-neutralizing antibodies (23, 25, 28). Existing picornavirus vaccines (23, 25), in addition to current strategies using recombinant-attenuated and protein-subunit vaccines (27, 32), are designed to elicit a protective neutralizing antibody response to capsid determinants. Indeed, serum-neutralizing titers are used to evaluate host immune status to a particular picornavirus pathogen.Mengo virus and EMCV are members of a single cardiovirus serotype and are indistinguishable by immune sera (42). The dramatic attenuation of Mengo virus by a truncation in the 5′-noncoding-region poly(C) tract preserves complete integrity of all virally encoded proteins (10), allowing in vivo exposure of structural and nonstructural proteins that may elicit an immune response. Normal immunocompetent mice immunized with an attenuated strain of Mengo virus (vMC₂₄) elicit high serum-neutralizing antibody titers and are protected from lethal EMCV challenge (9, 29). In addition to invoking a potent humoral response, vMC₂₄ is also capable of eliciting a cell-mediated immune (CMI) response (29) as an immunodominant CD8⁺ cytotoxic T-lymphocyte (CTL) epitope has been recently identified in the VP2 capsid protein in vMC₂₄-immunized C57BL/6 mice (11).Earlier investigations of CMI responses to cardioviruses in T-cell deficiency models vacillated between elucidating the immunopathologic role that these cells may contribute in disease and discerning the beneficial aspects that T cells may mediate in protection. T-cell subset depletion of BALB/c mice with anti-CD4 or anti-CD8 antibodies prior to EMCV infection ameliorated clinical disease and reduced the frequency of demyelination (44), suggesting a participatory role for T cells in pathology. Conversely, mice rendered CD4 deficient prior to infection with Theiler’s murine encephalomyelitis virus (TMEV), another murine cardiovirus, failed to produce neutralizing antibodies; consequently, they were unable to clear virus from the central nervous system (CNS) and died from overwhelming encephalitis (49). Similarly, infection of major histocompatibility complex (MHC) class I (β₂-microglobulin)-deficient (β₂m^−/−) mice with TMEV indicates a requisite role for CD8⁺ T cells in viral clearance and suggests that CD8⁺ T cells are not major mediators in demyelination or disease (13, 39).More recently, researchers have begun to unveil the beneficial role that CD8⁺ T cells may have in resolving infection and immune protection. An early and abundant TMEV-specific CD8⁺ T-cell response is critical in determining the balance between viral persistence or resolution of infection (6, 22, 30). Using vMC₂₄-immunized C57BL/6 mice, Escriou et al identified an immunodominant CD8⁺ CTL epitope (11) that is cross-reactive to the same VP2 epitope of TMEV (5), although VP2 epitope-immunized C57BL/6 mice were not fully protected from subsequent lethal Mengo virus challenge.The present study is a direct extension of our earlier observation (29) that vMC₂₄-specific CD4⁺ T cells are capable of adoptively transferring immune protection against lethal EMCV challenge in the absence of prophylactic levels of serum-neutralizing antibodies. Using MHC class II-deficient mice that lack CD4⁺ T cells and are incapable of T-cell-dependent humoral responses (15), we obtained evidence demonstrating CD8⁺ T cell-dependent protection against lethal EMCV infection in the absence of serum-neutralizing antibodies.     

Dendritic Cells Efficiently Induce Protective Antiviral Immunity

Cytotoxic T lymphocytes (CTL) are essential for effective immunity to various viral infections. Because of the high speed of viral replication, control of viral infections imposes demanding functional and qualitative requirements on protective T-cell responses. Dendritic cells (DC) have been shown to efficiently acquire, transport, and present antigens to naive CTL in vitro and in vivo. In this study, we assessed the potential of DC, either pulsed with the lymphocytic choriomeningitis virus (LCMV)-specific peptide GP33-41 or constitutively expressing the respective epitope, to induce LCMV-specific antiviral immunity in vivo. Comparing different application routes, we found that only 100 to 1,000 DC had to reach the spleen to achieve protective levels of CTL activation. The DC-induced antiviral immune response developed rapidly and was long lasting. Already at day 2 after a single intravenous immunization with high doses of DC (1 × 10⁵ to 5 × 10⁵), mice were fully protected against LCMV challenge infection, and direct ex vivo cytotoxicity was detectable at day 4 after DC immunization. At day 60, mice were still protected against LCMV challenge infection. Importantly, priming with DC also conferred protection against infections in which the homing of CTL into peripheral organs is essential: DC-immunized mice rapidly cleared an infection with recombinant vaccinia virus-LCMV from the ovaries and eliminated LCMV from the brain, thereby avoiding lethal choriomeningitis. A comparison of DC constitutively expressing the GP33-41 epitope with exogenously peptide-pulsed DC showed that in vivo CTL priming with peptide-loaded DC is not limited by turnover of peptide-major histocompatibility complex class I complexes. We conclude that the priming of antiviral CTL responses with DC is highly efficient, rapid, and long lasting. Therefore, the use of DC should be considered as an efficient means of immunization for antiviral vaccination strategies.Dendritic cells (DC) derived from bone marrow (bmDC) are found as antigen-presenting cells (APC) scattered throughout nonlymphoid tissues. After antigen acquisition and processing, DC migrate via lymph vessels or blood to the T-cell areas of regional secondary lymphoid organs, where they present major histocompatibility complex (MHC) class I- and II-restricted peptides to naive T cells (42). The combination of these properties, i.e., antigen uptake, processing, transport, and presentation, makes DC particularly suitable as vehicles for antigens in immunotherapy. DC pulsed with tumor-derived peptides (29, 39, 46), tumor-associated proteins (16), or tumor cell RNA (9) have been shown to activate tumor-specific cytotoxic T lymphocytes (CTL) and to reduce tumor load.The high efficiency of DC in eliciting T-cell responses against infectious agents has been demonstrated with different experimental systems; e.g., DC can prime immune responses against mycobacteria (19), Borrelia burgdorferi (30), and Leishmania major (31). Induction of virus-specific CTL by DC in vitro with virus peptides or virus proteins has been shown for influenza virus (27), Sendai virus (22), and human immunodeficiency virus (26). In vivo, adoptive transfer of human immunodeficiency virus peptide-pulsed DC (43) or hepatitis B virus protein-pulsed DC (10) elicits virus-specific CTL responses. Whether these CTL responses reflect the ability to provide antiviral protection in vivo, however, has not been addressed in these studies. This aspect is particularly important in viral infections, since the high speed of replication and amplification imposes demanding quantitative and functional requirements on protective CTL responses (6, 15, 24).Infection of mice with lymphocytic choriomeningitis virus (LCMV) is a well-characterized model system for studying CTL responses in vivo. Control of an acute virus infection is almost exclusively achieved by CD8⁺ T-cell-mediated, perforin-dependent cytotoxicity (21). Various in vivo and in vitro assays with graded sensitivities allow assessment of the efficiency and biological relevance of an immunization strategy (5, 12). In addition, several CTL immunization strategies, including peptide vaccination (1, 2, 41) and priming with recombinant vaccinia virus (17), have been well characterized for this experimental system. Therefore, the study of DC-induced immunity against LCMV infection allows an interesting qualitative comparison with other vaccination approaches.In the present study, we evaluated the potential of DC to induce CTL-mediated antiviral immunity in vivo using bone marrow-derived DC either pulsed with peptides or obtained from transgenic mice constitutively expressing the immunodominant epitope (GP33-41; hereafter referred to as GP33) of the LCMV (WE strain) glycoprotein (14a). Immunization with these cells allowed us to address the following questions. (i) How many DC are needed to induce protection against a viral infection? (ii) What are the in vivo kinetics of DC-induced CTL activation? (iii) Do DC-induced antiviral CTL emigrate to peripheral tissues to resolve viral infections, and are they protective against virus-induced immunopathology? (iv) Does turnover of peptide-MHC class I complexes influence DC immunogenicity?     

Neutralization Sensitivity of Human Immunodeficiency Virus Type 1 Primary Isolates to Antibodies and CD4-Based Reagents Is Independent of Coreceptor Usage

We have investigated whether the identity of the coreceptor (CCR5, CXCR4, or both) used by primary human immunodeficiency virus type 1 (HIV-1) isolates to enter CD4⁺ cells influences the sensitivity of these isolates to neutralization by monoclonal antibodies and CD4-based agents. Coreceptor usage was not an important determinant of neutralization titer for primary isolates in peripheral blood mononuclear cells. We also studied whether dualtropic primary isolates (able to use both CCR5 and CXCR4) were differentially sensitive to neutralization by the same antibodies when entering U87MG-CD4 cells stably expressing either CCR5 or CXCR4. Again, we found that the coreceptor used by a virus did not greatly affect its neutralization sensitivity. Similar results were obtained for CCR5- or CXCR4-expressing HOS cell lines engineered to express green fluorescent protein as a reporter of HIV-1 entry. Neutralizing antibodies are therefore unlikely to be the major selection pressure which drives the phenotypic evolution (change in coreceptor usage) of HIV-1 that can occur in vivo. In addition, the increase in neutralization sensitivity found when primary isolates adapt to growth in transformed cell lines in vitro has little to do with alterations in coreceptor usage.Human immunodeficiency virus type 1 (HIV-1) enters CD4⁺ T cells via an interaction with CD4 and coreceptor molecules, the most important of which yet identified are the chemokine receptors CXCR4 and CCR5 (4, 12, 23, 26, 28, 32). CXCR4 is used by T-cell line-tropic (T-tropic) primary isolates or T-cell line-adapted (TCLA) lab strains, whereas CCR5 is used by primary isolates of the macrophage-tropic (M-tropic) phenotype (4, 12, 23, 26, 28, 32). Most T-tropic isolates and some TCLA strains are actually dualtropic in that they can use both CXCR4 and CCR5 (and often other coreceptors such as CCR3, Bonzo/STRL33, and BOB/gpr15), at least in coreceptor-transfected cells (18, 24, 30, 54, 89). The M-tropic and T-tropic/dualtropic nomenclature has often been used interchangeably with the terms “non-syncytium-inducing” (NSI) and “syncytium-inducing” (SI), although it is semantically imprecise to do so.M-tropic viruses are those most commonly transmitted sexually (3, 33, 87, 106) and from mother to infant (2, 72, 81). If T-tropic strains are transmitted, or when they emerge, this is associated with a more rapid course of disease in both adults (17, 37, 46, 51, 52, 76, 78, 82, 92, 101) and children (6, 45, 84, 90). However, T-tropic viruses emerge in only about 40% of infected people, usually only several years after infection (76, 78). A well-documented, albeit anecdotal, study found that when a T-tropic strain was transmitted by direct transfer of blood, its replication was rapidly suppressed: the T-tropic virus was eliminated from the body, and M-tropic strains predominated (20). These results suggest that there is a counterselection pressure against the emergence of T-tropic strains during the early stages of HIV-1 infection in most people. But what is this pressure?Since the M-tropic and T-tropic phenotypes are properties mediated by the envelope glycoproteins whose function is to associate with CD4 and the coreceptors, a selection pressure differentially exerted on M- and T-tropic viruses could, in principle, act at the level of virus entry. In other words, neutralizing antibodies to the envelope glycoproteins, or the chemokine ligands of the coreceptors, could theoretically interfere more potently with the interactions of T-tropic strains with CXCR4 than with M-tropic viruses and CCR5. A differential effect of this nature could suppress the emergence of T-tropic viruses. Consistent with this possibility, neutralizing antibodies are capable of preventing the CD4-dependent association of gp120 with CCR5 (42, 94, 103), and chemokines can also prevent the coreceptor interactions of HIV-1 (8, 13, 23, 28, 70).Here, we explore whether the efficiency of HIV-1 neutralization is affected by coreceptor usage. Although earlier studies have not found T-tropic strains to be inherently more neutralization sensitive than M-tropic ones (20, 40, 44), previously available reagents and techniques may not have been adequate to fully address this question. One major problem is that even single residue changes can drastically affect both antibody binding to neutralization epitopes and the HIV-1 phenotype (25, 55, 62, 67, 83, 91), and so studies using relatively unrelated viruses and a fixed antibody (polyclonal or monoclonal) preparation have two variables to contend with: the viral phenotype (coreceptor use) and the antigenic structure of the virus and hence the efficiency of the antibody-virion interaction.We have used a new experimental strategy to explore whether coreceptor usage affects neutralization sensitivity in the absence of other confounding variables: the use of dualtropic viruses able to enter CD4⁺ cells via either CCR5 or CXCR4. By using a constant HIV-1 isolate or clone and the same monoclonal antibodies (MAbs) or CD4-based reagents as neutralizing agents, we can ensure that the only variable under study in the neutralization reaction is the nature of the coreceptor used for entry. Our major conclusion is that there is no strong association between coreceptor usage and neutralization sensitivity for primary HIV-1 isolates. Independent studies have reached the same conclusion (53a, 59). The emergence of T-tropic (SI) viruses in vivo may be unlikely to be due to escape from antibody-mediated selection pressure.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

A Peptide Mimic of a Protective Epitope of Respiratory Syncytial Virus Selected from a Combinatorial Library Induces Virus-Neutralizing Antibodies and Reduces Viral Load In Vivo

Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children worldwide. As yet, there is no effective vaccine against RSV infection, and previous attempts to develop a formalin-inactivated vaccine resulted in exacerbated disease in recipients subsequently exposed to the virus. In the work described here, a combinatorial solid-phase peptide library was screened with a protective monoclonal antibody (MAb 19) to identify peptide mimics (mimotopes) of a conserved and conformationally-determined epitope of RSV fusion (F) protein. Two sequences identified (S1 [HWYISKPQ] and S2 [HWYDAEVL]) reacted specifically with MAb 19 when they were presented as solid-phase peptides. Furthermore, after amino acid substitution analyses, three sequences derived from S1 (S1S [HWSISKPQ], S1K [KWYISKPQ], and S1P [HPYISKPQ]), presented as multiple antigen peptides (MAPs), also showed strong reactivity with MAb 19. The affinity constants of the binding of MAb 19, determined by surface plasmon resonance analyses, were 1.19 × 10⁹ and 4.93 × 10⁹ M⁻¹ for S1 and S1S, respectively. Immunization of BALB/c mice with these mimotopes, presented as MAPs, resulted in the induction of anti-peptide antibodies that inhibited the binding of MAb 19 to RSV and neutralized viral infection in vitro, with titers equivalent to those in sera from RSV-infected animals. Following RSV challenge of S1S mimotope-immunized mice, a 98.7% reduction in the titer of virus in the lungs was observed. Furthermore, there was a greatly reduced cell infiltration in the lungs of immunized mice compared to that in controls. These results indicate the potential of peptide mimotopes to protect against RSV infection without exacerbating pulmonary pathology.Respiratory syncytial virus (RSV) is the major cause of serious lower respiratory tract illness in infants and immunosuppressed individuals worldwide and is estimated to be responsible for 65 million infections and 1 million deaths annually (12, 19). Although the severity of disease declines with repeated infection, previous infection with RSV does not prevent illness in subsequent infections, and it is apparent that immunity is incomplete. Furthermore, attempts to develop a vaccine against RSV have encountered a series of problems. In the late 1960s, a formalin-inactivated vaccine not only failed to protect infants against RSV but also induced exacerbated disease during a subsequent epidemic (5, 19). Retrospective analysis of the sera demonstrated that the inactivated vaccine induced high anti-F (fusion) protein antibody titers, but with poor neutralizing activity, suggesting that the inactivation treatment had denatured or modified epitopes which were the target for neutralizing antibodies (19, 25). Live attenuated vaccines were also investigated as candidates; however, these were either poorly immunogenic (overattenuated) or genetically unstable (5, 45). Although new attenuated vaccines have given encouraging results in animal models (10, 11), there is an urgent need for alternative approaches to the development of an effective vaccine.Studies with experimental animals have provided evidence that RSV-specific neutralizing antibodies can prevent infection in the lungs when administered prophylactically (29). Intravenous administration of pooled human immunoglobulin G (IgG) containing a high titer of neutralizing antibodies prevented serious RSV lower respiratory tract illness in high-risk children (18). Both the F and G (attachment) glycoproteins of RSV play a major role in eliciting this humoral immunity. The conserved F glycoprotein induces protective immune responses (8, 36), and passive immunization with neutralizing anti-F monoclonal antibodies (MAbs) or recombinant Fab protects small animals from RSV infection (12, 38, 39). Based on the results of these studies, a neutralizing and protective MAb (MAb 19) has been reshaped and humanized (as MAb RSHZ19) and has been demonstrated to have protective properties superior to those of anti-RSV polyclonal antibodies in a rodent model (46). Its safety and efficacy have recently been assessed in human volunteers (13).The immunochemical characterization of the epitopes recognized by these protective antibodies is critical for the development of a vaccine against RSV, but the conformational constraints associated with the protective epitopes in RSV F protein make it unlikely that they will be identified from analysis of the primary amino acid sequence. Indeed, earlier attempts in our laboratory and elsewhere with synthetic peptides representing continuous sequences of the F protein (amino acids [aa] 205 to 225, 221 to 237, 261 to 273, 215 to 275, 417 to 438, and 481 to 491) failed to generate protective humoral responses (4, 23, 32, 42). The use of peptides (3), antigen fragments (24), and antibody escape mutants (39) has confirmed the involvement of discontinuous residues within the F protein in epitopes recognized by protective MAbs (39). Thus, the use of alternative approaches for the identification of these epitopes is essential.Solid-phase and bacteriophage combinatorial peptide libraries (22, 31) have been used for identification of peptide mimics (mimotopes) of ligands, and the identification of peptide sequences that mimic the conformational structure of protective epitopes would have great potential for the development of a synthetic peptide vaccine. Recently, using a solid-phase combinatorial peptide library, we identified 8-mer peptides that mimic an epitope recognized by a monoclonal anti-measles virus F protein antibody. The mimotopes identified by this approach did not bear any primary sequence relationship to sequences in the viral protein but mimicked its conformation. When used as an immunogen, one of the mimotopes induced virus-neutralizing and protective antibody responses (35). Phage-displayed peptide libraries have been used to delineate sequences which mimic a discontinuous epitope of hepatitis B surface antigen (7), the secondary structure of a neutralizing epitope of human immunodeficiency virus type 1 (15), and peptides that reacted with MAbs specific for polysaccharide antigens (43).In this paper, we describe the identification of mimotopes of a protective epitope of RSV F protein, as defined by the protective MAb 19, following the screening of a solid-phase combinatorial peptide library. One of these mimotopes induced a virus-neutralizing antibody response equivalent to that in RSV-immunized animals, which significantly reduced the viral load in RSV-challenged mice. These findings indicate the potential of synthetic peptide mimotopes for the development of a novel vaccine against RSV.     

The Cell Tropism of Human Immunodeficiency Virus Type 1 Determines the Kinetics of Plasma Viremia in SCID Mice Reconstituted with Human Peripheral Blood Leukocytes

Most individuals infected with human immunodeficiency virus type 1 (HIV-1) initially harbor macrophage-tropic, non-syncytium-inducing (M-tropic, NSI) viruses that may evolve into T-cell-tropic, syncytium-inducing viruses (T-tropic, SI) after several years. The reasons for the more efficient transmission of M-tropic, NSI viruses and the slow evolution of T-tropic, SI viruses remain unclear, although they may be linked to expression of appropriate chemokine coreceptors for virus entry. We have examined plasma viral RNA levels and the extent of CD4⁺ T-cell depletion in SCID mice reconstituted with human peripheral blood leukocytes following infection with M-tropic, dual-tropic, or T-tropic HIV-1 isolates. The cell tropism was found to determine the course of viremia, with M-tropic viruses producing sustained high viral RNA levels and sparing some CD4⁺ T cells, dual-tropic viruses producing a transient and lower viral RNA spike and extremely rapid depletion of CD4⁺ T cells, and T-tropic viruses causing similarly lower viral RNA levels and rapid-intermediate rates of CD4⁺ T-cell depletion. A single amino acid change in the V3 region of gp120 was sufficient to cause one isolate to switch from M-tropic to dual-tropic and acquire the ability to rapidly deplete all CD4⁺ T cells.The envelope gene of human immunodeficiency virus type 1 (HIV-1) determines the cell tropism of the virus (11, 32, 47, 62), the use of chemokine receptors as cofactors for viral entry (4, 17), and the ability of the virus to induce syncytia in infected cells (55, 60). Cell tropism is closely linked to but probably not exclusively determined by the ability of different HIV-1 envelopes to bind CD4 and the CC or the CXC chemokine receptors and initiate viral fusion with the target cell. Macrophage-tropic (M-tropic) viruses infect primary cultures of macrophages and CD4⁺ T cells and use CCR5 as the preferred coreceptor (2, 5, 15, 23, 26, 31). T-cell-tropic (T-tropic) viruses can infect primary cultures of CD4⁺ T cells and established T-cell lines, but not primary macrophages. T-tropic viruses use CXCR4 as a coreceptor for viral entry (27). Dual-tropic viruses have both of these properties and can use either CCR5 or CXCR4 (and infrequently other chemokine receptors [25]) for viral entry (24, 37, 57). M-tropic viruses are most frequently transmitted during primary infection of humans and persist throughout the duration of the infection (63). Many, but not all, infected individuals show an evolution of virus cell tropism from M-tropic to dual-tropic and finally to T-tropic with increasing time after infection (21, 38, 57). Increases in replicative capacity of viruses from patients with long-term infection have also been noted (22), and the switch to the syncytium-inducing (SI) phenotype in T-tropic or dual-tropic isolates is associated with more rapid disease progression (10, 20, 60). Primary infection with dual-tropic or T-tropic HIV, although infrequent, often leads to rapid disease progression (16, 51). The viral and host factors that determine the higher transmission rate of M-tropic HIV-1 and the slow evolution of dual- or T-tropic variants remain to be elucidated (4).These observations suggest that infection with T-tropic, SI virus isolates in animal model systems with SCID mice grafted with human lymphoid cells or tissue should lead to a rapid course of disease (1, 8, 44–46). While some studies in SCID mice grafted with fetal thymus and liver are in agreement with this concept (33, 34), our previous studies with the human peripheral blood leukocyte-SCID (hu-PBL-SCID) mouse model have shown that infection with M-tropic isolates (e.g., SF162) causes more rapid CD4⁺ T-cell depletion than infection with T-tropic, SI isolates (e.g., SF33), despite similar proviral copy numbers, and that this property mapped to envelope (28, 41, 43). However, the dual-tropic 89.6 isolate (19) caused extremely rapid CD4⁺ T-cell depletion in infected hu-PBL-SCID mice that was associated with an early and transient increase in HIV-1 plasma viral RNA (29). The relationship between cell tropism of the virus isolate and the pattern of disease in hu-PBL-SCID mice is thus uncertain. We have extended these studies by determining the kinetics of HIV-1 RNA levels in serial plasma samples of hu-PBL-SCID mice infected with primary patient isolates or laboratory stocks that differ in cell tropism and SI properties. The results showed significant differences in the kinetics of HIV-1 replication and CD4⁺ T-cell depletion that are determined by the cell tropism of the virus isolate.     

Immunization with a Single Major Histocompatibility Complex Class I-Restricted Cytotoxic T-Lymphocyte Recognition Epitope of Herpes Simplex Virus Type 2 Confers Protective Immunity

We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8⁺ cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2K^b-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8⁺ T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8⁺ CTL in HSV vaccine design.

Both humoral and cell-mediated components of the immune response are involved in controlling herpes simplex virus (HSV) infection (51, 61). Studies of humans and of mice have implicated a role for both CD8⁺ (6, 25, 32, 33, 47, 65–67) and CD4⁺ (27, 37–39, 52, 53) T-lymphocyte subsets in mediating protection against HSV infection. For example, CD8⁺ T cells have been shown to be important in limiting replication of HSV in the footpad (6) and colonization of the spinal dorsal root ganglia (6, 66). In contrast, other studies using a zosteriform model of infection have primarily indicated a role for CD4⁺ T cells in the clearance of HSV (37–39). Both CD4⁺ and CD8⁺ (56, 72, 74–76) HSV-specific T lymphocytes have been detected in humans seropositive for HSV. However, the contribution of each subset in the control of HSV infection has not been clearly defined. This illustrates the controversy regarding the relative roles of each subset in the resolution of HSV infection.To address the role of the CD8⁺ T-cell subset in providing acquired immunity to HSV infection, we examined the protection afforded by HSV-specific, CD8⁺ cytotoxic T lymphocytes (CTL) directed to a single CTL recognition epitope. In previous studies by others, immunization with single CTL epitopes has been effective in controlling viral pathogens including lymphocytic choriomeningitis virus (14, 54, 62, 73), murine cytomegalovirus (15), influenza virus (55), and Sendai virus (28). Although HSV-encoded CTL recognition epitopes have been identified by their ability to serve as targets for HSV-specific CTL (3, 8, 24, 64), the ability of CTL directed to these individual epitopes to confer protection against HSV infection has not been determined. We have designed two separate vaccination strategies which permit the exclusive induction of a single HSV epitope-specific, CD8⁺ T-lymphocyte response and have evaluated the ability of this response to confer protective immunity to HSV infection.Hanke et al. (24) broadly identified an immunodominant, H-2K^b-restricted epitope within HSV glycoprotein B (gB). The minimal amino acid sequence of this epitope, gB498-505 (SSIEFARL), was demonstrated by Bonneau et al. (8), using synthetic peptides and an epitope-specific CTL clone. The amino acid sequence, SSIEFARL, is identical in both HSV type 1 (HSV-1) (gB498-505) and HSV-2 (gB496-503) (11). CTL specific for gB498-505 are readily induced by immunization with synthetic peptide (8), a cell line expressing gB498-505 in the context of simian virus 40 (SV40) T antigen (5), and a recombinant viral vector expressing this epitope in the context of a cellular protein (19). In the present study, two recombinant vaccinia viruses (rVV-ES-gB498-505 and rVV-gB498-505) and a recombinant influenza virus (WSN/NA/gB) were generated to express a single HSV-encoded epitope, HSV-1 gB498-505, and were characterized for the ability to induce a potent, HSV-specific CTL response upon mucosal immunization. To determine the protection afforded by immunization with each of the individual recombinant viruses, we used a lethal model of HSV-2 encephalitis. Our findings suggest that the induction of a CTL response directed against a single HSV-specific CTL recognition epitope is sufficient to confer significant protective immunity to HSV infection.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.