Human parainfluenza virus type 1 C proteins are nonessential proteins that inhibit the host interferon and apoptotic responses and are required for efficient replication in nonhuman primates.

Recombinant human parainfluenza virus type 1 (rHPIV1) was modified to create rHPIV1-P(C-), a virus in which expression of the C proteins (C', C, Y1, and Y2) was silenced without affecting the amino acid sequence of the P protein. Infectious rHPIV1-P(C-) was readily recovered from cDNA, indicating that the four C proteins were not essential for virus replication. Early during infection in vitro, rHPIV1-P(C-) replicated as efficiently as wild-type (wt) HPIV1, but its titer subsequently decreased coincident with the onset of an extensive cytopathic effect not observed with wt rHPIV1. rHPIV1-P(C-) infection, but not wt rHPIV1 infection, induced caspase 3 activation and nuclear fragmentation in LLC-MK2 cells, identifying the HPIV1 C proteins as inhibitors of apoptosis. In contrast to wt rHPIV1, rHPIV1-P(C-) and rHPIV1-C(F170S), a mutant encoding an F170S substitution in C, induced interferon (IFN) and did not inhibit IFN signaling in vitro. However, only rHPIV1-P(C-) induced apoptosis. Thus, the anti-IFN and antiapoptosis activities of HPIV1 were separable: both activities are disabled in rHPIV1-P(C-), whereas only the anti-IFN activity is disabled in rHPIV1-C(F170S). In African green monkeys (AGMs), rHPIV1-P(C-) was considerably more attenuated than rHPIV1-C(F170S), suggesting that disabling the anti-IFN and antiapoptotic activities of HPIV1 had additive effects on attenuation in vivo. Although rHPIV1-P(C-) protected against challenge with wt HPIV1, its highly restricted replication in AGMs and in primary human airway epithelial cell cultures suggests that it might be overattenuated for use as a vaccine. Thus, the C proteins of HPIV1 are nonessential but have anti-IFN and antiapoptosis activities required for virulence in primates.

Role of interferon in the replication of human parainfluenza virus type 1 wild type and mutant viruses in human ciliated airway epithelium.

Human parainfluenza virus type 1 (HPIV1) is a significant cause of pediatric respiratory disease in the upper and lower airways. An in vitro model of human ciliated airway epithelium (HAE), a useful tool for studying respiratory virus-host interactions, was used in this study to show that HPIV1 selectively infects ciliated cells within the HAE and that progeny virus is released from the apical surface with little apparent gross cytopathology. In HAE, type I interferon (IFN) is induced following infection with an HPIV1 mutant expressing defective C proteins with an F170S amino acid substitution, rHPIV1-C(F170S), but not following infection with wild-type HPIV1. IFN induction coincided with a 100- to 1,000-fold reduction in virus titer, supporting the hypothesis that the HPIV1 C proteins are critical for the inhibition of the innate immune response. Two recently characterized live attenuated HPIV1 vaccine candidates expressing mutant C proteins were also evaluated in HAE. The vaccine candidates, rHPIV1-C(R84G/Delta170)HN(T553A)L(Y942A) and rHPIV1-C(R84G/Delta170)HN(T553A)L(Delta1710-11), which contain temperature-sensitive (ts) attenuating (att) and non-ts att mutations, were highly restricted in growth in HAE at permissive (32 degrees C) and restrictive (37 degrees C) temperatures. The viruses grew slightly better at 37 degrees C than at 32 degrees C, and rHPIV1-C(R84G/Delta170)HN(T553A)L(Y942A) was less attenuated than rHPIV1-C(R84G/Delta170)HN(T553A)L(Delta1710-11). The level of replication in HAE correlated with that previously observed for African green monkeys, suggesting that the HAE model has potential as a tool for the preclinical evaluation of HPIV1 vaccines, although how these in vitro data will correlate with vaccine virus replication in seronegative human subjects remains to be seen.

CD25(+)CD4(+) regulatory T cells from the peripheral blood of asymptomatic HIV-infected individuals regulate CD4(+) and CD8(+) HIV-specific T cell immune responses in vitro and are associated with favorable clinical markers of disease status.

Human immunodeficiency virus (HIV) disease is associated with loss of CD4(+) T cells, chronic immune activation, and progressive immune dysfunction. HIV-specific responses, particularly those of CD4(+) T cells, become impaired early after infection, before the loss of responses directed against other antigens; the basis for this diminution has not been elucidated fully. The potential role of CD25(+)CD4(+) regulatory T cells (T reg cells), previously shown to inhibit immune responses directed against numerous pathogens, as suppressors of HIV-specific T cell responses was investigated. In the majority of healthy HIV-infected individuals, CD25(+)CD4(+) T cells significantly suppressed cellular proliferation and cytokine production by CD4(+) and CD8(+) T cells in response to HIV antigens/peptides in vitro; these effects were cell contact dependent and IL-10 and TGF-beta independent. Individuals with strong HIV-specific CD25(+) T reg cell function in vitro had significantly lower levels of plasma viremia and higher CD4(+): CD8(+) T cell ratios than did those individuals in whom this activity could not be detected. These in vitro data suggest that CD25(+)CD4(+) T reg cells may contribute to the diminution of HIV-specific T cell immune responses in vivo in the early stages of HIV disease.