A novel coronavirus (CoV) has been described in association with cases of severe acute respiratory syndrome (SARS). The virus, SARS-CoV, differs from the previously described human coronaviruses, 229E and OC43. 229E was previously shown to productively infect human monocytes/macrophages, whereas OC43 poorly infected the cells. In this study, we examined whether SARS-CoV could productively infect purified monocytes/macrophages (PM) derived from human donor cells. Unlike 229E-infected cells, which produced viral titers of 10(3.5) to 10(6)TCID50/ml, SARS-CoV replicated poorly in PM, producing titers of 10(1.75) to 10(2)TCID50/ml. This finding was similar to results reported for OC43-infected cells, with titers ranging from 10(1.2) to 10(2.7)TCID50/ml. Of interest, SARS-CoV proteins were detected only in PM that did not produce significant amounts of interferon (IFN)-alpha, and in one such case, preliminary electron microscope studies demonstrated that SARS-CoV-like particles could enter the cells, possibly via phagocytosis. These results suggest that SARS-CoV, like human CoV OC43, poorly infects human PM, and production of IFN-alpha by these cells further limits the infection. Given the importance of monocytes/macrophages to the immune response, it is possible that their infection by SARS-CoV and alteration of this infection by IFN-alpha may be important to the course of the infection in humans.
Rab proteins and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. By using gene trap insertional mutagenesis, we identified Rab9, which mediates lateendosome-to-trans-Golgi-network trafficking, among several candidate host genes whose disruption allowed the survival of Marburg virus-infected cells, suggesting that Rab9 is utilized in Marburg replication. Although Rab9 has not been implicated in human immunodeficiency virus (HIV) replication, previous reports suggested that the late endosome is an initiation site for HIV assembly and that TIP47-dependent trafficking out of the late endosome to the trans-Golgi network facilitates the sorting of HIV Env into virions budding at the plasma membrane. We examined the role of Rab9 in the life cycles of HIV and several unrelated viruses, using small interfering RNA (siRNA) to silence Rab9 expression before viral infection. Silencing Rab9 expression dramatically inhibited HIV replication, as did silencing the host genes encoding TIP47, p40, and PIKfyve, which also facilitate late-endosome-to-trans-Golgi vesicular transport. In addition, silencing studies revealed that HIV replication was dependent on the expression of Rab11A, which mediates trans-Golgi-to-plasma-membrane transport, and that increased HIV Gag was sequestered in a CD63؉ endocytic compartment in a cell line stably expressing Rab9 siRNA. Replication of the enveloped Ebola, Marburg, and measles viruses was inhibited with Rab9 siRNA, although the nonenveloped reovirus was insensitive to Rab9 silencing. These results suggest that Rab9 is an important cellular target for inhibiting diverse viruses and help to define a late-endosome-toplasma-membrane vesicular transport pathway important in viral assembly.Most developed antiviral drugs have been designed to inhibit the function of viral proteins. In the case of human immunodeficiency virus (HIV), an unfortunate consequence of using drugs that target viral proteins has been the emergence of drug-resistant virions having compensatory genetic mutations (8,20). An alternative approach is to identify cellular genes essential for the viral life cycle, but not essential for the more genetically diverse host cell, and then to develop agents that inhibit their expression or function. In principle, such an approach may pose an insurmountable barrier against viral replication, as resistance would arise from a complex adaptation to use a different cellular protein for viral replication. There are several examples where a partial or complete resistance to pathogens results from the loss of expression or function of a critical host gene. Examples include the high degree of protection against HIV transmission afforded to individuals homozygous for a dysfunctional allele of CCR5 (a major HIV coreceptor) and the complete resistance to Plasmodium vivax malaria infection of individuals lacking expression of the erythrocyte receptor DARC (26,38).We have used gene trap insertional mutagenesis (58) as a high-throughput for...
We have examined trafficking of major histocompatibility complex (MHC) class II molecules in human B cells exposed to concanamycin B, a highly specific inhibitor of the vacuolar H(+)‐ATPases required for acidification of the vacuolar system and for early to late endosomal transport. Neutralization of vacuolar compartments prevents breakdown of the invariant chain (Ii) and blocks conversion of MHC class II molecules to peptide‐loaded, SDS‐stable alpha beta dimers. Ii remains associated with alpha beta and this complex accumulates internally, as ascertained biochemically and by morphological methods. In concanamycin B‐treated cells, a slow increase (> 20‐fold) in surface expression of Ii, mostly complexed with alpha beta, is detected. This surface‐disposed fraction of alpha beta Ii is nevertheless a minor population that reaches the cell surface directly, or is routed via early endosomes as intermediary stations. In inhibitor‐treated cells, the bulk of newly synthesized alpha beta Ii is no longer accessible to fluid phase endocytic markers. It is concluded that the majority of alpha beta Ii is targeted directly from the trans‐Golgi network to the compartment for peptide loading, bypassing the cell surface and early endosomes en route to the endocytic pathway.
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