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...

Background: Host genes serving potential roles in virus replication may be exploited as novel antiviral targets. Methods: Small interfering RNA (siRNA)-mediated knockdown of host gene expression was used to validate candidate genes in screens against six unrelated viruses, most importantly influenza. A mouse model of influenza A virus infection was used to evaluate the efficacy of a candidate FDA-approved drug identified in the screening effort. Results: Several genes in the PI3K-AKT-mTOR pathway were found to support broad-spectrum viral replication in vitro by RNA interference. This led to the discovery that everolimus, an mTOR inhibitor, showed in vitro antiviral activity against cowpox, dengue type 2, influenza A, rhino-and respiratory syncytial viruses. In a lethal mouse infection model of influenza A (H1N1 and H5N1) virus infection, everolimus treatment (1 mg/ kg/day) significantly delayed death but could not prevent mortality. Fourteen days of treatment was more beneficial in delaying the time to death than treatment for seven days. Pathological findings in everolimustreated mice showed reduced lung haemorrhage and lung weights in response to infection. Conclusions: These results provide proof of concept that cellular targets can be identified by gene knockout methods, and highlight the importance of the PI3K-AKT-mTOR pathway in supporting viral infections.

Two genetically distinct lineages of H1N1 influenza A viruses, circulated worldwide before 1994, were antigenically indistinguishable. In 1994, viruses emerged in China, including A/Beijing/262/ 95, with profound antigenic differences from the contemporary circulating H1N1 strains. Haemagglutinin sequence comparisons of either a predecessor virus, A/Hebei/52/94, or one representative of the cocirculating A/Bayern/7/95-like clade, A/Shenzhen/227/95, revealed a deletion of K at position 134 (H3 numbering) in the antigenic variants. The K134 deletion conferred a selective advantage to the Chinese deletion lineage, such that it eventually gave rise to currently circulating H1 viruses. Using reverse genetics to generate viruses with either an insertion or deletion of aa 134, we have confirmed that the K134 deletion, rather than a constellation of sublineage specific amino acid changes, was sufficient for the antigenic difference observed in the Chinese deletion lineage, and reinsertion of K134 revealed the requirement of a compatible neuraminidase surface glycoprotein for viral growth.Approximately 20 % of the world's population is infected by influenza A each year, resulting in significant mortality and morbidity (Stohr, 2002). The high incidence of influenza cases is attributable to the ability of the influenza virus to escape immunity induced by prior infection or vaccination. This escape is potentiated by the accumulation of mutations in the surface glycoproteins haemagglutinin (HA), and to a lesser extent neuraminidase (NA), which confer antigenic change to the virus. This phenomenon, known as antigenic drift, necessitates annual vaccine updates to confer protection against the currently circulating strains.Mutations in the HA molecule are considered to contribute almost entirely to the antigenic drift observed among influenza A viruses (Wilson & Cox, 1990). Those sites at the distal tip of the H1 HA molecule and on the side of the globular head near the receptor-binding pocket appear to be the main targets of the human immune response (Cox & Brokstad, 1999;Raymond et al., 1986;Sato et al., 2004). Influenza A viruses bind to sialic acids on the surface of target cells via a depression in the distal surface of the globular head of the HA molecule (Weis et al., 1988;Wilson et al., 1981). Several residues within this depression are highly conserved across the HA subtypes, including residues 98, 134, 138, 153 and 183 (H3 numbering) (Nobusawa et al., 1991). Amino acid substitutions within the receptor-binding pocket or the 'second shell' residues, including 190, 225 and 158, may alter the specificity toward certain types of galactosidic linkages, namely a2-6Gal or a2-3Gal linkages (Aytay & Schulze, 1991; Matrosovich et al., 2000). Because of its location on the HA threedimensional structure, mutations in the receptor-binding pocket or second shell residues can alter the antigenicity of a virus in addition to, or instead of, modifying receptor specificity or affinity (Daniels et al., 1984).The HA1 domains of the HA g...

No abstract