Quantitative Proteomic Analysis of Host-virus Interactions Reveals a Role for Golgi Brefeldin A Resistance Factor 1 (GBF1) in Dengue Infection

Dengue virus is considered to be the most important mosquito-borne virus worldwide and poses formidable economic and health care burdens on many tropical and subtropical countries. Dengue infection induces drastic rearrangement of host endoplasmic reticulum membranes into complex membranous structures housing replication complexes; the contribution(s) of host proteins and pathways to this process is poorly understood but is likely to be mediated by protein-protein interactions. We have developed an approach for obtaining high confidence protein-protein interaction data by employing affinity tags and quantitative proteomics, in the context of viral infection, followed by robust statistical analysis. Using this approach, we identified high confidence interactors of NS5, the viral polymerase, and NS3, the helicase/protease. Quantitative proteomics allowed us to exclude a large number of presumably nonspecific interactors from our data sets and imparted a high level of confidence to our resulting data sets. We identified 53 host proteins reproducibly associated with NS5 and 41 with NS3, with 13 of these candidates present in both data sets. The host factors identified have diverse functions, including retrograde Golgi-to-endoplasmic reticulum transport, biosynthesis of long-chain fatty-acyl-coenzyme As, and in the unfolded protein response. We selected GBF1, a guanine nucleotide exchange factor responsible for ARF activation, from the NS5 data set for follow up and functional validation. We show that GBF1 plays a critical role early in dengue infection that is independent of its role in the maintenance of Golgi structure. Importantly, the approach described here can be applied to virtually any organism/system as a tool for better understanding its molecular interactions.Viruses modify the intracellular environment of infected host cells in a number of important ways, including subverting the antiviral response, reorganizing host membranes, and manipulating host signaling pathways to create an environment more favorable for infection. For example, some viral proteins co-opt host proteins to degrade host interferon signaling components, thus antagonizing the antiviral response (1, 2); other viral proteins recruit metabolic enzymes that are potentially involved in the biogenesis of replication complexes (RCs)¹ (3); and some viral proteins interact with host regulatory proteins to block the cellular stress response (4). These examples illustrate only a few of the ways in which viral-host protein-protein interactions (PPIs) enable the viral life cycle and drive pathogenicity. Because of the limited coding capacity of many viral genomes, in particular RNA virus genomes, viral-host PPIs generally occur between a remarkably small number of viral proteins and a much larger number of host proteins (5). The study of these extensive interactions necessitates comprehensive and quantitative approaches, the development and validation of which will potentially contribute to: 1) our understanding of the mechanisms by which viruses subvert cellular pathways to their own advantage; 2) our understanding of fundamental cell biology; 3) the choice of potential drug targets and the rational design of such drugs; and 4) our understanding of the host response to infection.Dengue virus (DENV) is a positive-sense, single stranded RNA virus in the family Flaviviridae that is transmitted by the bite of an infected Aedes mosquito (6). DENV is an important emerging pathogen that is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, diseases which cumulatively pose formidable economic and health care burdens in many tropical and subtropical countries worldwide (7). Recent estimates of the global burden of DENV infection have revealed that DENV infection is ∼threefold more prevalent than previously estimated, with ∼400 million annual incidences worldwide (8). Moreover, development of an anti-DENV vaccine has been hindered by the existence of four antigenically distinct DENV serotypes (DENV-1, -2, -3, and -4), each of which is capable of producing the full spectrum of DENV-induced disease (9). DENV is also related to other flaviviruses that cause significant human disease, including yellow fever virus, West Nile virus, and Japanese encephalitis virus (10). Thus, insights into DENV biology may be applicable to other flaviviruses of medical importance.The flavivirus genome encodes only three structural (C, pr/M, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), and is translated as a single polyprotein, which is later cleaved into the mature viral proteins (6). The three structural proteins, capsid (C), membrane (M), and envelope (E) comprise the virion, whereas the NS proteins are mainly responsible for carrying out genome replication in infected cells. Among the seven NS proteins, NS5 and NS3 are the two largest and most highly conserved proteins (11); moreover, each possesses multiple enzymatic activities. NS5 contains an RNA-dependent RNA polymerase domain as well as a nucleoside-2′-O-methyltransferase domain; both of these activities are essential for replication (12, 13). NS3, on the other hand, possesses an N-terminal serine protease domain, which is responsible for cleaving the viral polyprotein at several sites (along with its cofactor, NS2B) (14). The C-terminal domain of NS3 has 5′ RNA triphosphatase, nucleoside triphosphatase, and helicase activities (15–17). NS5 and NS3 have been shown to interact in infected cells (18), most likely in the RC. The precise composition and biogenesis mechanisms of RCs are poorly understood, but likely involve host proteins as well as viral proteins. As with other viruses, DENV-host PPIs have been interrogated by a number of high-throughput yeast two-hybrid assays (19–31) and approaches coupling either affinity purification (AP), immunoprecipitation, or immunoaffinity purification (IP) with MS (32–35). These approaches have yielded a number of putative DENV-host PPIs; however, considering the large repertoire of interactions undertaken by other viruses (36–41), our knowledge of DENV-host PPIs is likely incomplete. One advantage of IP/MS approaches is their potential to comprehensively reveal bona fide time-resolved interactions from the environment of an infected cell; however, the extremely high sensitivity of modern mass spectrometers highlights the need to develop IP/MS workflows capable of reliably discriminating between genuine interactors and nonspecific contaminants (42). Here, we present a workflow incorporating immunoaffinity purification and quantitative proteomics from infected cells, followed by robust statistical analysis to identify high confidence interactors of virtually any protein of interest, and apply this workflow to DENV NS5 and NS3.     

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.