Gary R. Whittaker

Dissecting virus entry via endocytosis

Numerous virus families utilize endocytosis to infect host cells, mediating virus internalization as well as trafficking to the site of replication. Recent research has demonstrated that viruses employ the full endocytic capabilities of the cell. The endocytic pathways utilized include clathrin-mediated endocytosis, caveolae, macropinocytosis and novel non-clathrin, non-caveolae pathways. The tools to study endocytosis and, consequently, virus entry are becoming more effective and specific as the amount of information on endocytic component structure and function increases. The use of inhibitory drugs, although still quite common, often leads to non-specific disruptions in the cell. Molecular inhibitors in the form of dominant-negative proteins have surpassed the use of chemical inhibitors in terms of specificity to individual pathways. Dominant-negative molecules are derived from both structural proteins of endocytosis, such as dynamin and caveolin, and regulatory proteins, primarily small GTPases and kinases. This review focuses on the experimental approaches taken to examine virus entry and provides both classic examples and recent research on a variety of virus families.

Influenza Virus Can Enter and Infect Cells in the Absence of Clathrin-Mediated Endocytosis

ABSTRACT Influenza virus has been described to enter host cells via clathrin-mediated endocytosis. However, it has also been suggested that other endocytic routes may provide additional entry pathways. Here we show that influenza virus may enter and infect HeLa cells that are unable to take up ligands by clathrin-mediated endocytosis. By overexpressing a dominant-negative form of the Eps15 protein to inhibit clathrin-mediated endocytosis, we demonstrate that while transferrin uptake and Semliki Forest virus infection were prevented, influenza virus could enter and infect cells expressing Eps15Δ95/295. This finding is supported by the successful infection of cells with influenza virus in the presence of chemical treatments that block endocytosis, namely, chlorpromazine and potassium depletion. We show also that influenza virus may infect cells incapable of uptake by caveolae. Treatment with the inhibitors nystatin, methyl-β-cyclodextrin, and genistein, as well as transfection of cells with dominant-negative caveolin-1, had no effect on influenza virus infection. By combining inhibitory methods to block both clathrin-mediated endocytosis and uptake by caveolae in the same cell, we demonstrate that influenza virus may infect cells by an additional non-clathrin-dependent, non-caveola-dependent endocytic pathway. We believe this to be the first conclusive analysis of virus entry via such a non-clathrin-dependent pathway, in addition to the traditional clathrin-dependent route.

Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses.

Enveloped viruses often enter cells via endocytosis; however, specific endocytic trafficking pathway(s) for many viruses have not been determined. Here we demonstrate, through the use of dominant‐negative Rab5 and Rab7, that influenza virus (Influenza A/WSN/33 (H1N1) and A/X‐31 (H3N2)) requires both early and late endosomes for entry and subsequent infection in HeLa cells. Time‐course experiments, monitoring viral ribonucleoprotein colocalization with endosomal markers, indicated that influenza exhibits a conventional endocytic uptake pattern – reaching early endosomes after approximately 10 min, and late endosomes after 40 min. Detection with conformation‐specific hemagglutinin antibodies indicated that hemagglutinin did not reach a fusion‐competent form until the virus had trafficked beyond early endosomes. We also examined two other enveloped viruses that are also pH‐dependent for entry – Semliki Forest virus and vesicular stomatitis virus. In contrast to influenza virus, infection with both Semliki Forest virus and vesicular stomatitis virus was inhibited only by the expression of dominant negative Rab5 and not by dominant negative Rab7, indicating an independence of late endosome function for infection by these viruses. As a whole, these data provide a definitive characterization of influenza virus endocytic trafficking and show differential requirements for endocytic trafficking between pH‐dependent enveloped viruses.

Rab GTPases Are Recruited to Chlamydial Inclusions in Both a Species-Dependent and Species-Independent Manner

ABSTRACT Chlamydiae are obligate intracellular bacteria that replicate within an inclusion that is trafficked to the peri-Golgi region where it fuses with exocytic vesicles. The host and chlamydial proteins that regulate the trafficking of the inclusion have not been identified. Since Rab GTPases are key regulators of membrane trafficking, we examined the intracellular localization of several green fluorescent protein (GFP)-tagged Rab GTPases in chlamydia-infected HeLa cells. GFP-Rab4 and GFP-Rab11, which function in receptor recycling, and GFP-Rab1, which functions in endoplasmic reticulum (ER)-to-Golgi trafficking, are recruited to Chlamydia trachomatis, Chlamydia muridarum, and Chlamydia pneumoniae inclusions, whereas GFP-Rab5, GFP-Rab7, and GFP-Rab9, markers of early and late endosomes, are not. In contrast, GFP-Rab6, which functions in Golgi-to-ER and endosome-to-Golgi trafficking, is associated with C. trachomatis inclusions but not with C. pneumoniae or C. muridarum inclusions, while the opposite was observed for the Golgi-localized GFP-Rab10. Colocalization studies between transferrin and GFP-Rab11 demonstrate that a portion of GFP-Rab11 that localizes to inclusions does not colocalize with transferrin, which suggests that GFP-Rab11s association with the inclusion is not mediated solely through Rab11s association with transferrin-containing recycling endosomes. Finally, GFP-Rab GTPases remain associated with the inclusion even after disassembly of microtubules, which disperses recycling endosomes and the Golgi apparatus within the cytoplasm, suggesting a specific interaction with the inclusion membrane. Consistent with this, GFP-Rab11 colocalizes with C. trachomatis IncG at the inclusion membrane. Therefore, chlamydiae recruit key regulators of membrane trafficking to the inclusion, which may function to regulate the trafficking or fusogenic properties of the inclusion.

Role of the Influenza Virus M1 Protein in Nuclear Export of Viral Ribonucleoproteins

ABSTRACT The protein kinase inhibitor H7 blocks influenza virus replication, inhibits production of the matrix protein (M1), and leads to a retention of the viral ribonucleoproteins (vRNPs) in the nucleus at late times of infection (K. Martin and A. Helenius, Cell 67:117–130, 1991). We show here that production of assembled vRNPs occurs normally in H7-treated cells, and we have used H7 as a biochemical tool to trap vRNPs in the nucleus. When H7 was removed from the cells, vRNP export was specifically induced in a CHO cell line stably expressing recombinant M1. Similarly, fusion of cells expressing recombinant M1 from a Semliki Forest virus vector allowed nuclear export of vRNPs. However, export was not rescued when H7 was present in the cells, implying an additional role for phosphorylation in this process. The viral NS2 protein was undetectable in these systems. We conclude that influenza virus M1 is required to induce vRNP nuclear export but that cellular phosphorylation is an additional factor.

Influenza virus entry and infection require host cell N-linked glycoprotein

A widely held view of influenza virus infection is that the viral receptor consists of cell surface carbohydrate sialic acid, which can be present as glycoprotein or glycolipid. Here, we examined influenza virus entry and infection in Lec1 cells, a mutant CHO cell line deficient in terminal N-linked glycosylation caused by a mutation in the N-acetylglucosaminyltransferase I (GnT1) gene. We show that influenza virus cannot infect Lec1 cells, despite having full capacity to undergo virus binding and fusion. Lec1 cells also show no virus replication defect, and infection was restored in Lec1 cells expressing wild-type GnT1. Viruses were apparently arrested at the level of internalization from the plasma membrane and were not endocytosed. Lec1 cells were refractory to infection by several strains of influenza virus, including H1 and H3 strains of influenza A, as well as influenza B virus. Finally, cleavage of N-glycans from wild-type CHO cells markedly reduced infection by influenza virus. We suggest that influenza virus specifically requires N-linked glycoprotein for entry into cells, and that sialic acid, although acting as an efficient attachment factor, is not sufficient as an influenza virus receptor in vivo.

Mechanisms of Coronavirus Cell Entry Mediated by the Viral Spike Protein

Coronaviruses are enveloped positive-stranded RNA viruses that replicate in the cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their envelope with the host cell membrane. The spike glycoprotein (S) mediates virus entry and is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion protein, and is responsible for binding to the receptor on the host cell as well as mediating the fusion of host and viral membranes—A process driven by major conformational changes of the S protein. This review discusses coronavirus entry mechanisms focusing on the different triggers used by coronaviruses to initiate the conformational change of the S protein: receptor binding, low pH exposure and proteolytic activation. We also highlight commonalities between coronavirus S proteins and other class I viral fusion proteins, as well as distinctive features that confer distinct tropism, pathogenicity and host interspecies transmission characteristics to coronaviruses.

Role for Influenza Virus Envelope Cholesterol in Virus Entry and Infection

ABSTRACT Enveloped viruses are highly dependent on their lipid envelopes for entry into and infection of host cells. Here, we have examined the role of cholesterol in the virus envelope, using methyl-β-cyclodextrin depletion. Pretreatment of virions with methyl-β-cyclodextrin efficiently depleted envelope cholesterol from influenza virus and significantly reduced virus infectivity in a dose-dependent manner. A nonenveloped virus, simian virus 40, was not affected by methyl-β-cyclodextrin treatment. In the case of influenza virus, infectivity could be partially rescued by the addition of exogenous cholesterol. Influenza virus morphology, binding, and internalization were not affected by methyl-β-cyclodextrin depletion, whereas envelope cholesterol depletion markedly affected influenza virus fusion, as measured by a specific reduction in the infectivity of viruses induced to fuse at the cell surface and by fluorescence-dequenching assays. These data suggest that envelope cholesterol is a critical factor in the fusion process of influenza virus.

The role of nuclear import and export in influenza virus infection

Infection with influenza virus involves a complex series of nuclear import and export events. Early in infection, incoming viral ribonucleoproteins (vRNPs) are imported into the nucleus. Later, viral transcripts are exported from the nucleus, newly synthesized structural proteins are transported back into the nucleus and, finally, newly assembled vRNPs are exported. All these import and export steps, and, in particular, the bidirectional traffic of vRNPs rely on the transport machinery of the cell, but are regulated both by viral and cellular factors. The viral MI protein serves as the master organizer in determining the directionality of vRNP transport.

Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites

The coronavirus spike protein (S) plays a key role in the early steps of viral infection, with the S1 domain responsible for receptor binding and the S2 domain mediating membrane fusion. In some cases, the S protein is proteolytically cleaved at the S1–S2 boundary. In the case of the severe acute respiratory syndrome coronavirus (SARS-CoV), it has been shown that virus entry requires the endosomal protease cathepsin L; however, it was also found that infection of SARS-CoV could be strongly induced by trypsin treatment. Overall, in terms of how cleavage might activate membrane fusion, proteolytic processing of the SARS-CoV S protein remains unclear. Here, we identify a proteolytic cleavage site within the SARS-CoV S2 domain (S2′, R797). Mutation of R797 specifically inhibited trypsin-dependent fusion in both cell–cell fusion and pseudovirion entry assays. We also introduced a furin cleavage site at both the S2′ cleavage site within S2 793-KPTKR-797 (S2′), as well as at the junction of S1 and S2. Introduction of a furin cleavage site at the S2′ position allowed trypsin-independent cell–cell fusion, which was strongly increased by the presence of a second furin cleavage site at the S1–S2 position. Taken together, these data suggest a novel priming mechanism for a viral fusion protein, with a critical proteolytic cleavage event on the SARS-CoV S protein at position 797 (S2′), acting in concert with the S1–S2 cleavage site to mediate membrane fusion and virus infectivity.