The budded virus (BV) of the Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV) infects insect cells and transduces mammalian cells mainly through the endocytosis pathway. However, this study revealed that the treatment of the virus bound to Sf9 cells at low pH could efficiently rescue the infectivity of AcMNPV in the presence of endocytosis pathway inhibitors. A colocalization assay of the major capsid protein VP39 with the early endosome marker EEA1 showed that at low pH, AcMNPV entered Sf9 cells via an endosome independent pathway. Using a fluorescent probe (R18), we showed that at low pH, the viral nucleocapsid entered Sf9 cells via direct fusion at the cell surface. By using the myosin specific inhibitor 2, 3-butanedione monoxime (BDM) and microtubule inhibitor nocodazole, the low pH triggered direct fusion was demonstrated to be dependent on myosin-like proteins and independent of microtubules. RT-PCR of the IE1 gene as a marker for viral entry showed that the kinetics of AcMNPV in cells triggered by low pH was similar to that of the normal entry via endocytosis. The low pH mediated infection assay and VP39 and EEA1 co-localization assay also demonstrated that AcMNPV could efficiently transduce mammalian cells via direct membrane fusion at the cell surface. More importantly, we found that a low pH trigger could significantly improve the transduction efficiency of AcMNPV in mammalian cells, leading to the potential application of this method when using baculovirus as a vector for heterologous gene expression and for gene therapy.

Interferon-stimulated expression and conjugation of the ubiquitin-like modifier ISG15 restricts replication of several viruses. Here, we established complete E1-activating, E2-conjugating, and E3-ligase dependent expression systems for assaying both human and mouse ISGylation. We confirm that human HerC5, but not human HerC6, has ISG15 E3 ligase activity, and identify mouse HerC6 as a bona fide ISG15 E3 ligase. Furthermore, we demonstrate that influenza B virus NS1 protein potently antagonizes human, but not mouse ISGylation, a property dependent on B/NS1 binding the N-terminal domain of human, but not mouse, ISG15. Using chimeric human/mouse ISG15 constructs we show that the B/NS1:ISG15 interaction is both necessary and sufficient to inhibit ISGylation regardless of the ligation machinery used. Inability to block ISGylation in certain species may contribute to limiting influenza B virus host-range.

The complex natural cycle of vectored viruses that transition between host species, such as between insects and mammals, makes understanding the full life cycle of the virus an incredibly complex problem. Sindbis virus, an Arbovirus and prototypic alphavirus having an inner protein shell and an outer glycoprotein coat separated by a lipid membrane, is one example of a vectored virus that transitions between vertebrate and insect hosts. While evidence of host-specific differences in Sindbis has been observed, no work has been performed to characterize the impact of the host species on the structure of the virus. Here, we report the first study of the structural differences between Sindbis grown in mammalian and insect cells, which were determined by small-angle neutron scattering (SANS), a non-destructive technique that did not decrease the infectivity of the Sindbis virus particles studied. The scattering data and modeling show that while the radial position of the lipid bilayer does not change significantly, it was possible to conclude that it does have significantly more cholesterol when grown in mammalian cells. Additionally, the outer protein coat was found to be more extended in the mammalian Sindbis. The SANS data also demonstrated that the RNA and nucleocapsid protein share a closer interaction in the mammalian-grown virus than in the virus from insect cells.

The replication protein NS1 is essential for genome replication and protein production in parvoviral infection. Many of its functions, including recognition and site-specific nicking of the viral genome, helicase activity, and transactivation of the viral capsid promoter, are dependent on ATP. An ATP-binding pocket resides in the middle of the modular NS1 protein in a superfamily 3 helicase domain. Here, we have identified key ATP binding amino acid residues in canine parvovirus (CPV) NS1 protein and mutated amino acids from conserved A motif (K406), B motif (E444 and E445), and positively charged region (R508 and R510). All mutations prevented the formation of the infectious viruses. When provided in trans, all except R508A, reduced infectivity in a dominant-negative manner, possibly by hindering genome replication. These results suggest that the conserved R510, but not R508, is the arginine finger sensory element of CPV NS1. Moreover, the fluorescence recovery after photobleaching technique (FRAP), complemented by computer simulations, was used to assess the binding properties of mutated fluorescent fusion proteins. These experiments identified ATP dependent and independent binding modes for NS1 in living cells. Only the K406M mutant had a single binding site, which was concluded to indicate ATP-independent binding. Furthermore, our data suggests that DNA binding of NS1 is dependent on its ability to both bind and hydrolyze ATP.

Poxvirus based HIV vaccine candidates are currently under evaluation in preclinical and clinical trials. Modified Vaccinia Virus Ankara (MVA) vectors have excellent safety and immunogenicity records, but their behaviour in human cell cultures remains only partly characterized. We studied here various virological and immunological aspects of the interactions of MVA-HIV, a vaccine candidate developed by the ANRS, with primary human cells. We report that MVA-HIV infects and drives Gag expression in primary macrophages, DCs, epithelial and muscle cells. MVA-HIV infected DCs matured, efficiently presented Gag, Pol and Nef antigens and activated HIV-specific CTLs. As expected with this type of vector, infection was cytopathic and lead to DC apoptosis. Coculture of MVA-HIV infected epithelial cells or myotubes with DCs promoted efficient Gag antigen MHC-I cross-presentation without inducing direct infection and death of DCs. APCs infected with MVA-HIV also activated HIV-specific CD4+ T cells. Moreover, exposure of DCs to MVA-HIV, or to MVA-HIV infected myotubes, induced type-I IFN production, and inhibited subsequent HIV replication and transfer to lymphocytes. Altogether, these results show that MVA-HIV promotes efficient MHC-I- and MHC-II-presentation of HIV antigens by APCs without facilitating HIV replication. Deciphering the immune responses to MVA in culture experiments will help design innovative vaccine strategies.

Recently, mutations in the connection subdomain (CN) and RNase H domain (RH) of HIV-1 reverse transcriptase (RT) were observed to exhibit dual resistance to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs). To elucidate the mechanism by which CN and RH mutations confer resistance to NNRTIs, we hypothesized that these mutations reduce RNase H cleavage and provide more time for the NNRTI to dissociate from the RT, resulting in the resumption of DNA synthesis and enhanced NNRTI resistance. We observed that the effect of the reduction in RNase H cleavage on NNRTI resistance is dependent upon the affinity of each NNRTI to the RT and further influenced by the presence of NNRTI-binding pocket (BP) mutants. Mutants D549N, Q475A and Y501A, which reduce RNase H cleavage, enhance resistance to nevirapine (NVP) and delavirdine (DLV), but not to efavirenz (EFV) and etravirine (ETR), consistent with their increase in affinity for RT. Combining D549N with NNRTI BP mutants further increases NNRTI resistance from 3- to 30-fold, supporting the role of NNRTI-RT affinity in our NNRTI resistance model. We also demonstrated that CNs from treatment-experienced patients, previously reported to enhance NRTI resistance, also reduce RNase H cleavage and enhance NNRTI resistance in the context of the patient RT pol domain or a wild-type pol domain. Together, these results confirm key predictions of our NNRTI resistance model and provide support for a unifying mechanism by which CN and RH mutations can exhibit dual NRTI and NNRTI resistance.

Most HIV-infected individuals develop antibodies to the gp120 and gp41 components of the viral spike; however, only a fraction of these mount a broadly neutralizing serum response against HIV. We have cloned anti-HIV antibodies from the memory B cell compartment of six individuals with variable viral loads and high titers of broadly neutralizing antibodies. Here we report on the features of the anti-gp41 response in these patients. Competition experiments with previously characterized antibodies targeting defined epitopes on the gp41 ectodomain showed antibodies directed against the "immunodominant region" (cluster I), the carboxy-terminal heptad repeat (cluster II), and the membrane proximal external region (cluster IV). On the other hand, antibodies directed against the amino-terminal part of the molecule, including the fusion peptide, polar region and the N-terminal heptad repeat were not detected. When all patients' data was combined, unique B cell clones targeting the cluster I, II and IV accounted for 32%, 49% and 53% of all anti-gp41 reactive B cells, respectively; therefore, no single region was truly immunodominant. Finally, although we found no new neutralizing epitopes or HIV-1 neutralizing activity by any of the gp41 antibodies at concentrations of up to 50 mug/ml, high concentrations of 7 out of 15 anti-cluster I antibodies neutralized tier 2 viruses.

Extensive cell culture passage of the virulent Bucyrus (VB) strain of equine arteritis virus (EAV) to produce the modified live virus (MLV) vaccine strain has altered its tropism for equine CD3(+) T lymphocytes and CD14(+) monocytes. The VB strain primarily infects CD14(+) monocytes and a small subpopulation of CD3(+) T lymphocytes (predominantly CD4(+) T) as determined by dual-color flow cytometry. In contrast, the MLV vaccine strain has significantly reduced ability to infect CD14(+) monocytes and has lost its capability to infect CD3(+) T lymphocytes. Using a panel of five recombinant chimeric viruses, we demonstrated that interactions between GP2, GP3, GP4, GP5 and M envelope proteins play a major role in determining the CD14(+) monocyte tropism while the tropism of CD3(+) T lymphocytes is determined by GP2, GP4, GP5 and M envelope proteins but not the GP3 protein. The data clearly suggest that there are intricate interactions among these envelope proteins that affect binding of EAV to different cell receptors on CD3(+) T lymphocytes and CD14(+) monocytes. This study shows for the first time that CD3(+) T lymphocytes may play an important role in the pathogenesis of equine viral arteritis when horses are infected with the virulent strains of EAV.

Rhesus lymphocryptovirus (rLCV) and Epstein-Barr virus (EBV) are closely related gamma-herpesviruses that infect and cause disease in rhesus monkeys and humans, respectively. Thus, rLCV is an important model system for EBV pathogenesis. Both rLCV and EBV express microRNAs (miRNAs), several conserved in sequence and genomic location. We have applied deep sequencing technology to obtain an inventory of rLCV miRNA expression in latently rLCV-infected monkey B-cells. Our data confirm the presence of all previously identified mature rLCV miRNAs and have resulted in the discovery of 21 new mature miRNAs arising from previously identified precursor (pre)-miRNAs, as well as two novel pre-miRNAs (rL1-34 and rL1-35) that together generate four new mature miRNAs. Thus, the total number of rLCV-encoded pre-miRNAs is 35, and the total number of rLCV mature miRNAs is 68, the most of any virus examined. The exact 5' and 3' ends of all mature rLCV miRNAs were pinpointed, many showing marked sequence and length heterogeneity that could modulate function. We further demonstrate that rLCV mature miRNAs associate with Argonaute proteins in rLCV-infected B-cells.

KSHV is a B-lymphotropic herpesvirus strongly linked to both lymphoproliferative diseases and to Kaposi's sarcoma. The viral latency program of KSHV is central to persistent infection and plays important roles in the pathogenesis of KSHV-related tumors. Up to six polypeptides and 18 microRNAs are known to be expressed in latency, but it is unclear if all major latency genes have been identified. Here we have employed array-based transcript profiling and limiting dilution RT-PCR methodologies to explore this issue in several KSHV-infected cell lines. Our results show that RNAs encoding the K1 protein are found at low levels in most latently infected cell lines. The gene encoding vIL6 is also expressed as a latent transcript in some contexts. Both genes encode powerful signaling molecules with particular relevance to B cell biology: K1 mimics signaling through the B cell receptor, and vIL6 promotes B cell survival. These data resolve earlier controversies about K1 and vIL6 expression and indicate that, in addition to core latency genes, some transcripts can be expressed in KSHV latency in a context-dependent manner.

The late expression factor 2 (lef-2) gene of baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been identified as one of the factors essential for origin-dependent DNA replication in transient expression assays and has been shown to be involved in the late/very late gene expression. To study the function of lef-2 in the life cycle of AcMNPV, a lef-2 knockout mutant and repair bacmids were generated by homologous recombination in Escherichia coli. Growth curve analysis showed that lef-2 was essential for virus production. Interestingly, DNA replication assay indicated that lef-2 is not required for the initiation of viral DNA replication; rather, it is required for the amplification of DNA replication. lef-2 is also required for the expression of late and very late genes, as the expression of these genes were abolished in the lef-2 knockout bacmid. Temporal and spatial distribution of LEF-2 protein in infected cells was also analyzed and the data showed that LEF-2 protein was localized to the virogenic stroma in the nuclei of the infected cells. Analysis of purified virus particles revealed that LEF-2 is a new viral protein component of both budded and occlusion-derived virions, predominantly in the nucleocapsids of the virus particles. This observation suggests that LEF-2 may be required immediately after virus entry into host cells for efficient viral DNA replication.

APOBEC3F (A3F) and APBOBEC3G (A3G) are both host restriction factors that can potently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Their antiviral activities are at least partially mediated by cytidine deamination which causes lethal mutations of the viral genome. We recently showed that A3G blocks viral plus-strand DNA transfer and inhibits provirus establishment in the host genome. Here, we investigated whether A3F similarly interferes with HIV-1 provirus formation. We observed that both A3F and A3G inhibit viral DNA synthesis and integration, but A3F is more potent than A3G in preventing viral DNA integration. We further investigated the mechanisms by which A3F and A3G block viral DNA integration by analyzing their effects on viral cDNA processing using Southern blotting analysis. A3G generates a 6-bp extension at the viral U5 end of 3'-LTR, which is a poor substrate for integration; in contrast, A3F inhibits viral DNA integration by reducing 3' processing of viral DNA at both the U5 and U3 ends. Furthermore, we demonstrated that a functional C-terminal catalytic domain is more critical for A3G than A3F function in blocking HIV-1 provirus formation. Finally, we showed that A3F has a greater binding affinity for a viral 3'-LTR dsDNA oligonucleotide template than A3G. Taken together, we demonstrated that mechanisms utilized by A3F to prevent HIV-1 viral DNA integration were different from A3G, and that their target specificities and/or their affinities for dsDNA may contribute to their distinct mechanisms.

Hantaviruses (family Bunyaviridae) are rodent-borne emerging viruses causing a serious, worldwide threat to human health. Hantavirus diseases include hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. Virions are enveloped and contain a tripartite single-stranded negative-sense RNA genome. Two types of glycoproteins, GN and GC, are embedded in the viral membrane and form protrusions, or 'spikes'. The membrane encloses a ribonucleoprotein core, which consists of the RNA segments, the nucleocapsid protein and the RNA-dependent RNA polymerase. Detailed information on hantavirus virion structure and glycoprotein spike composition is scarce. Here, we have studied the structures of Tula hantavirus virions using electron cryo-microscopy and tomography. Three-dimensional density maps show how the hantavirus surface glycoproteins, membrane and ribonucleoprotein are organized. The structure of the GN-GC spike complexwas solved to 3.6 nm resolution by averaging of tomographic sub-volumes. Each spike complex is a square-shaped assembly with four-fold symmetry. Spike complexes formed ordered patches on the viral membrane by means of specific lateral interactions. These interactions may be sufficient for creating membrane curvature during virus budding. In conclusion, the structure and assembly principles of Tula hantavirus exemplify a unique assembly paradigm for enveloped viruses.

Borna disease virus (BDV) frequently persists in the brain of infected animals. To analyze viral dissemination in the mouse nervous system, we generated a mouse-adapted virus that expresses GFP. This viral vector supported GFP expression for up to 150 days and possessed an extraordinary staining capacity, visualizing complete dendritic arbors as well as individual axonal fibers of infected neurons. GFP-positive cells were first detected in cortical areas from where the virus disseminated through the entire CNS. Late in infection, GFP expression was found in the sciatic nerve, demonstrating viral spread from the central to the peripheral nervous system.

The E protein of most flaviviruses is modified by Asn-linked glycosylation at residue 153/154 and in the case of the four dengue virus (DENV) serotypes by a second glycan at residue 67. However, the absence of E protein glycosylation among numerous natural isolates of different flaviviruses suggests that the glycan, per se, is not critically important in the virus life cycle. Consistent with this notion, we show that ablation of both glycans from the DENV-2 E protein reduces, but does not prevent growth of the variant in mammalian and mosquito cells. We found a pronounced and opposing effect of glycan ablation on two stages of the virus growth cycle: infectivity and release. Loss of either of the two DENV E protein glycans markedly enhanced infectivity of variants for mosquito cells at the expense of efficient virion release. The variants also displayed reduced release in mammalian cells, which was more prominent for viruses lacking the Asn 67- than Asn 153-linked glycan, without a marked change in infectivity. Mutations, which compensated for the defect in virus morphogenesis associated with ablation of the Asn 67-linked glycan in mammalian cells, but interestingly not in mosquito cells, were identified at the glycosylation acceptor motif and a second site in E protein domain II. The duelling influences of infectivity and release on virus growth affected by the glycans may explain the plasticity in E protein glycosylation among the flaviviruses.

After membrane fusion with a target cell, the core of human immunodeficiency virus type 1 (HIV-1) enters into the cytoplasm where uncoating occurs. The cone-shaped core is composed of the viral capsid protein (CA), which disassembles during uncoating. The underlying factors and mechanisms governing uncoating are poorly understood. Several CA mutations can cause changes in core stability and a block at reverse transcription, demonstrating the requirement for optimal core stability during viral replication. HIV-1 integrase (IN) catalyzes the insertion of the viral cDNA into the host genome, and certain IN mutations are pleiotropic. Similar to some CA mutants, two IN mutants, one with a complete deletion of IN (NL-DeltaIN) and the other with a Cys to Ser substitution (NL-C130S), were noninfectious with a replication block at reverse transcription. When compared to wildtype (WT), the cytoplasmic CA levels of the IN mutants in infected cells were reduced, suggesting an accelerated uncoating. The role of IN during uncoating was examined by isolating and characterizing cores from NL-DeltaIN and NL-C130S. Both IN mutants could form functional cores, but the core yield and stability were decreased. Also, virion incorporation of cyclophilin A (CypA), a cellular peptidyl-prolyl isomerase that binds specifically to CA, was decreased in the IN mutants. Cores isolated from WT virus depleted of CypA had an unstable core phenotype, confirming a role of CypA in promoting optimal core stability. Taken together, our results indicate that IN is required during uncoating by maintaining CypA-CA interaction, which promotes optimal stability of the viral core.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer in sheep that shares similarities with human bronchioloalveolar carcinoma (BAC). JSRV is unique because the envelope gene (env) is the oncogene, as it can transform cells in culture and induce tumors in animals. The PI3K-Akt-mTOR and H/N-Ras-MEK-MAPK pathways have been shown to be critical for Env transformation. However, the question still remains how disruption of these pathways relate to tumor formation. To address this, JSRV Env transformation was studied in the context of epithelial structure using the polarized Madin-Darby Canine Kidney epithelial cell (MDCK) 3-D culture system. Results indicate that JSRV Env transformed MDCK acini in that the structures were larger in size and had full or multiple lumens in contrast to single lumens observed in controls. The altered phenotype was largely mediated by an increase in proliferation in addition to overcoming the proliferative suppression signal. JSRV Env was not found to disrupt polarity, tight junctions or inhibit lumen apoptosis. The PI3K-Akt-mTOR pathway was important for Env transformation in MDCK cells although the mechanisms of action differed in 3-D compared to monolayer culture. PI3K-dependent signaling to mTOR occurred in monolayer while PI3K-independent signaling to mTOR occurred in 3-D culture. In contrast, the H/N-Ras-MEK-MAPK pathway was found to be inhibitory to transformation in both normal and transformed MDCK cells in 3-D culture. However, in monolayer culture inhibition of MEK reverted the transformed phenotype suggesting different mechanism(s) of action in monolayer vs. 3-D culture.

The genomic RNA of retroviruses and retrovirus-like transposons must be sequestered from the cellular translational machinery so that it can be packaged into viral particles. Eukaryotic mRNA processing bodies (P bodies) play a central role in segregating cellular mRNAs from the translational machinery for storage or decay. In this work, we provide evidence that the RNA of the yeast Ty1 retrotransposon is packaged into virus-like particles (VLPs) in P bodies. Ty1 RNA is translationally repressed and Ty1 Gag, the capsid and RNA binding protein, accumulates in discrete cytoplasmic foci, a subset of which localize to P bodies. Human APOBEC3G, a potent Ty1 restriction factor that is packaged into Ty1 VLPs via an interaction with Gag, also localizes to P bodies. The association of APOBEC3G with P bodies does not require Ty1 element expression, suggesting that P body localization of APOBEC3G and Ty1 Gag precedes VLP assembly. Additionally, we report that two P body-associated 5' to 3' mRNA decay pathways, deadenylation-dependent decay (DDD) and nonsense-mediated decay (NMD) stimulate Ty1 retrotransposition. The additive contributions of DDD and NMD explain the strong requirement for general 5' to 3' mRNA degradation factors, Dcp1, Dcp2 and Xrn1 in Ty1 retromobility. 5' to 3' decay factors act at a posttranslational step in retrotransposition, and Ty1 RNA packaging into VLPs is abolished in the absence of the 5' to 3' exonuclease, Xrn1. Together, the results suggest that VLPs assemble in P bodies and that 5' to 3' mRNA decay is essential for packaging of Ty1 RNA in VLPs.

Human papillomaviruses (HPV) link their life cycles to epithelial differentiation and induce productive replication of viral DNA in suprabasal cells. Viral DNA amplification requires cells to remain active in the cell cycle upon differentiation. This is in contrast to normal cells, which lose proliferative capability upon differentiation. One factor that negatively regulates proliferative capability upon differentiation is microRNA-203 (miR-203), which is expressed primarily in suprabasal epithelial cells. Although HPVs do not encode their own microRNAs (miRNAs), they modulate expression of cellular miRNAs to regulate the activity of cellular proteins. We show that the E7 protein downregulates expression of miR-203 expression upon differentiation which may occurs through the MAP kinase/PKC pathway. One target of miR-203 is the p63 family of transcription factors and we demonstrate that HPV positive cells maintain significantly higher levels of these factors upon differentiation than in normal keratinocytes. Several downstream targets of p63, CARM-1, p21 and Bax, were also increased in E7 expressing cells and their levels inversely correlated with amounts of miR-203. Introduction of expression vectors for miR-203 into keratinocytes that stably maintain HPV episomes resulted in short term elevation of HPV genome copy numbers but these were rapidly lost upon subsequent passage. When HPV positive cells expressing high levels of miR-203 were induced to differentiate in methylcellulose, impaired genome amplification was observed. We conclude that high levels of miR-203 are inhibitory to HPV amplification and that HPV proteins act to suppress expression of this microRNA to allow for productive replication in differentiating cells.

APOBEC3G (A3G) is a host cytidine deaminase that serves as a potent intrinsic inhibitor of retroviral replication. A3G is packaged into human immunodeficiency virus type 1virions and deaminates deoxycytidine to deoxyuridine on nascent minus-strand retroviral cDNA, leading to hyper deoxyguanine to deoxyadenine mutations on positive strand cDNA and inhibition of viral replication. The antiviral activity of A3G is suppressed by Vif, a lentiviral accessory protein that prevents encapsidation of A3G. In this study, we identified dominant negative mutants of Vif that interfered with the ability of wild type Vif to inhibit encapsidation and antiviral activity of A3G. These mutants were non-functional due to mutations in the highly conserved HCCH and/or SOCS-box motifs, which are required for assembly of a functional Cul5 E3 ubiquitin ligase complex. Similarly, mutation or deletion of a PPLP motif, which was previously reported to be important for Vif dimerization, induced a dominant-negative phenotype. Expression of dominant-negative Vif counteracted the Vif-induced reduction of intracellular A3G levels presumably by preventing Vif-induced A3G degradation. Consequently, dominant-negative Vif interfered with wild type Vif's ability to exclude A3G from viral particles and reduced viral infectivity despite the presence of wild type Vif. The identification of dominant negative mutants of Vif presents exciting possibilities for the design of novel antiviral strategies.