Jitka Štokrová

Caveolae Are Involved in the Trafficking of Mouse Polyomavirus Virions and Artificial VP1 Pseudocapsids toward Cell Nuclei

ABSTRACT Electron and confocal microscopy were used to observe the entry and the movement of polyomavirus virions and artificial virus-like particles (VP1 pseudocapsids) in mouse fibroblasts and epithelial cells. No visible differences in adsorption and internalization of virions and VP1 pseudocapsids (“empty” or containing DNA) were observed. Viral particles entered cells internalized in smooth monopinocytic vesicles, often in the proximity of larger, caveola-like invaginations. Both “empty” vesicles derived from caveolae and vesicles containing viral particles were stained with the anti-caveolin-1 antibody, and the two types of vesicles often fused in the cytoplasm. Colocalization of VP1 with caveolin-1 was observed during viral particle movement from the plasma membrane throughout the cytoplasm to the perinuclear area. Empty vesicles and vesicles with viral particles moved predominantly along microfilaments. Particle movement was accompanied by transient disorganization of actin stress fibers. Microfilaments decorated by the VP1 immunofluorescent signal could be seen as concentric curves, apparently along membrane structures that probably represent endoplasmic reticulum. Colocalization of VP1 with tubulin was mostly observed in areas close to the cell nuclei and on mitotic tubulin structures. By 3 h postinfection, a strong signal of the VP1 (but no viral particles) had accumulated in the proximity of nuclei, around the outer nuclear membrane. However, the vast majority of VP1 pseudocapsids did not enter the nuclei.

Mouse Polyomavirus Enters Early Endosomes, Requires Their Acidic pH for Productive Infection, and Meets Transferrin Cargo in Rab11-Positive Endosomes

ABSTRACT Mouse polyomavirus (PyV) virions enter cells by internalization into smooth monopinocytic vesicles, which fuse under the cell membrane with larger endosomes. Caveolin-1 was detected on monopinocytic vesicles carrying PyV particles in mouse fibroblasts and epithelial cells (33). Here, we show that PyV can be efficiently internalized by Jurkat cells, which do not express caveolin-1 and lack caveolae, and that overexpression of a caveolin-1 dominant-negative mutant in mouse epithelial cells does not prevent their productive infection. Strong colocalization of VP1 with early endosome antigen 1 (EEA1) and of EEA1 with caveolin-1 in mouse fibroblasts and epithelial cells suggests that the monopinocytic vesicles carrying the virus (and vesicles containing caveolin-1) fuse with EEA1-positive early endosomes. In contrast to SV40, PyV infection is dependent on the acidic pH of endosomes. Bafilomycin A1 abolished PyV infection, and an increase in endosomal pH by NH4Cl markedly reduced its efficiency when drugs were applied during virion transport towards the cell nucleus. The block of acidification resulted in the retention of a fraction of virions in early endosomes. To monitor further trafficking of PyV, we used fluorescent resonance energy transfer (FRET) to determine mutual localization of PyV VP1 with transferrin and Rab11 GTPase at a 2- to 10-nm resolution. Positive FRET between PyV VP1 and transferrin cargo and between PyV VP1 and Rab11 suggests that during later times postinfection (1.5 to 3 h), the virus meets up with transferrin in the Rab11-positive recycling endosome. These results point to a convergence of the virus and the cargo internalized by different pathways in common transitional compartments.

Polyomavirus EGFP-pseudocapsids: Analysis of model particles for introduction of proteins and peptides into mammalian cells

A vector for preparation of mouse polyomavirus capsid‐like particles for transfer of foreign peptides or proteins into cells was constructed. Model pseudocapsids carrying EGFP fused with the C‐terminal part of the VP3 minor protein (EGFP‐VLPs) have been prepared and analysed for their ability to be internalised and processed by mouse cells and to activate mouse and human dendritic cells (DC) in vitro. EGFP‐VLPs entered mouse epithelial cells, fibroblasts and human and mouse DC efficiently and were processed by both, lysosomes and proteasomes. Surprisingly, they did not induce upregulation of DC co‐stimulation molecules or maturation markers in vitro; however, they did induce interleukin 12 secretion.

Interactions of heterologous DNA with polyomavirus major structural protein, VP1

‘Empty polyomavirus pseudocapsids, self‐assembled from the major structural protein VP1, bind DNA non‐specifically and can deliver it into the nuclei of mammalian cells for expression [Forstová et al. (1995) Hum. Gene Ther. 6, 297–306]. Formation of suitable VP1‐DNA complexes appears to be the limiting step in this route of gene delivery. Here, the character of VP1‐DNA interactions has been studied in detail. Electron microscopy revealed that VP1 pseudocapsids can create in vitro at least two types of interactions with double‐stranded DNA: (i) highly stable complexes, requiring free DNA ends, where the DNA is partially encapsidated; and, (ii) weaker interactions of pseudocapsids with internal parts of the DNA chain.

Analysis of mouse polyomavirus mutants with lesions in the minor capsid proteins

Polyomavirus mutants E, Q and H, expressing non-myristylated VP2, were generated by replacing the N-terminal glycine residue with glutamic acid, glutamine or histidine, respectively. Viruses mutated in either VP2 or VP3 translation initiation codons were also prepared. All mutated genomes, when transfected into murine host cells, gave rise to viral particles. Infectivity of VP2- and VP3- viruses, as measured by the number of cells expressing viral antigens, was dramatically diminished, indicative of defects in the early stages of infection. In contrast, the absence of a myristyl moiety on VP2 did not substantially affect the early steps of virus infection. No differences in numbers of cells expressing early or late viral antigens were observed between wild-type (wt) and E or Q myr- viruses during the course of a life cycle. Furthermore, no delay in virus DNA replication was detected. However, when cells were left for longer in culture, the number of infected cells, measured by typical virus bursts, was much lower when mutant rather than wt genomes were used. In situ, cell fractionation studies revealed differences in the interaction of viral particles with host cell structures. The infectivity of mutants was affected not only by loss of the myristyl group on VP2, but also, and to a greater extent, by alterations of the N-terminal amino acid composition.

Production of polyomavirus structural protein VP1 in yeast cells and its interaction with cell structures.

The gene for mouse polyomavirus major structural protein VP1 was expressed in Saccharomyces cerevisiae from the inducible GAL7 promoter. VP1 pseudocapsids were purified from cell lysates. Their subpopulation contained fragments of host DNA, which, in contrast to those of VP1 pseudocapsids produced in insect cells, did not assemble with cellular histones into pseudonucleocores. VP1 pseudocapsids accumulated in the yeast cell nuclei. A strong interaction of VP1 with tubulin fibres of the mitotic spindle was observed. The fibres of spindles were larger in diameter, apparently due to tight VP1 binding. Substantial growth inhibition of yeast cells producing VP1 was observed.

Effect of Dimerizing Domains and Basic Residues on In Vitro and In Vivo Assembly of Mason-Pfizer Monkey Virus and Human Immunodeficiency Virus

ABSTRACT Assembly of immature retroviral particles is a complex process involving interactions of several specific domains of the Gag polyprotein localized mainly within capsid protein (CA), spacer peptide (SP), and nucleocapsid protein (NC). In the present work we focus on the contribution of NC to the oligomerization of CA leading to assembly of Mason-Pfizer monkey virus (M-PMV) and HIV-1. Analyzing in vitro assembly of substitution and deletion mutants of ΔProCANC, we identified a “spacer-like” sequence (NC15) at the M-PMV NC N terminus. This NC15 domain is indispensable for the assembly and cannot be replaced with oligomerization domains of GCN4 or CREB proteins. Although the M-PMV NC15 occupies a position analogous to that of the HIV-1 spacer peptide, it could not be replaced by the latter one. To induce the assembly, both M-PMV NC15 and HIV-1 SP1 must be followed by a short peptide that is rich in basic residues. This region either can be specific, i.e., derived from the downstream NC sequence, or can be a nonspecific positively charged peptide. However, it cannot be replaced by heterologous interaction domains either from GCN4 or from CREB. In summary, we report here a novel M-PMV spacer-like domain that is functionally similar to other retroviral spacer peptides and contributes to the assembly of immature-virus-like particles.

Minor capsid proteins of mouse polyomavirus are inducers of apoptosis when produced individually but are only moderate contributors to cell death during the late phase of viral infection.

Minor structural proteins of mouse polyomavirus (MPyV) are essential for virus infection. To study their properties and possible contributions to cell death induction, fusion variants of these proteins, created by linking enhanced green fluorescent protein (EGFP) to their C‐ or N‐termini, were prepared and tested in the absence of other MPyV gene products, namely the tumor antigens and the major capsid protein, VP1. The minor proteins linked to EGFP at their C‐terminus (VP2–EGFP, VP3–EGFP) were found to display properties similar to their nonfused, wild‐type versions: they killed mouse 3T3 cells quickly when expressed individually. Carrying nuclear localization signals at their common C‐terminus, the minor capsid proteins were detected in the nucleus. However, a substantial subpopulation of both VP2 and VP3 proteins, as well as of the fusion proteins VP2–EGFP and VP3–EGFP, was detected in the cytoplasm, co‐localizing with intracellular membranes. Truncated VP3 protein, composed of 103 C‐terminal amino acids, exhibited reduced affinity for intracellular membranes and cytotoxicity. Biochemical studies proved each of the minor proteins to be a very potent inducer of apoptosis, which was dependent on caspase activation. Immuno‐electron microscopy showed the minor proteins to be associated with damaged membranes of the endoplasmic reticulum, nuclear envelope and mitochondria as soon as 5u2003h post‐transfection. Analysis of apoptotic markers and cell death kinetics in cells transfected with the wild‐type MPyV genome and the genome mutated in both VP2 and VP3 translation start codons revealed that the minor proteins contribute moderately to apoptotic processes in the late phase of infection and both are dispensable for cell destruction at the end of the virus replication cycle.

The Noncanonical Gag Domains p8 and n Are Critical for Assembly and Release of Mouse Mammary Tumor Virus

ABSTRACT The mouse mammary tumor virus (MMTV) Gag contains the unique domains pp21, p3, p8, and n. We investigated the contribution of these domains to particle assembly and found that the region spanning the p8 and n domains is critical for shape determination and assembly. Deletion of pp21 and p3 reduced the number of released particles, but deletion of the n domain resulted in frequent formation of aberrant particles, while deletion of p8 severely impaired assembly. Further investigation of p8 revealed that both the basic and the proline-rich motifs within p8 contribute to MMTV assembly.

In vitro assembly of xenotropic murine leukemia virus-related virus CA-NC protein

Using in vitro expression/assembly system we studied the formation of virus-like particles Xenotropic Murine Leukemia Virus-related virus (XMRV). XMRV is novel human gammaretrovirus discovered in association with human prostate tumors. The genome organization is typical for gammaretroviruses consisting of two overlapping ORFs coding for Gag-Pro-Pol and Env polyproteins. The predicted Gag polyprotein consists of 536 amino acids and is separated from the Pro-Pol sequence by UAG stop codon. n nBased on the amino acids similarities between MLV and XMRV Gag polyproteins, we designed primers bordering CA-NC region of Gag. Resulting PCR fragment was cloned into pET22b vector for expression of CA-NC in E. coli. We found that purified XMRV full-length CANC, starting with the conserved proline residue at the N-terminus of CA, was not able to assemble into particles. However, a modification of the N-terminus of CANC (modCANC) enabled formation of spherical particles. Moreover, the negative staining of the in vitro assembled particles of XMRV modCANC revealed different organization of protein layers in comparison to CA-NC of M-PMV.