No abstract

The murine gammaherpesvirus-68 genome encodes 73 protein-coding open reading frames with extensive similarities to human c 2 herpesviruses, as well as unique genes and cellular homologues. We performed transcriptome analysis of stage-specific viral RNA during permissive infection using an oligonucleotide-based microarray. Using this approach, M4, K3, ORF38, ORF50, ORF57 and ORF73 were designated as immediate-early genes based on cycloheximide treatment. The microarray analysis also identified 10 transcripts with early expression kinetics, 32 transcripts with early-late expression kinetics and 29 transcripts with late expression kinetics. The latter group consisted mainly of structural proteins, and showed high expression levels relative to other viral transcripts. Moreover, we detected all eight tRNA-like transcripts in the presence of cycloheximide and phosphonoacetic acid. Lytic infection with MHV-68 also resulted in a significant reduction in the expression of cellular transcripts included in the DNA chip. This global approach to viral transcript analysis offers a powerful system for examining molecular transitions between lytic and latent virus infections associated with disease pathogenesis.

Hitherto, all enveloped viruses were thought to shed their lipid membrane during entry into cells by membrane fusion. The extracellular form of Vaccinia virus has two lipid envelopes surrounding the virus core, and consequently a single fusion event will not deliver a naked core into the cell. Here we report a previously underscribed mechanism in which the outer viral membrane is disrupted by a ligand-induced nonfusogenic reaction, followed by the fusion of the inner viral membrane with the plasma membrane and penetration of the virus core into the cytoplasm. The dissolution of the outer envelope depends on interactions with cellular polyanionic molecules and requires the virus glycoproteins A34 and B5. This discovery represents a remarkable example of how viruses manipulate biological membranes, solves the topological problem of how a double-enveloped virus enters cells, reveals a new effect of polyanions on viruses, and provides a therapeutic approach for treatment of poxvirus infections, such as smallpox.antiviral therapy ͉ extracellular enveloped virus ͉ membrane dissolution ͉ Vaccinia virus ͉ virus entry H itherto, membrane fusion was the only known mechanism by which enveloped viruses overcome the lipid barrier to enter and replicate in cells (1, 2). Here we show that the extracellular enveloped virus (EEV) of Vaccinia virus (VACV) sheds its outer lipid membrane by a ligand-dependent nonfusogenic mechanism.VACV replication produces several distinct virions: the intracellular mature virus (IMV), intracellular enveloped virus, cellassociated enveloped virus (CEV), and EEV (3, 4). IMV is surrounded by one lipid membrane (5-9) and is physically robust to aid virus transmission between hosts. CEV and EEV are IMV particles wrapped with an additional membrane derived from the trans-Golgi network (10) or endosomes (11) and are responsible for virus dissemination within the host (4). This extra lipid envelope (EEV membrane) and the associated virus and host membrane proteins serve to protect the IMV particle within from immune surveillance and may contribute to a broader cell tropism of the virus (4). Recently, we provided unequivocal electron micrographs showing that IMV enters by fusion with the plasma membrane (8), consistent with previous reports (6,12,13), and the recent genetic evidence for entry by fusion (14-17). Once the naked core has entered the cytoplasm, it moves deeper into the cell on microtubules (18). However, for VACV EEV, the additional EEV membrane presents an unexplained topological problem for entry, because fusion of the EEV outer envelope with the plasma membrane or the membrane of an intracellular vesicle will release only an IMV, instead of a naked core, into the cytosol. For a recent review of VACV entry, see ref.19.Here we studied the entry of EEV by immuno-EM and demonstrated that the EEV outer membrane is disrupted at the point of cell contact after binding. This enables the IMV within to enter the cell by fusion with the plasma membrane. The ligands required for membrane rupture were ...

No abstract

Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE), we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37°C), avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human proximal airways (32°C). These data support the hypothesis that avian influenza viruses, ordinarily adapted to the temperature of the avian enteric tract (40°C), rarely infect humans, in part due to differences in host airway regional temperatures. Previously, a critical residue at position 627 in the avian influenza virus polymerase subunit, PB2, was identified as conferring temperature-dependency in mammalian cells. Here, we use reverse genetics to show that avianization of residue 627 attenuates a human virus, but does not account for the different infection between 32°C and 37°C. To determine the mechanism of temperature restriction of avian influenza viruses in HAE at 32°C, we generated recombinant human influenza viruses in either the A/Victoria/3/75 (H3N2) or A/PR/8/34 (H1N1) genetic background that contained avian or avian-like glycoproteins. Two of these viruses, A/Victoria/3/75 with L226Q and S228G mutations in hemagglutinin (HA) and neuraminidase (NA) from A/Chick/Italy/1347/99 and A/PR/8/34 containing the H7 and N1 from A/Chick/Italy/1347/99, exhibited temperature restriction approaching that of wholly avian influenza viruses. These data suggest that influenza viruses bearing avian or avian-like surface glycoproteins have a reduced capacity to establish productive infection at the temperature of the human proximal airways. This temperature restriction may limit zoonotic transmission of avian influenza viruses and suggests that adaptation of avian influenza viruses to efficient infection at 32°C may represent a critical evolutionary step enabling human-to-human transmission.