The ruminants are one of the most successful mammalian lineages, exhibiting morphological and habitat diversity and containing several key livestock species. To better understand their evolution, we generated and analyzed de novo assembled genomes of 44 ruminant species, representing all six Ruminantia families. We used these genomes to create a time-calibrated phylogeny to resolve topological controversies, overcoming the challenges of incomplete lineage sorting. Population dynamic analyses show that population declines commenced between 100,000 and 50,000 years ago, which is concomitant with expansion in human populations. We also reveal genes and regulatory elements that possibly contribute to the evolution of the digestive system, cranial appendages, immune system, metabolism, body size, cursorial locomotion, and dentition of the ruminants.

Mutations in the S region of the hepatitis B virus (HBV) envelope gene are associated with immune escape, occult infection, and resistance to therapy. We previously identified naturally occurring mutations in the S gene that alter HBV virion secretion. Here we used transcomplementation assay to confirm that the I110M, G119E, and R169P mutations in the S domain of viral envelope proteins impair virion secretion and that an M133T mutation rescues virion secretion of the I110M and G119E mutants. The G119E mutation impaired detection of secreted hepatitis B surface antigen (HBsAg), suggesting immune escape. The R169P mutant protein is defective in HBsAg secretion as well and has a dominant negative effect when it is coexpressed with wild-type envelope proteins. Although the S domain is present in all three envelope proteins, the I110M, G119E, and R169P mutations impair virion secretion through the small envelope protein. Conversely, coexpression of just the small envelope protein of the M133T mutant could rescue virion secretion. The M133T mutation could also overcome the secretion defect caused by the G145R immune-escape mutation or mutation at N146, the site of N-linked glycosylation. In fact, the M133T mutation creates a novel N-linked glycosylation site ( 131 NST 133 ). Destroying this site by N131Q/T mutation or preventing glycosylation by tunicamycin treatment of transfected cells abrogated the effect of the M133T mutation. Our findings demonstrate that N-linked glycosylation of HBV envelope proteins is critical for virion secretion and that the secretion defect caused by mutations in the S protein can be rescued by an extra glycosylation site.The hepatitis B virus (HBV) is an enveloped DNA virus with a tropism for the liver. The 3.2-kb HBV genome harbors genes encoding core protein and its secreted version (called HBeAg), DNA polymerase, the transcriptional transactivator HBx, and envelope proteins. The envelope gene, which is completely overlapped by the polymerase gene, has three in-frame AUG codons that can serve as alternative translation initiation sites. This leads to the expression of three coterminal envelope proteins: large (L), middle (M), and small (S). The sequence unique to the L protein is called the pre-S1 domain, while a downstream sequence shared with the M protein is called the pre-S2 domain. The S domain is present in all three envelope proteins. The S and M proteins are translated from a 2.1-kb subgenomic RNA with a heterogeneous 5Ј end, while the L protein is expressed from a longer (2.4-kb) subgenomic RNA. The S protein is the most abundantly expressed envelope protein. The L and S proteins exist in nonglycosylated and monoglycosylated forms (L protein, p39 and gp42, respectively; S protein, p24 and gp27, respectively) due to a facultative Nlinked glycosylation site (N-X-S/T) at N146 of the S domain. The M protein contains an extra, constitutive N-linked glycosylation site at position 4 in the pre-S2 domain and consequently exists in monoglycosylated (gp33) and diglycosylated (gp36) form...

Hepatitis B virus (HBV) contains three coterminal envelope proteins on the virion surface: large (L), middle (M), and small (S). The M and S proteins are also secreted as empty "subviral particles," which exceed virions by at least 1,000-fold. The S protein serves as the morphogenic factor for both types of particles, while the L protein is required only for virion formation. We found that cotransfecting replication constructs with a small dose of the expression construct for the missing L, M, and S proteins reconstituted efficient virion secretion but only 5 to 10% of subviral particles. The L protein inhibited secretion of subviral particles in a dose-dependent manner, whereas a too-high or too-low L/S protein ratio inhibited virion secretion. Consistent with the results of cotransfection experiments, a point mutation at the ؊3 position of the S gene AUG codon reduced HBsAg secretion by 60 to 70% but maintained efficient virion secretion. Surprisingly, ablating M protein expression reduced virion secretion but markedly increased the maturity of virion-associated genomes, which could be reversed by providing in trans both L and M proteins but not just M protein. M protein stability was dependent on the coexpression of S protein. Our findings suggest that efficient HBV virion secretion could be maintained despite drastic reduction in subviral particle production, which supports the recent demonstration of separate secretion pathways adopted by the two types of particles. The M protein appears to facilitate core particle envelopment, thus shortening the window of plus strand DNA elongation.Hepatitis B virus (HBV) is a small enveloped DNA virus that infects only humans and higher primates (17). It is estimated that 2 billion people worldwide have been exposed to HBV and more than 350 million are chronically infected. Inside the 42-nm virion is a nucleocapsid or core particle that shields the relaxed circular double stranded DNA genome. The minus-strand DNA is full length (3.2-kb) and terminally redundant, whereas the plus-strand DNA is incomplete. Upon infection of hepatocytes, the genome is repaired to form the unit-length, fully double-stranded covalently closed circular DNA in the nucleus (54), which serves as the template for transcription. The four major transcripts of 3.5, 2.4, 2.1, and 0.7 kb are termed pre-C/C, pre-S1, preS2/S, and X, respectively, all sharing the same 3Ј end but different 5Ј initiation sites (4). The mRNAs are exported to cytoplasm for protein translation and genome replication. The 3.5-kb transcript is over the genome length and therefore contains all of the genetic information of the viral genome. The longer and less abundant version (preC RNA) directs the expression of hepatitis B e antigen (HBeAg), a secreted viral protein with immunomodulatory function (37). The shorter one or pregenomic RNA (pgRNA) serves as the messenger for core protein and viral polymerase, as well as the template for DNA synthesis. To this end, the nascent core protein assembles into core particle, packaging bot...