Antonito T. Panganiban

Circles with two tandem LTRs are precursors to integrated retrovirus DNA.

Infection of susceptible cells by retroviruses results in the synthesis of linear DNA with two long terminal repeats (LTRs), circular DNA with a single LTR, and circular DNA with two tandem LTRs. To determine which of these unintegrated molecules serves as the precursor to the provirus, we inserted into a retrovirus vector a 49 bp fragment containing the junction formed by in vivo blunt-end ligation of two LTRs. Infection of chicken embryo fibroblasts with virus recovered from this vector and subsequent characterization of the proviral DNA revealed that efficient integration can occur from this introduced junction sequence. Therefore, circular DNA with two tandem LTRs is a precursor to the provirus.

Small glutamine-rich protein/viral protein U–binding protein is a novel cochaperone that affects heat shock protein 70 activity

Abstract Molecular chaperone complexes containing heat shock protein (Hsp) 70 and Hsp90 are regulated by cochaperones, including a subclass of regulators, such as Hsp70 interacting protein (Hip), C-terminus of Hsp70 interacting protein (CHIP), and Hsp70-Hsp90 organizing factor (Hop), that contain tetratricopeptide repeats (TPRs), where Hsp70 refers to Hsp70 and its nearly identical constitutive counterpart, Hsc70, together. These proteins interact with the Hsp70 to regulate adenosine triphosphatase (ATPase) and folding activities or to generate the chaperone complex. Here we provide evidence that small glutamine-rich protein/viral protein U–binding protein (SGT/UBP) is a cochaperone that negatively regulates Hsp70. By “Far-Western” and pull-down assays, SGT/UBP was shown to interact directly with Hsp70 and weakly with Hsp90. The interaction of SGT/UBP with both these protein chaperones was mapped to 3 TPRs in SGT/UBP (amino acids 95–195) that are flanked by charged residues. Moreover, SGT/UBP caused an approximately 30% reduction in both the intrinsic ATPase activity of Hsc70 and the ability of Hsc70 to refold denatured luciferase in vitro. This negative effect of SGT/UBP on Hsc70 is similar in magnitude to that observed for the cochaperone CHIP. A role for SGT/UBP in protein folding is also supported by evidence that a yeast strain containing a deletion in the yeast homolog to SGT/UBP (ΔSGT/UBP) displays a 50-fold reduction in recovery from heat shock compared with the wild type parent. Together, these results are consistent with a regulatory role for SGT/UBP in the chaperone complex.

Cell type-dependence for Vpu function

Human immunodeficiency virus type 1 Vpu has been shown to facilitate virus release from HeLa cells. We demonstrated that Vpu expression is not required for efficient virus release from Cos 1 and CV‐1 cells. A yeast GAL4 transcriptional activation system was used to screen for cellular proteins that may interact with Vpu. One such protein was identified which we provisionally designate “Vpu interactive protein” or VIP.

Duplication of the primary encapsidation and dimer linkage region of human immunodeficiency virus type 1 RNA results in the appearance of monomeric RNA in virions.

ABSTRACT The dimerization initiation site (DIS) and the dimer linkage sequences (DLS) of human immunodeficiency virus type 1 have been shown to mediate in vitro dimerization of genomic RNA. However, the precise role of the DIS-DLS region in virion assembly and RNA dimerization in virus particles has not been fully elucidated, since deletion or mutation of the DIS-DLS region also abolishes the packaging ability of genomic RNA. To characterize the DIS-DLS region without altering packaging ability, we generated mutant constructs carrying a duplication of approximately 1,000 bases including the encapsidation signal and DIS-DLS (E/DLS) region. We found that duplication of the E/DLS region resulted in the appearance of monomeric RNA in virus particles. No monomers were observed in virions of mutants carrying the E/DLS region only at ectopic positions. Monomers were not observed whenpol or env regions were duplicated, indicating an absolute need for two intact E/DLS regions on the same RNA for generating particles with monomeric RNA. These monomeric RNAs were most likely generated by intramolecular interaction between two E/DLS regions on one genome. Moreover, incomplete genome dimerization did not affect RNA packaging and virion formation. Examination of intramolecular interaction between E/DLS regions could be a convenient tool for characterizing the E/DLS region in virion assembly and RNA dimerization within virus particles.

The Triplet Repeats of the Sin Nombre Hantavirus 5′ Untranslated Region Are Sufficient in cis for Nucleocapsid-Mediated Translation Initiation

ABSTRACT Hantavirus nucleocapsid protein (N) can replace the cellular cap-binding complex, eukaryotic initiation factor 4F (eIF4F), to mediate translation initiation. Although N can augment translation initiation of nonviral mRNA, initiation of viral mRNA by N is superior. All members of the Bunyaviridae family, including the species of the hantavirus genus, express either three or four primary mRNAs from their tripartite negative-sense genomes. The 5′ ends of the mRNAs contain nonviral heterologous oligonucleotides that originate from endonucleolytic cleavage of cellular mRNA during the process of cap snatching. In the hantaviruses these caps terminate with a 3′ G residue followed by nucleotides arising from the viral template. Further, the 5′ untranslated region (UTR) of viral mRNA uniformly contains, near the 5′ end, either two or three copies of the triplet repeat sequence, UAGUAG or UAGUAGUAG. Through analysis of a panel of mutants with mutations in the viral UTR, we found that the sequence GUAGUAG is sufficient for preferential N-mediated translation initiation and for high-affinity binding of N to the UTR. This heptanucleotide sequence is present in viral mRNA containing either two or three copies of the triplet repeat.

Bunyavirus N: eIF4F surrogate and cap-guardian

Hantaviruses comprise a genus of the bunyavirus family of viruses. Viruses of this family, along with the arenaviruses, and the orthomyxoviruses, including influenza, contain a negative sense, segmented RNA genome. Viral nucleocapsid proteins play a well-established role in the formation of intracellular and virion-associated nucleocapsids that harbor and shield viral genomic RNA. However, recent observations indicate that hantavirus nucleocapsid protein (N) has additional unexpected biological activities that interface with both the cellular mRNA translation and mRNA degradation apparatus. N has an activity that mimics or circumvents the cellular cap-binding complex, eIF4F, in the initial stages of translation initiation. As a consequence of its translation initiation activity, N can augment translational expression. In addition to its ability to enhance translation initiation, N co-localizes with the cellular peptides that mediate mRNA decay. mRNA decay often takes place in cytoplasmic processing bodies (P-bodies), and N is abundant in P bodies. The association of N with P bodies enables cap-snatching for viral transcription initiation. It is likely that these two surprising new activities of N function in concert during bunyavirus gene expression. All the activities of N revolve around the ability of N to recognize RNA in a correct, context-dependent manner.

N-linked glycosylation and reticuloendotheliosis retrovirus envelope glycoprotein function

Different properties of the spleen necrosis virus (SNV) envelope glycoprotein were analyzed following biosynthesis in the presence of glycosylation inhibitors. Tunicamycin, which inhibits all asparagine N-linked glycosylation, prevented intracellular processing and translocation to the cell surface of the envelope protein. In contrast, castanospermine or deoxymannojirimycin, which block glycosidase trimming of the early high-mannose chains and subsequent complex type N-glycosylation, did not inhibit proteolytic cleavage or cellular translocation. The ability of unglycosylated and partially glycosylated envelope protein to bind the viral receptor was assayed using an infection interference assay. Tunicamycin abrogated SNV envelope glycoprotein-induced receptor interference, whereas the trimming glycosidase inhibitors had no effect on interference. Similarly, tunicamycin but not the glycosidase inhibitors reduced the titers of released virus 100-fold. We conclude that carbohydrate trimming and complex N-glycosylation are not essential for envelope glycoprotein translocation, proteolytic cleavage, receptor binding, or infectivity, whereas cotranslational high-mannose N-glycosylation is essential for all of the SNV envelope glycoprotein properties tested. Syncytia formation can be induced following transfection into D17 cells of an envelope glycoprotein expression plasmid. Unlike virus particle infectivity, cell fusion is strongly inhibited by the glycosidase inhibitors.

Strand Switching During Retroviral Reverse Transcription

A distinctive feature of the retroviral life cycle is the process of reverse transcription which results in the conversion of the single stranded RNA genome into a linear double stranded DNA copy. A principal area of research in our lab has centered on template switching during reverse transcription and the consequences of this translocation depending on whether switching occurs intermolecularly or intramolecularly. For these studies we have examined the process of nucleic acid replication in replication-competent avian reticuloendotheliosis viruses (REV).