Hepadnavirus cores

Details Hepadnavirus cores Hepadnavirus cores

1997-07-11

2003-02-11

Woodward, Michael P. (Department: 1631)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S320100, C435S235100

Reexamination Certificate

active

06518014

ABSTRACT:

The present invention relates to cell-free hepadnavirus-derived core particles comprising hepadnavirus core proteins, hepadnavirus polymerase and a nucleic acid such as RNA that is a template for initiation, initial chain elongation or other steps in the replication of a nucleic acid strand encoding at least a portion of the viral genome.
Hepadnaviruses such as hepatitis B virus (“HBV”) replicate by a unique pathway that has proved difficult to reconstitute in vitro. The viral genome is a partially duplex DNA. Covalently attached to the 5′ end of the (−) strand is a copy of the viral-encoded polymerase enzyme (P) involved in viral replication. The (−) strand contains the entire viral genome. The ends of the (−) strand are not ligated, but instead are held in proximity to one another by an overlapping (+) strand which, depending on the viral isolate, comprises about 20% to about 80% of the length of the (−) strand. The (+) strand has a short, capped segment of RNA covalently attached at the 5′ end. After infection, the viral genome is believed to migrate to the nucleus of the infected cell, where cellular DNA repair processes are believed to convert it to a closed, circular form, termed “cccDNA”. The closed, circular form is then transcribed to create messenger RNA's encoding viral proteins, including the hepadnavirus polymerase and the hepadnavirus core protein (C) that forms the core particle (i.e., capsid) that encapsidates the viral genetic information (thereby becoming a nucleocapsid). Some of the RNA transcripts are full-length and serve as the template for the replication of the viral genome; these RNA transcripts are known as pregenomic RNA (pgRNA). Hepadnavirus polymerases function (a) as reverse transcriptases, synthesizing the (−) strand of the genomic DNA using the pgRNA as a template, (b) as DNA polymerases, synthesizing the (+) strand of genomic DNA, and (c) as RNase Hs, sequentially digesting portions of the pgRNA template immediately after they have been reverse transcribed. See, Ganem et al. Infectious Agents and Disease 3: 85093, 1994, for a review of the literature on hepadnavirus replication.
The pgRNA typically has the following properties: (a) it is capped; (b) it is greater than genome length; (c) each end (both the 5′ and the 3′ end) has a repeat that includes (i) a sequence element termed “DR1” whose 3′ copy is the apparent replication origin of the virus and (ii) a stem-loop-bulge sequence element, termed “&egr;”, that contains the true replication origin; and (d) just to the 5′ side of the 3′ repeat there is another copy of the replication origin sequence designated the “DR2” element. According to current understanding, in the cytoplasm the hepadnavirus polymerase and pgRNA are encapsidated into the core particle formed from multiple copies of C. Hepadnavirus polymerase interacts with the 5′&egr; element and reverse transcribes a 3 to 4 base oligomer from the template provided by a sequence within the bulge of the stem-loop-bulge sequence. The first covalent bond in this reverse transcription is formed between a tyrosine hydroxyl of hepadnavirus polymerase and the 5′ phosphate of a deoxynucleotide specified as complementary to a nucleotide of the stem-loop bulge template (see step 1 illustrated in FIG.
1
). Thus, in a sense, hepadnavirus polymerase is the “primer” for the initial reverse transcript. This initial bond formation and the subsequent formation of the initial three to four-mer is termed the “priming” reaction. The protein and covalently attached oligomer then migrate to a complementary sequence found in the 3′ DR1 element. This migration step is termed the “translocation” reaction. From the three or four-mer now base-paired at the 3′ DR1, the polymerase reverse transcribes through to the 5′ end of the pgRNA (see step 2 illustrated in FIG.
1
), thereby synthesizing the (−) strand of the viral genome. This reverse transcription step is here termed the “(−) strand elongation” reaction. Concurrently with catalyzing the reverse transcription, a separate domain of hepadnavirus polymerase exhibits a RNase H activity that digests the RNA after it has been reverse transcribed into DNA (see step 3 illustrated in FIG.
1
). Upon completion of the synthesis of the (−) strand, a 17 to 18 base residue of the 5′ end of the pgRNA including the DR1 sequence remains (see step 4 illustrated in FIG.
1
). This residue is translocated to the complementary DR2 element of the (−) strand, and then serves as the primer for the synthesis, again mediated by hepadnavirus polymerase, of a (+) strand priming fragment of the (+) strand complementary to the 5′ end of the (−) strand (see step 5 illustrated in FIG.
1
). A portion of this (+) strand priming fragment is also complementary to the 3′ end of the (−) strand and, through this complementarity, the (+) strand priming fragment is used to create a non-covalent bridge linking the two ends of the (−) strand (see step 6 illustrated in FIG.
1
). Once the bridge is formed, further (+) strand synthesis proceeds.
In infected cells, hepadnavirus replication occurs inside the viral nucleocapsid. Moreover, genetic studies have implicated C as critical to the process of viral replication in vivo. Nassal, J. Virol. 66: 4107-4116, 1992; Schlicht et al., J. Virol. 63: 2995-3000, 1989; Yu and Summers, J. Virol. 65: 2511-2517, 1991. Nonetheless, it has proved possible, after substantial initial difficulty, to measure some initial replicative activity in vitro—outside of the core particles—using copies of hepadnavirus polymerase produced by a variety of molecular biology-based techniques. See, for example, Seifer and Standring, J. Virol. 67: 4513-4520; Tavis and Ganem, Proc. Natl. Acad. Sci. USA 90: 4107-4111, 1993; Lanford, J. Virol. 69: 4431-4439, 1995; Seeger, U.S. Pat. No. 5,334,525. However, given the importance of C and core particles to replication in vivo, it is clear that such systems do not faithfully reflect the authentic replication environment and are thus of only limited value as tools for identifying antiviral agents that disrupt viral replication. Furthermore, it is believed that these systems have only a limited capability to elongate minus-strand DNA chains, and that these systems at least in vitro have not been shown to elongate de novo chains of more than, for example, 200 nucleotides.
Others have transfected mammalian cells in “trans”, meaning two separate expression vectors were used to express C and polymerase and thus create core particles. The cells were transfected with (a) an expression vector specifying a pgRNA which encodes hepadnavirus polymerase but has a frame-shift mutation making it deficient for the production of C and (b) an expression vector encoding C. See, for example, Bartenschlager et al., J. Virol. 64: 5324-5332, 1990 and Hirsch et al., Nature, 344: 552-555, 1990, both of which articles report mutational studies indicating that hepadnavirus polymerase is needed to correctly package the pgRNA into viral core particles. What this prior work has not done is isolate core particles that are “frozen” in an early stage of the replication process such that the core particles can be used in an in vitro assay that reproduces the intra-core particle environment in which the replication process occurs in vivo. The core particles of this prior work are also believed to have replicated more extensively and have completed much of there (−)-strand synthesis. These prior art core particles thus have reduced reverse transcriptase activity in vitro relative to core particles frozen in a early stage of replication. I
What is needed for determining whether a test compound inhibits early genomic replication mediated by hepadnavirus polymerase is an in vitro system wherein hepadnavirus polymerase operates within the core particle, its natural operative environment, and wherein hepadnavirus polymerase o

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