Method of detecting cytomegalovirus (CMV)

Details Method of detecting cytomegalovirus (CMV) Method of detecting cytomegalovirus (CMV)

1997-11-21

2001-02-27

Riley, Jezia (Department: 1656)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C530S350000, C436S022000, C435S006120

Reexamination Certificate

active

06194542

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to diagnostic and therapeutic methods employing latent transcripts and promoters of human cytomegalovirus.
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BACKGROUND OF THE INVENTION
Human cytomegalovirus (CMV), a ubiquitous species-specific herpesvirus and significant pathogen in immunocompromised individuals and neonates (Ho; Alford and Britt, 1993), is the best studied member of the betaherpesviruses (Morarski, 1993). Latent infection is a hallmark of all herpesviruses, and the neuronal site of latency of the alphaherpesviruses (such as herpes simplex virus-1) as well as the lymphoid site of latency of the gammaherpesviruses (such as Epstein-Barr virus) have been well-studied (Roizman and Sears, 1993; Liebowitz and Kieff, 1993). However, although latent infection by CMV is widespread and reactivation of latent virus after either immunosuppression or progressive immunodeficiency is the single most important contributor to emergence of CMV disease, the site(s) of viral latency remain poorly characterized (Mocarski, 1993).
Viral DNA has been detected in peripheral blood cells of healthy seropositive carriers (Bevan, et al., Taylor-Wiedeman, et al., 1991), and monocytes have been implicated as the most likely cell type harboring latent viral genomes (Taylor-Wiedeman, et al., 1991). Although the CMV genome persists in monocytes, virus does not reactivate during cultivation under conditions that stimulate growth and differentiation (Taylor-Wiedeman, et al., 1994). Thus, it has remained unclear whether monocytes, or other mononuclear cell types in peripheral blood, correspond to true sites of latency or simply reflect an occasional depository of viral DNA during sporadic reactivation or persistent infection (Ibanez, et al., Lathey and Spector, Schrier, et al.).
Two well-studied human CMV (HCMV) strains—Towne and AD169—have been sequenced (Stenberg, et al., 1984; Stenberg, et al., 1985; Boxhart, et al., 1985; Stenberg, et al., 1989; Chee, et al., 1990).
SUMMARY OF THE INVENTION
The present invention includes a purified polypeptide that is (i) encoded by cytomegalovirus (CMV) DNA sequences and (ii) produced specifically during latent infection. In one embodiment, such polypeptides (herein referred to as latency-associated polypeptides) contain an amino acid sequence encoded by sequences derived from the CMV genomic region approximately 500 bp 5′ to the CMV PSS ie1/ie2 transcription start site.
Further, a polypeptide of the present invention can be encoded by an RNA whose transcription start site is located within an approximately 500 base pair region of the CMV genome 5′ to the PSS CMV ie1/ie2 transcription start site. In one embodiment, the transcription start site is contained within the ie1/ie2 enhancer region, for example, an RNA whose transcription start site is SEQ ID NO:36 or SEQ ID NO:37. Exemplary latency-associated polypeptides include, but are not limited to, SEQ ID NO:59, SEQ ID NO:61 and SEQ ID NO:63.
In a further embodiment, the polypeptide of the present invention is encoded by an RNA transcribed from the strand complementary to the coding strand (i.e., antisense) for CMV ie1/ie2 transcripts, where the location of said RNA overlaps introns 2 and 3 of the ie1/ie2 gene: the DNA sequence presented as SEQ ID NO:57 corresponds to one such RNA. Exemplary latency-associated polypeptides encoded by antisense transcripts include, but are not limited to SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71 and SEQ ID NO:73.
In addition, the polypeptides of the present invention include translation products derived from cytomegalovirus latency transcripts.
The present invention also includes methods for making the polypeptides of the present invention, such as, recombinant or synthetic production. Further, the invention includes vectors capable of expressing the latency-associated polypeptides in a selected host. Exemplary coding sequences for latency-associated polypeptides include, but are not lim

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