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Review
. 2008 Jun;134(1-2):39-63.
doi: 10.1016/j.virusres.2007.12.006. Epub 2008 Feb 14.

Nucleocapsid protein function in early infection processes

James A Thomas  1 , Robert J Gorelick
Affiliations
Review

Nucleocapsid protein function in early infection processes

James A Thomas et al. Virus Res. 2008 Jun.

Abstract

The role of nucleocapsid protein (NC) in the early steps of retroviral replication appears largely that of a facilitator for reverse transcription and integration. Using a wide variety of cell-free assay systems, the properties of mature NC proteins (e.g. HIV-1 p7(NC) or MLV p10(NC)) as nucleic acid chaperones have been extensively investigated. The effect of NC on tRNA annealing, reverse transcription initiation, minus-strand-transfer, processivity of reverse transcription, plus-strand-transfer, strand-displacement synthesis, 3' processing of viral DNA by integrase, and integrase-mediated strand-transfer has been determined by a large number of laboratories. Interestingly, these reactions can all be accomplished to varying degrees in the absence of NC; some are facilitated by both viral and non-viral proteins and peptides that may or may not be involved in vivo. What is one to conclude from the observation that NC is not strictly required for these necessary reactions to occur? NC likely enhances the efficiency of each of these steps, thereby vastly improving the productivity of infection. In other words, one of the major roles of NC is to enhance the effectiveness of early infection, thereby increasing the probability of productive replication and ultimately of retrovirus survival.

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Figures

Fig. 1
Fig. 1. Comparison of retroviral NC proteins
The identity, apparent molecular weight, genus, and relative size of several mature NC proteins are shown. A black box indicates the location of the N-terminal zinc finger, and a grey box indicates the location of the C-terminal zinc finger. Slashed boxes indicate the location of the three Gly-Arg boxes in PFV. Although the PFV NC domain of Gag is shown for comparison, it is not cleaved from the Gag precursor during maturation, and is consequently never a discrete protein (Linial, 1999).
Fig. 2
Fig. 2. Events that occur during HIV-1 replication are shown to illustrate the numerous places where NC functions
A structure of HIV-1 NC, rendered from NMR studies of NC in solution by Summers and coworkers (Lee et al., 1998), is shown in the upper right of the figure. The molecule was drawn using Cn3D version 4.1 (http://www.ncbi.nlm.nih.gov). The backbone of the molecule is shown as a tube structure, with the locations of basic and acidic amino acid residues colored blue and red, respectively. The side chains of aromatic residues are drawn as stick and ball diagrams. The side chains of Cys are shown as space filling pictures. The zinc ions are colored green. Many of the key processes of viral replication require NC. All events are shown in a single cell for convenience, with infection commencing with receptor binding and ending with budding and maturation. A legend for the viral proteins is presented in the upper left of the figure. Early events start with the virus binding to receptors, subsequent fusion of the virus and cell membranes then the core enters the cytoplasm. After cytoplasmic entry, the core begins to uncoat, a RTC forms with reverse transcription ensuing and it is transported through the cytoplasm to the nuclear membrane. The RTC is transformed via the reverse transcription process into a PIC, where it can be actively transported into the nucleus (in nondividing cells), then is targeted to species-specific regions in the chromosomes where integration occurs. The RTC and PIC are shown in the figure without NC or other factors for clarity. Late events commence with transcription of viral messages from the provirus then translation of viral proteins occurs. Subsequently, packaging of gRNA into assembling virus particles results relying on the ability of the NC domain of the Gag precursor to bind specific regions of the gRNA, forming a nucleation site for the multimerization of Gag upon the RNA scaffold. In this illustration, the assembly depicted to occur at the cell membrane has been described by Booth et al. (2006), Deneka et al. (2007), Finzi et al. (2007), Jolly et al. (2007), Jolly and Sattentau (2007), Jouvenet et al. (2006), Rudner et al. (2005), and Welsch et al. (2007). However, some reports indicate that assembly can occur in intracellular compartments (Jouve et al., 2007; Nydegger et al., 2003; Perlman and Resh, 2006; Sherer et al., 2003). The maturation of virions after budding entails several events that are likely interrelated, from the gRNA dimer maturation, which result in extensive intra- and intermolecular interactions, as well as the condensation of the nucleoprotein core. (Artwork courtesy of Louis E. Henderson, AIDS Vaccine Program, SAIC-Frederick, Inc., NCI-Frederick).
Fig. 3
Fig. 3. Proteolytic processing of HIV-1 Gag by PR
The pattern and order of PR cleavage sites in Gag are shown. For proteins other than NC, the different species that exist during processing are indicated. The initial cleavage occurs between SP1 and the NC domain, secondary cleavages occur at approximately 10-fold lower rates than the initial cut. The tertiary cleavages occur approximately 400-fold slower than the primary cleavage (Shehu-Xhilaga et al., 2001b). Complete processing of Gag into its mature proteins is efficient and rapid, so that almost immediately after budding, the intermediates are not typically detected by Western blotting (Kaplan et al., 1994b). During proteolysis, NC exists in two intermediate forms, p15NC (partial cleavage product containing NC/SP2/p6), p9NC (partial cleavage product containing NC/SP2), and the fully processed form, p7NC. All three of these proteins exhibit nucleic acid chaperone activities (Cruceanu et al., 2006).
Fig. 4
Fig. 4. Key steps of reverse transcription where NC has been shown to be involved (HIV-1 example presented)
The process of reverse transcription entails the conversion of a plus-strand RNA genome (A) to a double-strand DNA (H), with an LTR duplicated at each end (638 bp in the case of HIV-1) (Telesnitsky and Goff, 1997). The first requirement for reverse transcription is the annealing of a primer tRNA molecule onto the PBS of the gRNA (B). Once annealed, reverse transcriptase synthesizes vDNA to the end of the genome; this product is termed minus-strand strong-stop DNA (C). As RT continues synthesizing the vDNA, the RNase H activity of RT cleaves the gRNA involved in heteroduplex formation into small fragments (Schultz and Champoux, 2008) (C). RNase H digestion allows the transfer of the cDNA repeat ® region, facilitated by NC, to the RNA R region at the 3′ end of the genome, an event termed minus-strand transfer (D). Minus-strand transfer can occur intra- or intermolecularly, so that either genome can contribute genetic material to the final product. Upon transfer, minus-strand synthesis proceeds until it reaches the PBS at the 5′ end of the genome. As RT continues to synthesize the vDNA, RNase H functions to cleave the genome into small fragments. In HIV-1, two regions, the PPT and cPPT (E), are resistant to RNase H cleavage. They both serve as primers for plus-strand DNA synthesis, using the minus-strand DNA as template. As the plus-strand synthesis reaches the 5′ end of the minus-strand DNA, it copies the PBS from the tRNA that remains covalently bound to the 5′ end of the minus strand (E). The two complementary PBS regions on the minus- and plus-DNA strands anneal, forming a circular intermediate (F) (in the case of the intramolecular transfer that is presented in this example), enabling the synthesis of the full plus-strand DNA sequence. Two additional steps are important; completion of the 5′ LTR requires the strand-displacement synthesis activity of RT (G). In an additional region, strand-displacement synthesis occurs in HIV-1 resulting in the central flap, a 99 nt region of ssDNA generated by limited synthesis at the cPPT (not shown).
Fig. 5
Fig. 5. Integration
Integration of vDNA into the host chromosome is the final step of early infection (Brown, 1997). The substrate for this reaction is full-length double-strand vDNA (A). Each LTR contains an att site, necessary for the first enzymatic step performed by IN. In this step, 3′ processing of the vDNA ends occur, which generates a free 3′-CA end (B). The host chromosome is cut to generate staggered nicks, 4 nucleotides apart in the case of HIV-1. The 3′ hydroxyl of the processed vDNA joins to the 5′ PO4 in the second enzymatic step of integration, strand-transfer (C). This step results in a 2-nucleotide flap with the 5′ vDNA overhang. These 2 nucleotides are then removed, and the 6 nucleotide single-stranded region is repaired by nuclear enzymes to create direct repeats at each end (Brown, 1997; Yoder and Bushman, 2000). The resulting integrated vDNA is called the provirus (D).
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